The voltage-activation of contraction in skeletal muscle

Prog.Biophys.raolec.Biol.,Vol. 57, pp. 181-223, 1992. Printed in Great Britain.

0079-6107/92 $15.00 1992 PergamonPressLtd

THE VOLTAGE-ACTIVATION OF CONTRACTION IN SKELETAL MUSCLE ANGELAF. DULHUNTY John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra City, A C T 2601, Australia

CONTENTS I. INTRODUCTION II. STRUCTURESAND PROTEINS IN EC COUPLING 1. The T-system, Triad Junction and Junctional "Feet'" 2. Proteins Associated with the Junctional Foot Complex (a) The voltage sensitive molecule--a dihydropyridine receptor (b) The ryanodine receptor calcium release channel (c) Coupling proteins (d) Proteins that modulate calcium release (e) Cytoskeletal and other proteins 3. Questions about the Calcium Release Channels (a) Is there more than one type of calcium release channel? (b) Where is the inositol 1,4,5-triphosphate receptor? (c) Are all calcium release channels in junctional feet? III. PHYSIOLOGICALSTUDIES OF EC COUPLING 1. Voltage-activated Tension 2. Optical Measurements of Events in EC Coupling (a) Optical indicators (b) Calcium transients (c) The rate of calcium release 3. Electrical Signals from the Voltage Sensor (a) The origin of asymmetric charge movement (b) "Charge I" and "charge 2" (c) The two components of char#e 1 (d) Immobilization of charge movement 4. Properties of Single Calcium Release Channels (a) Activation and permeability of ryanodine and IPj receptors (b) Subconductance states of the calcium release channel (c) The "sarcobalr' technique IV. ACTIVATIONAND INACTIVATIONOF EC COUPLING 1. Depolarization-dependent Activation of Contraction (a) The voltage-dependence of tension is set by the voltage sensor (b) Tension-voltage curves: a labile property of muscle (c) Molecular regulation of voltage sensitivity 2. Inactivation of Contraction (a) Inactivation is related to a conformational state of the voltage sensor (b) Steady-state inactivation: shifts in voltage dependence (c) Molecular mechanisms underlying inactivation 3. The Kinetics of lnactivation (a) Slow time course of inactivation in mammals (b) A biphasic time course of inactivation: two inactivated states (c) A sequential model with two inactivated states (d) Pedestal tensions: independent or sequential activation and inactivation processes V. THE MECHANISMOF EC COUPLING VI. CONCLUDING COMMENTS ACKNOWLEDGEMENTS REFERENCES

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I. I N T R O D U C T I O N Tension in skeletal muscle is generated when action potentials invade the interior of individual fibres along the membranes of the transverse (T-) tubule system. Passage of the action potential is followed by the increase in free myoplasmic calcium concentration that activates troponin and allows cross-bridge cycling and contraction. The term "excitation181

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contraction (EC) coupling" refers to the events, triggered by depolarization, which translate the response of a "voltage sensor" in the T-tubule membrane into a massive efflux of calcium from internal stores contained within the sarcoplasmic reticulum. The coupling process enables voltage-dependent conformational changes of a protein in one membrane system to rapidly open or close ion channels in proteins located in a separate membrane system. The intermembrane "gating" process is unique and, although it is the subject of intense investigation, its mechanism remains unknown. Major advances in understanding the molecular basis for EC coupling have taken place in the past few years with the identification, isolation and sequencing of two of the key proteins: a dihydropyridine (DH P) receptor which senses the T-tubule membrane potential and a ryanodine receptor which contains the calcium release channel in the sarcoplasmic reticulum. The contribution of these and other closely related molecules to the coupling events will be considered and current concepts about the nature of the coupling process discussed. One aim of this article is to look at the results of physiological studies of contraction in the context of the new findings about the molecular biology of channels and proteins involving EC coupling. A second aim is to examine the contribution of each individual component of excitation-contraction coupling to the final tension response. The increase and decay of tension following depolarization depend on changes in calcium concentration within the myofilament lattice, calcium activation of troponin and cross-bridge cycling. The changes in calcium concentration alone depend on the interactions between at least six different processes: the T-tubule action potential; the response of the voltage sensor; the putative EC coupling mechanism; the properties of the calcium release channel; calcium diffusion to, and through, the myofibrils; calcium binding to myoplasmic calcium buffers and calcium removal by the sarcoplasmic reticulum calcium ATPase. In order to properly understand contraction, it is important to understand how each of these events influences the final contractile response. Many of the events can be measured independently and their contribution to the characteristics of the tension transient evaluated. The role of calcium release in determining the contractile properties of different types of mammalian muscle fibres will be discussed. The time-dependent and membrane potential-dependent properties of calcium release and tension during steady-state depolarization are of particular interest since it will be shown that they reflect either conformational changes in the voltage sensitive DHP receptor protein in the T-system or changes in the mechanism that couples the voltage sensor to the calcium release channel. II. STRUCTURES AND PROTEINS IN EC C O U P L I N G 1. The T-system, Triad Junction and Junctional "Feet"

EC coupling occurs at the "triad junction" where the membranes of the T-tubule and terminal cisternae are closely apposed. The T-system provides the key to rapid activation of skeletal muscle since the tubules penetrate the entire cross-section of fibres with diameters as great as 100 ktm. The action potential is actively propagated along the T-tubule membranes to the centre of the fibre. T-tubules develop as deep invaginations of the plasmalemma, their membranes remain continuous with the exterior surface membrane in adult muscle and the lumen is continuous with the extracellular space. The externally derived tubules are covered on two sides, for most of their length, by terminal sacs of sarcoplasmic reticulum. The resultant ternary complex of one T-tubule and two terminal cisternae forms a structure that has been appropriately named a "triad". The T-system and associated terminal cisternae form a disk-like network which wraps around the myofibrils and extends across the muscle fibre at regular intervals in register either with the Z-line in amphibia or the region of overlap between actin and myosin filaments in mammals. The total area of T-tubule and sarcoplasmic reticulum membranes is of the order of 10--50 times greater than the area of the fibre surface (Peachey, 1965; Mobley and Eisenberg, 1975; Eisenberg et al., 1974; Eisenberg and Kuda, 1976; Dulhunty et al., 1986) and depends on fibre diameter, the type of muscle fibre and animal species. T-tubules at the triad have a general dumbbell shape with a long axis parallel to the transverse dimension

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of the fibre and a short axis parallel to the length of the fibre (Fig. 1). The terminal cisternae are apposed to the narrowest part of the T tubule, i.e. the neck of the dumbbell.

A B longitudinal section

LONG AXIS

C

transverse section

SHORq" AXIS

FIG. 1. A schematic illustration of the structure of a T-tubule forming a triad junction with terminal cisternae---the structure of the bare tubules outside the triad is less regular--showing the distribution of the tetrameric junctional feet, as described by Block et al. (1988). The junctional feet (dense structures) are continuous with the junctional face membranes of the terminal cisternae which are not shown. A shows a three dimensional representation of the T-tubule. The long and short axes of the tubule cross-section are labelled, T indicates the transverse dimension of the fibre and the arrow points along the length of the tubule. B shows the appearance of the T-tubule and feet in electron micrographs of longitudinal sections of the triad and C shows the appearance in transverse sections of the triad. Both longitudinal and transverse sections of triads are seen in longitudinal sections of muscle fibres (modified from Dulhunty, 1989).

As a consequence of the geometry of the T-tubules and terminal cisternae, the action potential is able to initiate the release of calcium from stores that are located within only a few hundred nanometers of the surface of the target myofibrils. The maximum distance that calcium must diffuse to the centre of adjacent myofilaments is less than 1.0 #m. Myoplasmic calcium buffers (troponin, parvalbumin and calmodulin), and calcium removal by the calcium ATPase in the sarcoplasmic reticulum, limit the range of calcium action to myofibrils within one sarcomere immediately adjacent to the release site. T-tubule membrane depolarization is sensed by proteins in the T-tubule membrane and a signal transmitted across the triad junction to calcium release channels in the terminal cisternae membrane. The 12 nm gap between the T-tubule and the sarcoplasmic reticulum is only marginally wider than the 10 nm membranes in either side: this separation is greater than that of"gap"junctions where the membranes are separated by only 2 nm (Makowski et al., 1977). Although the calcium release channel has been likened to a gap junction protein (Ma et al., 1988), electrical coupling between the T-system and terminal cisternae has been dismissed on the grounds (a) that the specific capacitance of the fibre of 6 to 10/~F-cm 2 of surface membrane (Fatt and Katz, 1951; Falk and Fatt, 1964; Gage and Eisenberg, 1969; Dulhunty et al., 1984) is equal to that predicted from the known areas of exterior surface and T-tubule membranes and thus considerably less than would be expected if the sarcoplasmic reticulum membranes were electrically coupled to the T-system (Costantin, 1975) and (b) the ionic composition of the sarcoplasmic reticulum differs from that of the extracellular fluid but is similar to that of the myoplasm (Somlyo et al., 1977a,b). The triad junction contains a parallel row of electron dense "feet" (Franzini-Armstrong, 1970) with a distribution shown schematically in Fig. 1. The structures span most of the 12 nm gap and always appear to be continuous with the sarcoplasmic reticulum, but not necessarily with the T-tubule membrane. The feet are about 18 nm wide and are separated by an interval of about 12 nm. Other structures, including bridges (Somlyo, 1979) and pillars (Eisenberg and Gilai, 1979), have been described in the junctional gap. These structures, as well as the junctional feet, are now considered to be different reflections of one tetrameric foot complex (Franzini-Armstrong and Nunzi, 1983; Ferguson et al., 1984; Dulhunty, 1989). JPB 57:3-E

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It has long been thought that the junctional feet may be crucial for EC coupling because of their location between the T-tubules and the terminal cisternae and because voltageactivated contraction is observed only when the foot structure is normal. Disruption of the feet by mild glycerol-treatment is accompanied by a loss of EC coupling (Dulhunty et al., 1981) and their absence in dysgenic mice is associated with paralysis (Rieger et al., 1987). 2. Proteins Associated with the Junctional Foot C o m p l e x

If EC coupling was in some way associated with junctional foot complex, the voltage sensors in the junctional T-tubule membrane, and the calcium release channel in the sarcoplasmic reticulum, should be aligned with the feet, This prediction has been confirmed with the identification and localization of the voltage sensitive molecule and the calcium release channel. Many other proteins are isolated with triad membranes, or have been immunologically localized at the triad junction, and could also be involved directly in EC coupling or in modulation of calcium release. (a) The voltage sensitive m o l e c u l e - - a dihydropyridine receptor The voltage sensor in the T-system was unequivocally identified as an L-type calcium channel and D H P receptor protein when EC coupling and voltage dependent calcium currents were restored to dysgenic muscle fibres following microinjection of cDNA encoding the ~1 subunit of the skeletal muscle D H P receptor (Tanabe et al., 1987, 1990; Adams et al., 1990). However, D H P receptors were thought to be associated with the voltage sensor for several years before these definitive experiments. Skeletal muscle T-tubule membranes were recognized as the richest source of D H P receptor protein (Fosset et al., 1983; Galizzi et al., 1984). The D H P receptor was known to be a voltage-dependent calcium channel (Curtis and Catterall, 1983, 1986; Flockerzi et al., 1986; Kim et al., 1990a) but the requirement for such a high concentration of the proteins in skeletal muscle fibres was not clear. Fewer than 5 % of the D H P receptors represent functional calcium channels (Schwartz et al., 1985). The role of T-tubule calcium channels is in itself a puzzle because the calcium current is too slow to play a role in the action potential and, although some studies implied that external calcium might be necessary for, or enhance, EC coupling (Barrett and Barrett, 1978; Ildefonse et al., 1985; Kotsias et al., 1986), the bulk of available evidence showed that extracellular calcium is not important for the generation of twitches, tetanic contractions or K-contractures (Armstrong et al., 1972; Miledi et al., 1984; Dulhunty and Gage, 1988; Luttgau and Spiecker, 1979; Cota and Stefani, 1981; Dulhunty, 1991). The location of calcium currents and D H P receptors in the T-tubule membrane (Almers et al., 1981; Fosset et al., 1983; Jorgensen et al., 1989) suggested that the D H P receptors might be involved in some way in EC coupling. A close association between D H P receptors and the voltage sensor for EC coupling was indicated by the effects on contraction of calcium channel blockers such as D600 and diltiazem (Eisenberg et al., 1983; Gottschalk and Luttgau, 1985; McCleskey, 1985; Gallant and Goettl, 1985; Luttgau et al., 1987) and the dihydropyridine calcium channel blockers (Ildefonse et al., 1985; Gallant and Goettl, 1985; Avila-Sakar et al., 1986; Rios et al., 1986; Dulhunty and Gage, 1988; Gamboa-Aldeco et al., 1988; Neuhaus et al., 1990). In addition, the response of the voltage sensor to T-tubule depolarization, asymmetric charge movement (Schneider and Chandler, 1973), was reduced by calcium channel blockers or low external calcium (Hui et al., 1984; Lamb, 1986a; Rios et al., 1986; Luttgau et al., 1987; Lamb and Walsh, 1987; Rios and Brum, 1987; Brum et al., 1988a). Taken together, these observations precipitated the hypothesis that the voltage sensor for EC coupling was a D H P sensitive calcium channel protein (Rios et al., 1986; Beam et al., 1986; Lamb and Walsh, 1987; Rios and Brum, 1987; Pizarro et al., 1988; Dulhunty and Gage, 1988). The conclusion was supported by studies in dysgenic mice where muscle fibres were found to lack both EC coupling and D H P sensitive calcium channels (Beam et al., 1986). The subunit composition and amino acid sequence of the D H P receptor are well documented. The protein has a molecular mass of around 390 kDa and consists of ~ (185 kDa), c~2 (143 kDa), fl (54 kDa, ~5(26 kDa) and y (30 kDa) subunits (Takahashi et al., 1987). The ~1 subunit is the functional subunit, containing the receptor for dihydropyridines

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(Kim et al., 1990a), the calcium channel (Tanabe et al., 1987), the voltage sensor for EC coupling (Adams et al., 1990), as well as the regions critical for communication with the ryanodine receptor calcium release channel during EC coupling in skeletal muscle (Tanabe et al., 1988, 1990; Adams et al., 1990). There is a striking homology between the ~1 subunit of the D H P receptor and the voltage dependent sodium channel (Tanabe et al., 1987). Both polypeptides contain four membrane spanning segments: each of the four segments contains six putative transmembrane ~-helices and the fourth helix ($4) in each segment contains a regularly spaced array of positive charges that are thought to form the hypothetical voltagesensor for channel opening or for EC coupling (Noda et al., 1984, 1986; Stuhmer et al., 1989a; Adams et al., 1990; Adams and Beam, 1990). Synthetic peptides with the amino-acid sequence of the highly conserved third helix form D H P sensitive calcium channels in lipid bilayers, suggesting that this helix may be intimately involved in the channel pore (Grove et al., 1991). Contraction in primitive forms of skeletal muscle in invertebrates, and in cardiac and smooth muscle, depends on calcium influx through L-type, DHP-sensitive, channels. The extracellularly derived calcium ions either directly activate the contractile proteins or evoke further calcium release from internal stores by the calcium-induced calcium release mechanism. The channel protein has evolved in vertebrate skeletal muscle to form a T-tubule voltage sensor that regulates calcium release from internal stores without calcium influx across the plasmalemma. The portion of the protein that allows it to gate calcium release from the terminal cisternae is the cytoplasmic portion between the second and the third membrane spanning segment (Tanabe et al., 1990). The most striking difference between this region of the ~1 subunit from cardiac and skeletal muscle (Mikami et al., 1989) is a deletion of 11 amino acids from the skeletal muscle peptide: how this deletion relates to the difference between the EC coupling in cardiac and skeletal muscle is yet to be discovered. The ~ , ct2 and fl subunits of the D H P receptor have been localized in the triad junctions of skeletal muscle using immunostaining techniques (Jorgensen et al., 1989; Flucher et al., 1990). Clusters of four particles are seen in freeze-fracture replicas of the junctional T-tubule membrane in register with every second foot structure, and are thought to be formed by D H P receptor proteins (Block et al., 1988). This observation raises the possibilities that all ryanodine receptors may not be active in EC coupling at any one time, or that there may be two different activation mechanisms (Block et al., 1988) or that more than one ryanodine receptor can be gated by one D H P receptor complex (Kim et al., 1990b; Block et al., 1988). The isolated D H P receptor itself does not have a high affinity for the ryanodine receptor but, as will be discussed below, other smaller proteins with a high affinity for both molecules have been isolated (Brandt et al., 1990; Kim et al., 1990b). (b) The ryanodine receptor calcium release channel Ryanodine has been instrumental in the isolation and characterization of the calcium release channel (Fleischer et al., 1985; Pessah et al., 1985, 1986). Ryanodine is a neutral plant alkaloid and a naturally occurring insecticide (Jenden and Fairhurst, 1969) that (a) has a high affinity for sarcoplasmic reticulum calcium channels, (b) induces small maintained efflux of calcium from terminal cisternae vesicles at low concentrations (< 10 #M) and blocks calcium release at concentrations greater than 10/~M (Fleischer et al., 1985; Meissner, 1986a; Lattanzio et al., 1987), and (c) induces a permanently open subconductance state in single sarcoplasmic reticulum calcium release channels (Rousseau et al., 1987). The first high molecular weight protein isolated from the junctional membrane was the junctional foot protein (Kawamoto et al., 1986). The protein was further purified and shown to be a ryanodine receptor (Hymel et al., 1988; Lai et al., 1988) that consisted of four homomonomers, each containing 5032 amino acid residues and a molecular mass of 563,584 Da (Takeshima et al., 1989; Zorzato et al., 1990). Lower molecular masses of 360 and 450 kDa have previously been determined for the monomers using the less accurate sodium dodecyl sulphate gels polyacrylamide gel electrophoresis (Inui et al., 1987a; Lai et al., 1988; Imagawa et al., 1987; Wagenknecht et al., 1989). The first hydropathy analysis of the amino acid sequence of the ryanodine receptor protein

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predicted a structure that was thought to be reminiscent of the nicotinic acetylcholine receptor, with each monomer containing four membrane spanning helices, of 20 amino acids each, in the C-terminal tenth of the molecule (Takeshima et al., 1989). A later analysis predicted 10 transmembrane sequences in the C-terminal fifth of the molecule and an additional two transmembrane sequences located near the centre of the molecule (Zorzato et al., 1990). The transmembrane passages, with hairpin loops into the lumen of the terminal cisternae, are likely to form the calcium channel. The first 4000 amino-acids of the molecule, apart from the two short central hydrophobic regions (with a total of 28 residues), are predominantly hyrophilic and probably constitute the large cytoplasmic domain of the molecule. The three-dimensional structure of the ryanodine receptor has been reconstructed from negatively stained purified proteins (Wagenknecht et al., 1989). The four subunits form a 27 x 27 x 14 nm structure. A 14 × 14 × 4 nm base-plate is believed to be inserted into the terminal cisternae membrane while the remainder of the molecule is cytosolic and located in the junctional gap. The four to twelve transmembrane segments in each subunit probably come together to form the base-plate of the protein (Zorzato et al., 1990). It is suggested that calcium passes through a central channel in the base-plate and then into four radial channels between the cytoplasmic portions of the subunits, into the junctional gap (Wagenknecht et al., 1989). That the ryanodine receptor calcium release channel does release calcium during EC coupling is implied from its location and structure (Inui et al., 1987a,b; Lai et al., 1988), from the effect of ryanodine on EC coupling in intact muscle fibres (Fryer et al., 1989), and from the physiological and pharmacological properties of the calcium channel in sarcoplasmic reticulum vesicles (Endo, 1977; Nagasaki and Kasai, 1983; Ikemoto et al., 1985; Meissner et al., 1986a,b). The tetrameric protein is considered to form the foot structure seen in electron micrographs because of its ultrastructural appearance and dimensions (Inui et al., 1987a; Lai et al., 1988). In addition, it is isolated from the junctional-face membrane of the terminal cisternae (Inui et al., 1987a) and it has been immunologically localized in the region of the triad (Kawamoto et al., 1986; Airey et al., 1990). Ryanodine receptors have recently been recognized as a class of ion channel which might control calcium release from internal stores in endoplasmic reticulum of many cells. The protein is found in an increasingly large number of tissues including liver (Shoshan-Barmatz et al., 1990), central neurons (Ellisman et al., 1990; McPherson and Campbell, 1990), smooth muscle (Herrmann-Frank et al., 1990) and cardiac muscle (Rousseau et al., 1986; Inui et al., 1987a; Anderson et al., 1989). The ryanodine receptor from cardiac muscle demonstrates a 66% homology with the skeletal muscle protein and has the same hydropathy profile (Otsu et al., 1990). Functional similarities between ion channels formed by ryanodine receptors from different sources are discussed in Section IlI.4.a (below). (c) Coupling proteins The location of putative voltage sensors in the T-tubule membrane adjacent to the junctional feet (Block et al., 1988) has revived the mechanical hypothesis for EC coupling (Chandler et al., 1976a). However a direct communication between the proteins has not been demonstrated: isolated ryanodine receptors do not show an affinity for D H P receptors (Brandt et al., 1990) and it is thought that other small molecules might act as a link between the two macromolecules. Several proteins with high affinities for both the D H P receptor and the ryanodine receptor are potential candidates for the "missing link". A 60 to 70 kDa protein has been isolated with T-tubules and binds to purified ryanodine receptor protein (Chadwick et al., 1988). Aldolase (40 kDa), glyceraldehyde 3-phosphate dehydrogenase (GAPD, 35-40 kDa) and a 95 kDa protein, are isolated with triad junctions (Brandt et al., 1990). Aldolase binds to ryanodine receptors and is released by inositol phosphates (Thieleczek et al., 1989). GAPD promotes the formation of junctions between dissociated T-tubules and terminal cisternae (Corbett et al., 1985) and forms a ternary complex with the ryanodine receptor and the D H P receptor (Brandt et al., 1990). The 95 kDa protein is intrinsic to the junctional terminal cisternae membrane and binds strongly

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to the ryanodine receptor and to the 0c1 subunit of the D H P receptor (Kim et al., 1990b). The precise functions of these proteins remain to be discovered. (d) Proteins that modulate calcium release A surprising discovery has been the active role of calsequestrin in calcium release from terminal cisternae vesicles (Ikemoto et al., 1989; Collins et al., 1990). Calsequestrin is a 60 kDa, low affinity, high capacity calcium binding protein contained in the lumen of the terminal cisternae. The molecule has traditionally been thought to inertly "store" calcium ions when they are not needed to activate the contractile proteins. However a growing body of evidence suggests that this may not be the case. A component of calsequestrin is isolated with the junctional face membrane (Ikemoto et al., 1989) and has a high affinity for the ryanodine receptor protein (Kawamoto et al., 1986). The protein appears as electron dense material in thin sections of the terminal cisternae. The material is contained within the lumen of the cisternae and is closely associated with junctional face and extra-junctional membrane (Saito et al., 1984; Dulhunty, 1989). The amino acid residues of calsequestrin that are responsible for the protein binding to the junctional face membrane have been identified (Collins et al., 1990). Anchoring to the terminal cisternae membrane may occur via thin strands of material seen in deep-etched muscle (Franzini-Armstrong et al., 1987). Calcium-activated calcium release is abolished by dissociation of calsequestrin from the junctional face membrane (Ronjat and Ikemoto, 1989). The calcium release channel appears to be influenced by the conformational state of calsequestrin which changes dramatically with calcium binding (Ikemoto et al., 1974; Aaron et al., 1984; Ohnishi and Reithmeier, 1987). Calcium-dependent conformational changes in junctional face membrane proteins (including the ryanodine receptor) disappear when calsequestrin is dissociated from the membrane (Ikemoto et al., 1989). Although calcium is not the primary trigger for calcium release during voltage-activated contraction it is likely that the same calcium release channel can be activated either by calcium ions or by T-tubule depolarization (Lamb and Stephenson, 1990a) and that calsequestrin modulation of channel activity occurs in both cases. The precise role of the calcium binding protein in EC coupling remains to be established. However the protein might be directly involved in EC coupling, perhaps a part of the mechanical link between the voltage sensor and the calcium release channel. An alternative hypothesis is that a change in the affinity of calsequestrin for calcium, triggered by the T-tubule voltage sensor, results in an increased luminal calcium concentration and an efftux of calcium through open ryanodine receptor channels. Other endogenous proteins modulate EC coupling. Calmodulin-dependent phosphorylation of a 60 kDa protein reduces calcium-activated calcium release from sarcoplasmic reticulum vesicles (Kim and Ikemoto, 1986; Kim et al., 1988). Calmodulin alone depresses calcium-, caffeine- and AMP-induced calcium release from vesicles (Meissner, 1986a; Plank et al., 1988) and reduces the open time, but not the conductance, of single calcium release channels incorporated into lipid bilayers: inhibition of the channel opening depends on calcium concentration but not on ATP (Smith et al., 1989). In addition to protein modulation other agonists, such as adrenaline, increase the amplitude of calcium transients in amphibian muscle (Brum et al., 1990), possibly via stimulation of cyclic AMP which increases the activity of single calcium release channels. (e) Cytoskeletal and other proteins

Several additional proteins have been immunolocalized at the triad junction or isolated with triad and T-tubule fractions: specific roles for most have yet to be discovered and their precise location in the triad determined. Two cytoskeletal proteins, dystrophin and ankyrin (Hoffman et al., 1987; Flucher et al., 1990), could be involved in maintaining the geometry of the membranes at the junction and the strict relationship between the membranes and the myofilament lattice. Ankyrin is not co-localized with the D H P receptor and is probably not directly associated with EC coupling (Flucher et al., 1990). A 30 kDa peptide forms a major component of the T-tubule protein (Rosemblatt et al., 1981; Horgan and Kuypers, 1987) and a minor 28 kDa protein has been described

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(Jorgensen et al., 1990) as well as 38 kDa and 53 kDa proteins (Rosemblatt and Scales, 1989). Junctional terminal cisternae membranes contain several 25-50 kDa peptides (Damiani and Margreth, 1990), as well as a 170 kDa protein that may be involved in anchoring calsequestrin to the membrane (Kim et al., 1990b). Some of these proteins could be associated with "indentations" and "rods" in the terminal cisternae membrane (Rayns et al., 1975; Dulhunty and Valois, 1983; Dulhunty et al., 1983, 1984; Dulhunty, 1987). Both structures are more common in mammalian fast-twitch fibres than in slow-twitch fibres. It has been suggested that the indentations contain extra-junctional calcium release channels (Dulhunty et al., 1984) and that the rods may contain the 170 kDa protein (Kim et al., 1990b) and form an anchor for calsequestrin to the junctional and extra-junctional terminal cisternae membrane. 3. Questions about the Calcium Release Channels

(a) Is there more than one type of calcium release channel? Although the ryanodine receptor appears to be the calcium release channel specifically associated with the junctional feet, several lines of evidence suggest that there may be more than one type of calcium channel in the sarcoplasmic reticulum membrane. Calcium efflux from the sarcoplasmic reticulum can be activated through several independent mechanisms. Studies with isolated sarcoplasmic reticulum vesicles and skinned muscle fibres show that calcium release can be gated either by ionic depolarization of the T-tubule membrane or by calcium-activation of the calcium release channel (Donaldson, 1986; Lamb and Stephenson, 1990a). It is not clear whether there is more than one type of calcium channel, or whether the one channel has multiple regulatory sites. A third mechanism for calcium release is stimulated when sulfhydryl reagents and heavy metals including mercury, silver, copper, cadmium and zinc bind to a free sulfhydryl group on a calcium channel protein (Abramson et al., 1983). Other sulfhydryl reagents block twitches, and silver and potassium induced contractions in single frog muscle fibres, but do not alter caffeine contractures (Oba and Yamaguchi, 1990). The active sulfhydryl group is on a ryanodine receptor protein (Zaidi et al., 1989a), which has been described as having a molecular mass of 106 kDa and is thought not to be a subunit of the 563 kDa ryanodine receptor complex (Zaidi et al., 1989b). However, it should be noted that the putative transmembrane regions of the 563 kDa protein also contains three cysteine residues that would provide potential targets for the sulfhydryl reagents. Two isoforms ofryanodine receptor protein occur in avian, amphibian and piscine skeletal muscle, but not in mammalian muscle (Airey et al., 1990; Olivares et al., 1991). Two functional populations of ryanodine receptor calcium release channel are found in muscle from humans susceptible to malignant hyperthermia (Fill et al., 1991). It has recently been shown that of 50% of ryanodine receptor calcium channels in amphibian sarcoplasmic reticulum vesicles are specifically stimulated by inositol, 1,4,5-trisphosphate, IP 3 (Suarezisla et al., 1991). (b) Where is the inositol 1,4,5-trisphosphate receptor? IP 3 is a second messenger which regulates intracellular calcium concentrations in many tissues (Berridge and Irvine, 1989). IP 3 and the enzymes associated with its metabolism, have been isolated from skeletal muscle (Asotra and Vergara, 1986; Varsanyi et al., 1989; Lagos and Vergara, 1990) and IP 3, in the hands of many investigators, induces calcium release from the sarcoplasmic reticulum (Vergara et al., 1985; Volpe et al., 1985; Volpe and Stephenson, 1986; Donaldson et al., 1987; Rojas et al., 1987). The IP a induced tension response in skeletal muscle is slow (Walker et al., 1987) and, although the messenger is not thought to be the primary trigger for calcium release during normal voltage-activated contraction, IP 3 may modulate calcium release. That IP 3 does induce calcium release, however slow, implies that there is a specific IP 3 receptor in skeletal muscle. |t is not yet clear whether separate calcium release channels, activated by IP a, exist in the sarcoplasmic reticulum of skeletal muscle, and/or whether some ryanodine receptor calcium

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release channels are sensitive to IP 3. No high affinity IP 3 receptors have so far been detected in the sarcoplasmic reticulum of skeletal muscle (Rojas and Hidalgo, 1990). Studies with rabbit sarcoplasmic reticulum have failed to demonstrate IP 3 sensitivity of the ryanodine receptor calcium release channels, but show a strong phosphatidylinositol 4,5-bisphosphate (PIP2)-induced calcium release from terminal cisternae vesicles through a ryanodine sensitive pathway, and a 2 to 12-fold increase in the open probability of the channel in lipid bilayers with PIP 2 (Chu and Stefani, 1991). IP3-stimulated calcium channels have been found in sarcoplasmic reticulum vesicles from frog skeletal muscle (Suarez-isla et al., 1988), with a "bell" shaped dependence on cytoplasmic calcium concentration (Suarez-isla et al., 1991) normally associated with the ryanodine receptor calcium release channel. Calcium channels formed when purified ryanodine receptors are incorporated into lipid bilayers are stimulated by IP 3 (Liu et al., 1989b). The strong inhibition of IP 3 activation by micromolar or millimolar calcium concentrations may explain the frequently reported absence of IP 3 sensitivity (Suarez-isla et al., 1991). IP3 receptors have been isolated from many types of cells, and have mostly been studied in non-muscle tissue. Like the ryanodine receptor, the protein consists of four homomonomers, each with a molecular mass of 313,000 Da and showing some sequence homology with the ryanodine receptor, particularly in two of the putative membrane spanning segments (Marks et al., 1990). IP 3 receptor and ryanodine receptor calcium release channels co-exist in the same cells in brain (Ross et al., 1989; Ellisman et al., 1990; Walton et al., 1991; Bezprozvanny et al., 1991), liver (Shoshan-Barmatz et al., 1990) and smooth muscle (Herrmann-Frank et al., 1990). The IP 3 receptor calcium release channel in cerebellum has a very similar calcium dependence (Bezprozvanny et al., 1991) to the IP 3 sensitive calcium channel in the sarcoplasmic reticulum (Suarez-isla et al., 1991). However the IP 3 sensitive channels in skeletal muscle differ from the IP3-activated channels in smooth muscle and cerebellum in a number of significant ways (see Section III.4.a, below): it is likely that different types of calcium channel have IP3 binding sites and can be modulated by the second messenger. Indeed, it has been shown that D H P receptor calcium channels can be activated by IP 3 (Vilven and Coronado, 1988). (c) Are all calcium channels in junctional feet? The hypothesis that calcium release from the sarcoplasmic reticulum occurs through the foot structure is appealing since EC coupling is rapid and the location of the voltage sensor and the calcium release channels within the one macromolecular complex provides a physical basis for mechanical coupling between the two proteins (Chandler et al., 1976a). However, the possibility that all calcium required for contraction is released into the junctional gap has been questioned (Dulhunty, 1988; Dulhunty et al., 1992a; Ashley et al., 1991). The problem is that the free space in the junction is very small, 0.08% of the fibre volume in mammals (Dulhunty, 1988) or 0.013% in amphibia (Dulhunty et al., 1992a), and diffusion of calcium out of the gap is slowed by the physical bulk of the junctional feet and by calcium binding to negative fixed charges on the cytoplasmic surface of the T-tubule and terminal cisternae membranes and on the junctional foot proteins. If the concentration of calcium in the gap increased above 1 mM, as would occur if it has to accommodate a total efflux from the sarcoplasmic reticulum of 36 pM/mS (Baylor et al., 1983), the concentration gradient for calcium across the junctional terminal cisternae membrane would be reduced to zero (Ashley et al., 1991) and the calcium release channel would be inactivated (Smith et al., 1985; Bezprozvanny et al., 1991). The problem of calcium accumulation within the gap would be alleviated if some of the calcium release channels were located in the extrajunctional membranes of the terminal cisternae and released calcium directly into the myoplasm. Monoclonal antibodies to the ryanodine receptor calcium release channel can be used in immunoelectron microscopy with colloidal gold-conjugated second antibodies to localize the proteins. Gold particles marking the approximate sites of ryanodine receptor proteins are frequently seen on extra-junctional terminal cisternae membrane (Fig. 2). A minimum of 20% of terminal cisternae ryanodine receptors are extra-junctional (Dulhunty et al., 1992a). The precise number of extra-junctional ryanodine receptors remains

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7. 0

FIG. 2. Ryanodine receptors are located on both junctional and extra-junctional terminal cisternae membrane. The longitudinal sections of rat sternomastoid muscle fibres have been stained with a primary antibody to the ryanodine receptor (Dulhunty et al., 1992). The black dots on the micrographs are colloidal gold particles conjugated to the secondary antibody. A and C show examples of both junctional and extra-junctional receptors. B and D show clusters of particles extending from the junctional region into extra junctional membrane, t, denotes T-tubules; the arrows point along the junctional gap and the adjacent electron dense areas are terminal cisternae; e, extra-junctional receptors; *, junctional receptors; c, clusters extending from the junction into the extra-junctional membrane. The calibration bar is 100 nm. to be determined, as does their role in EC coupling. If the proteins do act as calcium channels during voltage-activated contraction, their gating mechanism may differ from that of the junctional calcium release channels. The distance between the voltage sensor and extrajunctional channels is too great for the direct coupling postulated for junctional channels (Chandler et al., 1976a). Alternative gating processes that might act over distances greater than 10 nm include calcium-activated calcium release, which may play a role in normal voltage-activation of skeletal muscle (Fabiato, 1985; Simon et al., 1989; Klein et al., 1990), or a mechanical link through calsequestrin which has physical connections to both the junctional and extra-junction terminal cisternae membrane and is functionally involved in EC coupling (Section II.2.d, above). III. PHYSIOLOGICAL

S T U D I E S O F EC C O U P L I N G

A complete description of the voltage-activation of contraction and EC coupling requires not only identification of the key proteins, but also the molecular interactions between the proteins, the functional properties of the proteins and the contribution of each protein to the characteristics of tension changes produced by depolarization of the surface membrane. Therefore, functional studies can be generally divided into two categories, those that look at the overall process of EC coupling, and those that look at its component parts. Measurements of tension, or myoplasmic calcium concentration, in response to depolarization fall into the first category. The second category includes studies of asymmetric charge movement, i.e. the capacitive current produced by conformational changes in the voltage sensor, as well as measurements of the single channel properties of ryanodine receptor

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proteins. Both types of study are essential to a full understanding of the mechanism of EC coupling. An important question is: which of the individual steps in EC coupling sets the voltage dependence of calcium release and the kinetics of the increase in myoplasmic calcium concentration. Twitch and tetanic contractions produced by action potentials (evoked singly or in bursts) are the most commonly studied tension responses in skeletal muscle fibres. The time course and amplitude of the twitch depend not only on the kinetics of calcium release and uptake by the sarcoplasmic reticulum, diffusion into the myofibrils and the response of the contractile proteins, but also on the rate of change of membrane potential and the transverse propagation of the action potential along the T-tubule. All these events become significant in the time course of contraction of fast-twitch fibres at 35-37°C, when the time to peak tension during isometric twitches can be as brief as 3.7 msec for inferior rectus and 6.9 msecs for exterior digitorum longus (Luff, 1981). The relationship between myoplasmic calcium concentration or tension and T-tubule membrane potential can only be rigorously studied in response to step changes in membrane potential to different steady-state levels. Isometric tension has been recorded in response to prolonged depolarizations induced by rapid changes in potassium concentration (K-contractures) (Hodgkin and Korowicz, 1960; Luttgau, 1963; Caputo, 1972; Dulhunty, 1980) or by voltage clamp pulses (Heistracher and Hunt, 1969a,b; Caputo et al., 1984; Caputo and De Bolanos, 1979; Luttgau et al., 1986) applied to single or small bundles of intact muscle fibres. Skinned muscle fibres (Nakajima and Endo, 1973) provide an alternative preparation in which contractures can be recorded and the T-tubule membrane depolarized by ionic substitution (Donaldson, 1985; Stephenson, 1985; Volpe and Stephenson, 1986; Fill and Best, 1988; Lamb and Stephenson, 1990a,b). The skinned fibre technique has an advantage in that the ionic composition of the myoplasm can be controlled and varied during the experiment. The drawback of the technique is that membrane potentials are not precisely known. 1. Voltage-activated Tension

The changes in tension during steady depolarization of the T-tubule membrane are independent of the means used to control membrane potential and are the same in intact and skinned fibres. A rapid increase in tension occurs as soon as the fibre is depolarized. Tension reaches a peak, or a plateau and then slowly and spontaneously decays if the depolarization is maintained for several seconds (Fig. 3). The amplitude of the contractures increases to a maximum value (that is slightly greater than tetanic tension) as the membrane potentials become more positive. The increase and the decay of tension are strongly voltage dependent and are faster at more depolarized membrane potentials (Fig. 3; Hodgkin and Horowicz, 1960). The changes in tension with depolarization are slower than the rise and decay of twitch or tetanic tension and are considered to reflect changes in myoplasmic calcium concentration and EC coupling. The rates of calcium binding to and dissociation from troponin, crossbridge cycling rates and calcium removal by the calcium ATPase in the sarcoplasmic reticulum are much faster, having millisecond time constants (see Section III.2.b and Stein et al., 1988 for discussion), and are not rate-limiting in these slow depolarization-induced contractures. Tension induced by steady-state depolarization is discussed in more detail in Section IV. The changes in tension during a K-contracture were originally thought to reflect the production and depletion of an activator substance (Hodgkin and Horowicz, 1960) but are now thought of in terms of the activation and inactivation properties of the voltage-sensitive D H P receptor molecule (Caputo and De Bolanos, 1979; Luttgau et al., 1986; Luttgau et al., 1987; Dulhunty, 1991). The more recent interpretation has evolved from studies of calcium release from the sarcoplasmic reticulum, the response of the voltage sensor to depolarization, and the characteristics of single calcium release channels described in the following sections. 2. Optical Measurements of Events in EC Coupling

The second type of experiment that looks at EC coupling as a whole uses calcium sensitive dyes as indicators of voltage-activated changes in myoplasmic calcium concentration during

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[,mN

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FIG. 3. Potassium (K) contractures in small bundles of 5 to 15 rat soleus muscle fibres. The series of contractures wererecordedin responseto step changesfrom 3.5 mMK + to: 40 mMK + ( - 35 mV) in A; 60mM K + (-30mV) in B; 80m~i K + (-26mV) in C; and 120mM K + (-19mV) in D. Maximum tension is achievedbetween 120 mMK ÷ ( - 19 mV) and 200 mu K ÷ (--7 mV) (modified from Dulhunty, 1991).

T-tubule depolarization. The technique essentially isolates the EC coupling events since it monitors calcium concentration rather than contraction. The rise and fall in calcium concentration and tension changes are identical during prolonged depolarizations whereas the calcium transient is faster than the tension transient following brief depolarization. The significance of these differences in time course is complicated by the fact that we still know little about the subcellular distribution of dyes, particularly whether or not the dye penetrates the myofilament lattice. (a) Optical indicators In some of the first studies of intracellular events occurring during EC coupling the inherent birefringence of muscle fibres following depolarization was recorded (Bozler and Cottrell, 1937; Baylor and Oetliker, 1975; Oetliker et al., 1975). The birefringence signal included a small early event and a large late event, both of which preceded contraction and were thought to arise from the T-system and sarcoplasmic reticulum membranes respectively (Baylor and Oetliker, 1977a,b). Measurements of birefringence provided an attractive noninvasive means of studying EC coupling. However the origin of the birefringence changes was difficult to pin-point and specific optical indicators for membrane potential, [ C a 2 + ] , [Mg 2 +3 and pH have since been developed. The disadvantage of these indicators is that most are lipid insoluble and must be injected into intact fibres or infiltrated into cut fibres. The majority of studies of EC coupling using optical indicators have concentrated on the changes in intracellular calcium. The luminescent protein aequorin, was one of the first calcium indicators developed (Endo and Blinks, 1973; Taylor et al., 1975). Arsenazo III and antipyrylazo III (Baylor et al., 1979, 1982) have been favoured tools for studying calcium transients; new purpurate indicators have recently been described (Hirota et al., 1989). These metallochromic dyes are not suitable for monitoring resting calcium concentrations and the higher affinity fluorescent calcium indicator fura-2, is frequently used simultaneously with antipyrylazo III (Klein et al., 1988; Simon et al., 1991) to monitor both resting calcium and calcium transients during activation. (b) Calcium transients The profile of the calcium transient during maintained depolarization is identical to the tension transient. Calcium concentration increases to maximum levels after 2-3 sec and then spontaneously decays if depolarization is maintained for a further I0-20 sec (Brum et al., 1988b). Thus inactivation of tension during steady-state depolarization reflects a fall in

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myoplasmic calcium concentration and, is therefore, a decay in the efficacy of EC coupling rather than an inability of the contractile proteins to produce tension. A question that is frequently asked is whether it is EC coupling or the response of the contractile proteins that limits the rate of rise of tension during isometric contractions. It should be possible to answer this question by comparing calcium transients with the time course of twitches and tetanic contractions. Calcium concentration increases and decays more rapidly than tension (Endo and Blinks, 1973; Taylor et al., 1975): the delay between the calcium and tension transients may reflect the kinetics of calcium binding to troponin and the rate of cross-bridge formation and detachment (Stein et al., 1988). However, it could equally well be argued that the calcium transient reflects changes in calcium concentration near the release sites ("hot-spots") and that the delay between the changes in calcium concentration and tension reflects the time required for calcium to equilibrate in the myofilament space. Not only does the largest signal come from the area of highest calcium concentration, but calcium indicators being highly negatively charged are probably excluded from the myofilament lattice (Stephenson et al., 1981). Calcium is released within 1/~m of the centre of the myofilaments and the first mechanical event occurs simultaneously with the initial increase in calcium concentration (Close and Lannergren, 1984). The first mechanical event registers the arrival of calcium at the periphery of the filaments, not its equilibration within the fibril: it is likely that calcium diffusion through the myofilaments is significantly slower than free diffusion because of calcium buffering by troponin. Times taken for calcium equilibration within the myofilaments vary from 4.5 msec at a sarcomere length of 2.2/~m to 8.5 msec at 3.2 #m (Close, 1981). These times represent a significant fraction of the contraction time in fast-twitch fibres at temperatures above 25°C: as mentioned previously, the time to peak tension in the inferior rectus of the rat is 3.7-5.1 msec at 35°C (Close and Luff, 1974; Luff, 1981). Laser flash photolysis of caged calcium allows a study of the rate of tension in response to a step increase in calcium concentration. The half time for force development at 12°C is 40 msec and close to the half time for the tension increase during an isometric tetanus at the same temperature (see Ashley et al., 1991 for review). Therefore the response of the contractile proteins may be rate limiting for tension development, like tension decay (Fryer and Neering, 1989) at low temperatures (< 18°C). However at temperatures above 20°C, the decay of tension and calcium concentration have the same Qlo, suggesting that there is a close relationship between the two events (Fryer and Neering, 1986). Similar experiments have not been performed on the rate of increase of calcium concentration and tension. However plots of contraction time against temperature show a strong break point near 20°C (Stein et al., 1982). It is possible that the rate limiting step in contraction at 12°C may not be the same as the rate limiting step at 25°C or 35°C. Several experimental observations can be explained only by postulating a strong influence of the time course of EC coupling on the rate of isometric contraction at temperatures above 20°C. Firstly, twitch contraction times of fast-twitch inferior rectus muscles are one half that of extensor digitorum longus (EDL) muscles, yet the force/velocity properties of the muscles are the same (Close and Luff, 1974; Luff, 1981). Secondly, chronic experimental manipulations which alter the expression of contractile characteristics invariably produce rapid and parallel changes in EC coupling and isometric contraction times followed by changes in myosin and isotonic shortening velocity that develop more slowly (see Dulhunty et al., 1987 for discussion). It is likely that the faster calcium transients recorded from fasttwitch mammalian skeletal muscle fibres (Eusebi et al., 1985; Fryer and Neering, 1986, 1989) contribute to the faster contraction times in these fibres. (c) The rate of calcium release Calcium transients have been used to calculate the rate of calcium release from the sarcoplasmic reticulum, taking into account calcium binding to the indicator dye and to myoplasmic calcium buffers and calcium removal by the sarcoplasmic reticulum ATPase (Melzer et al., 1984). In amphibian muscle, the rate of release rises to a maximum of 36 #M/msec after 5 msec and then declines within 50-100 msec to a much lower sustained

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level (Baylor et al., 1983; Schneider and Simon, 1988). The early decline in the rate of release is due to calcium inactivation of calcium release (Schneider and Simon, 1988; Simon et al., 1991), a phenomenon also seen in skinned muscle fibres (Kwok and Best, 1987), in isolated sarcoplasmic reticulum vesicles (Meissner et al., 1986), in the calcium release channel (Smith et al., 1985), and in intact fibres where contraction threshold increases immediately after a brief subthreshold depolarization (Dulhunty, 1982). It is important to stress that the early calcium-inactivation of calcium release slows the rate of increase in myoplasmic calcium concentration, but does not reduce calcium concentration: both the total calcium concentration and tension continue to rise despite the fall in release rate. Calciuminactivation of the rate of calcium release should not be confused with the inactivation process that occurs over a period of seconds and reduces both myoplasmic calcium concentrations and tension to zero. 3. Electrical Signals from the Voltage Sensor A major break-through in understanding EC coupling was made in the 1970S with the discovery of gating currents, small asymmetric or non-linear capacitive currents that are generated in voltage sensitive molecules following changes in membrane potential. The discovery of charge movement provided the first opportunity to study an isolated event in EC coupling in intact fibres. Gating currents for sodium channels (Armstrong and Bezanilla, 1973; Keynes and Rojas, 1973), calcium channels (Adams and Gage, 1976) and EC coupling (Schneider and Chandler, 1973) were reported in the space of three years. The asymmetric charge movements were thought to reflect conformational changes in proteins associated with opening of ion channels or with calcium release from the sarcoplasmic reticulum. Gating currents were recorded under voltage clamp conditions in cells in which all ionic currents had been blocked and linear capacitive currents removed by subtraction. A transient outward "ON" current occurred upon depolarization and a transient inward "OFF" current was seen with repolarization. The current was not ionic because the charges carried in the ON and OFF components were equal. The charge movement was asymmetric because it was initiated by depolarizing but not hyperpolarizing voltage steps. The slow asymmetric capacitive current in amphibian skeletal muscle rose to a peak at 5-10 msec and decayed during the following 30-50 msec. (a) The origin of asymmetric charge movement Gating currents for sodium channels can also be recorded from skeletal muscle but are much faster than the slow asymmetric charge movement, reaching a peak in 50 psec and decaying in 2-3 msec (Campbell, 1983). The slow charge movement was intially thought to reflect either the activation of delayed rectifier potassium channels or the activation of EC coupling (Chandler et al., 1975). Charge movement was reversibly inactivated during prolonged depolarization (Adrian and Almers, 1976a,b; Adrian et al., 1976; Chandler et al., 1976a; Rakowski, 1981) and was significantly reduced by glycerol treatment (Chandler et al., 1976a; Dulhunty et al., 1981), properties shared with contraction but not with potassium channels. That asymmetric charge movement is generated by a DHP receptor protein was unequivocally established when asymmetric currents were restored to dysgenic muscle by injection of DHP receptor cDNA (Adams et al., 1990). However it is not clear whether the charge movement is associated with the opening of DHP-sensitive calcium channels or with EC coupling. The time course of non-linear capacitive currents that gate calcium channels in Aplysia neurons (Adams and Gage, 1976) is similar to that of the slow asymmetric charge movement in skeletal muscle. About 30% of the charge movement in skeletal muscle is sensitive to low concentrations of dihydropyridines and may be associated with DHP sensitive ion channels (Lamb, 1986a; Lamb and Walsh, 1987). A question that is yet to be resolved is whether one DHP receptor protein can act simultaneously as an ion channel and as a voltage sensor for EC coupling (Lamb, 1991) and, if it can, whether one conformational change gates both processes. It is simpler to imagine that DHP receptors that gate EC

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coupling are not functional calcium channels. Indeed, problems of calcium accumulation in the junctional gap during EC coupling (Section II.3.c, above) would be accentuated by a simultaneous influx of calcium across the T-tubule membrane. Comparisons of the voltage-dependence of charge movement, tension and calcium conductance suggest that most of the asymmetric charge movement in skeletal muscle is associated with EC coupling rather than calcium currents. Charge movement and tension are activated at more negative membrane potentials in mammalian slow-twitch fibres than in fast-twitch fibres (Gage and Dulhunty, 1981; Hollingworth and Marshall, 1981; Dulhunty and Gage, 1983, 1985); the relationship between DHP-sensitive calcium conductance and membrane potential, on the other hand, is similar in fast- and slow-twitch muscle (Lamb and Walsh, 1987). (b) "Charge 1" and "charge 2" The charge movement story is complicated by the suggestion that there are two species of charge that can be seen in different membrane potential ranges. "Charge 1" moves between - 9 0 mV and 0 mV in normally polarized fibres (Schneider and Chandler, 1973; Chandler et al., 1976a), while "charge 2" moves between - 2 0 0 mV and - 9 0 mV in depolarized fibres (Schneider and Chandler, 1973; Adrian and Almers, 1976a,b; Chandler et al., 1976a,b). Brum and Rios (1987) suggested that charge 1 and charge 2 interconvert when the membrane is conditioned at different potentials. Thus charge 1 occurs when the voltage sensor is activatable and charge 2 occurs when the molecule is in the inactive state. Adrian et al. (1976) had also considered an interconversion mechanism to explain charge 1 and charge 2, but they did not see a reduction in charge 2 during mechanical repriming and rejected the hypothesis. Later studies showed that the total amount of charge moving between - 140 mV and - 7 0 mV was identical in polarized and depolarized fibres (Lamb, 1987), again suggesting that charge is not converted from one form to another by depolarization. The physiological significance of a species of charge that moves between - 140 mV and - 70 mV remains a mystery. (c) The two components of charge I There are two distinguishable components of charge 1 that have been named Qa and Qr. Depolarization to potentials between - 90 mV and contraction threshold (about - 60 mV) produces an asymmetric capacitive current that decays exponentially and is called Q~. Further depolarization increases the amplitude of Qa and adds a slow, hump-like component, Qr to the exponential decay (Adrian and Peres, 1977, 1979). The origin of the two currents is the subject of some controversy. Q~ appears at contraction threshold (Horowicz and Schneider, 1981a,b; Huang, 1981; Adrian and Huang, 1984a,b; Lamb, 1986b) and, like contraction, is blocked by dantrolene and tetracaine (Huang, 1981; Hui, 1983a,b). Qp is resistant to block by tetracaine (Almers and Best, 1976; Huang, 1981; Hui, 1983a,b). It was initially suggested that Q~ and Q~ were separate species of charge (Hui, 1983a,b; Huang, 1987), located in the surface (Qp) and T-tubule (Q~) membranes (Adrian and Huang, 1984a,b) and that Q~ was the component most directly involved in calcium release from the terminal cisternae. Other experiments have indicated that Qr is a consequence of calcium release from the sarcoplasmic reticulum, rather than the causal event. Q~ is not always seen as a separate component of charge movement (Melzer et al., 1986; Hollingworth and Marshall, 1981; Dulhunty and Gage, 1983; Lamb, 1986b). Tetracaine blocks contraction in mammals (Dulhunty and Gage, 1983), but has no effect on charge movement in isotonic solutions (HoUingworth et al., 1990). Curiously, tetracaine does block a fraction of Qa when fibres are exposed to hypertonic solutions (Hollingworth et al., 1990). Qr is strongly influenced by factors that act specifically on the calcium release channel and on calcium release from the sarcoplasmic reticulum (Csernoch et al., 1991; Garcia et al., 1991; Szucs et al., 1991; Pizarro et al., 1991). It has been suggested that Qa is primarily responsible for calcium release from the terminal cisternae and that Qr is generated when calcium binds to negative fixed charges

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on the intracellular surface of the T-tubule membrane, producing additional depolarization and thus additional movement of charge in the same population of molecules that generated Qp. The origin of Qp and Qr remains to be finally resolved. Other experiments examining the Boltzmann distribution of the two components of charge are more consistent with the suggestion that "there are two separate species of intramembranous charge that appear to be able to move in a parallel and independent manner" (Hui and Chandler, 1991). (d) Immobilization of charge movement The spontaneous decay in myoplasmic calcium concentration and tension during long depolarizations is normally attributed to inactivation of the voltage sensor. There is compelling circumstantial evidence for this hypothesis (Section IV.2.a, below) and direct evidence is provided by the observation that charge movement is gradually depressed when depolarization is maintained. The time course of"immobilization" of charge is similar to that of inactivation of EC coupling. The decay in charge movement, like tension, is highly temperature dependent: the time constant in amphibian muscle fibres during depolarization to - 20 mV is 1.5 sec at 5°C (Rakowski, 1981) and 13-24 sec at 2°C (Chandler et al., 1976a). Both the rates of charge immobilization and tension decay are strongly voltage dependent: the time constant of charge immobilization at - 20 mV, 1.5 sec, is reduced to 0.28 sec at + 20 mV (Rakowski, 1981). Charge movement recovers when negative membrane potentials are restored and the rate of recovery of charge, like repriming of the fibre's ability to produce tension, is slow with a time constant of 24 to 53 sec at 2°C (Chandler et al., 1976a). 4. Properties of Single Calcium Release Channels

Another component of EC coupling that can be studied in isolation is the calcium release channel. The pioneering work on calcium release channels looked at calcium release from sarcoplasmic reticulum either in isolated vesicles (Weber and Herz, 1968; Nagasaki and Kasai, 1983) or in skinned muscle fibres (Ford and Podolsky, 1970; Endo et al., 1970). Calcium-activated calcium release has been extensively studied but a full review of the large volume of literature that exists on this topic is not possible here. Those studies provided the basis for experiments with single calcium release channels and the effects of a library of ligands that were thought to modulate the channel. Currents from single calcium channels were first recorded when sarcoplasmic reticulum vesicles were incorporated into lipid bilayers (Smith et al., 1985) or used to form a bilayer on the tip of a patch-clamp electrode (Suarez-isla et al., 1986). Sarcoplasmic reticulum calcium channels have also been studied under more physiological conditions in "sarcoballs", blebs of internal membrane that form on the surface of skinned muscle fibres (Stein and Palade, 1988). Calcium channels formed by purified ryanodine receptor proteins have been studied in lipid bilayers (Smith et al., 1988; Lai et al., 1988). So far it has not been possible to record single calcium channel activity under conditions in which the channel is gated by activation of the D H P receptor. Development of a preparation in which this is possible may provide the next major breakthrough in EC coupling. (a) Activation and permeability of ryanodine and IP 3 receptors The isolated purified ryanodine receptor forms a high conductance (100 to 170 pS) calcium channel in planar lipid bilayers (Smith et al., 1988; Lai et al., 1988). The channel has pharmacological properties that are similar to those of (a) calcium channels in heavy sarcoplasmic reticulum vesicles incorporated into bilayers (Smith et al., 1985, 1986) and (b) calcium release from terminal cisternae vesicles and skinned muscle fibres (Endo et al., 1970; Endo, 1977; Nagasaki and Kasai, 1983; Ikemoto et al., 1985; Meissner et al., 1986; Meissner, 1986b). Calcium release in all cases is stimulated by micromolar levels of calcium and by adenine nucleotides and is inhibited by millimolar cytoplasmic calcium or magnesium, and by calmodulin and ruthenium red. The skeletal muscle ryanodine receptor channel is highly selective for cations over anions. The channel is permeable to both monovalent and divalent

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cations. The Ca2+:Na ÷ permeability ratio is 5:1 (Liu et al., 1989a) and the Ca2+:K ÷ permeability ratio is 6.5 (Stein and Palade, 1988). However, the channel is two to three times more permeable to Cs ÷ than Ca 2÷ (Hamilton et al., 1989). Ryanodine receptors from different tissues form calcium channels in lipid bilayers with properties that are generally similar to the skeletal muscle calcium channel. The cardiac ryanodine receptor channel differs from the skeletal channel in its sensitivity to various ligands (Rousseau et al., 1986; Anderson et al., 1989). Calcium is essential for activation of the cardiac channel, whereas the skeletal channel may be partially activated by ATP at nanomolar calcium concentrations. The cardiac channel is less sensitive to inhibition by magnesium and ruthenium red. Ryanodine receptors isolated from whole brain and incorporated into lipid bilayers form a 107 pS channel that is activated by caffeine and blocked by ruthenium red and ryanodine and has a bell-shaped dependence on cytoplasmic calcium concentration with a maximum open probability between 10 #M and 100 ~M C a 2 + (McPherson et al., 1991a,b; Bezprozvanny et al., 1991). IP3-activated calcium currents were seen when sarcoplasmic reticulum vesicles from smooth muscles, or endoplasmic reticulum vesicles from cerebellum, were incorporated into lipid bilayers (Ehrlich and Watras, 1988; Meyer et al., 1990; Bezprozvanny et al., 1991). This channel is distinctly different from the ryanodine receptor calcium release channel. It has a relatively low conductance of about 10 pS in aortic arch smooth muscle (Ehrlich and Watras, 1988) or 80 pS in cerebellum (Bezprozvanny et al., 1991) and it is not activated by caffeine, it is not blocked by ruthenium red and it is not sensitive to ryanodine (Ehrlich and Watras, 1988; Bezprozvanny et al., 1991). Furthermore, it is blocked by 10 #g ml-1 heparin and the maximum probability of opening occurred with 0.2 #M Ca 2 +, with sharp decreases on either side of the maximum: the probability of channel opening becomes very low at 10/~M Ca 2 ÷ (Bezprozvanny et al., 1991). Thus the IP 3 receptor calcium channel is activated at a lower range calcium concentration than the ryanodine receptor calcium release channel. The steep calcium-dependence of IP 3 receptor channel activity is important to note since calciumactivated calcium release is often thought of as a specific property of the ryanodine receptor channel. The IP3-stimulated calcium channel in frog skeletal muscle is also different from IP3-stimulated channels isolated from smooth muscle and brain. It is activated by caffeine (Suarez-isla et al., 1991) and blocked by ruthenium red and by ryanodine (Suarez-isla et al., 1988). It seems likely that the IP3-stimulated channel in skeletal muscle is a subset of the ryanodine receptor calcium release channels and therefore a separate protein from the IP 3 receptor in smooth muscle and brain. Curiously, the calcium sensitivity of IP3-stimulation of the skeletal muscle calcium channel is very similar to the calcium sensitivity of IP 3 activated channel from cerebellum (Bezprozvanny et al., 1991 ). Perhaps the binding site for IP 3 has the same affinity for calcium in both types of protein. (b) Subconductance states of the calcium release channel The calcium release channel, like many other ion channels (Fox, 1987), demonstrates multiple conductance states. The molecular basis for subconductance states is not yet understood: they may reflect intramolecular changes within one channel or co-operativity between aggregated channels (Krouse et al., 1986). Stein and Palade (1988) describe two subconductance states in calcium channels in sarcoballs. Liu et al. (1989a) observed at least 4 subconductance states in the purified ryanodine receptor, while Hymel et al., (1988) report a multitude of states ranging in size from 3.8 pS to 120 pS implying that, if all multiples of the smallest conductance are possible, there may be 30 or more conductance levels. In this context, it is interesting to note that the base-plate of the ryanodine receptor may have as many as 48 membrane spanning segments (Section II.2.b), and could accommodate as many subconductance states if each segment provided a conducting pathway. In a parallel study, Hymel et al. (1988) found that D H P receptors incorporated into lipid bilayers also form calcium channels with multiple conductance levels. A minimum conductance of 0.9 pS was seen and other conductance levels that were integral multiples of 0.9 pS, up to 60 pS. Ma and Coronado (1988) found three conductance states of 3, 9 and 12 pS in the D H P receptor. It is suggested that the large number of subconductance states in

198

A.F. DULHUNTY

both the D H P receptor and the ryanodine receptor may reflect a common gating mechanism in which the ryanodine receptor is directly coupled to the dihydropyridine receptor (Hymel et al., 1988). Four conductance levels have been described in the IP 3 receptor calcium channels isolated from cerebellum (Bezprozvanny et al., 1991; Meyer et al., 1990) and correlated with the four subunits of the protein. (C) The "sarcoball" technique Until recently, all studies of ion channels in sarcoplasmic reticulum depended on the isolation of microsomal fractions of the muscle fibre and usually required the reincorporation of channels into artificial lipid bilayers. The sarcoball technique developed by Stein and Palade in 1988 provided a unique opportunity to study the channel with less experimental intervention. Sarcoballs (Fig. 4) form at the surface of skinned muscle fibres during unrestrained contractures when internal vesicles are extruded and coalesce (Stein and Palade, 1988; Lewis and Bretag, 1991).

FIG.4. The formationofsarcobaUson the surfaceofskinned the semitendinosusskeletalmusclefibres from Xenopus laevis. A shows a very small bleb (S) formingin an area where clusters of vesiclesare aggregated at the surface of the fibre: V is located within empty vesicles,probably of sarcoplasmic reticulumorigin;M is placedbesidemitochondria;T is placedbesidea triadjunction. B shows a small sarcoball (S) besidea mediumsizedsarcoball and C shows a large sarcoball,typicalof those used for patch clamp recoding.The calibration bar at the top of each micrograph is 2 #m (Lewis,Dulhunty, Junankar and Stanhope, unpublished observations). Sarcoplasmic reticulum, T-system and mitochondria are extruded from contracting skinned fibres (Fig. 4A) and the membranes of all organelles apparently fuse to form the foam-like structure of the sarcoball (Lewis, Dulhunty, Junankar and Stanhope, unpublished observations). Small blebs or sarcoballs initially form at the surface of the skinned muscle fibre (Fig. 4A) and these grow into larger sarcoballs (Fig. 4B), which reach maximum dimensions of 40 #m to 100/~m (Fig. 4C). Monoclonal antibodies to both the ryanodine receptor and the sarcoplasmic reticulum calcium ATPase bind to membranes on the surface and within the sarcoball, thus confirming the presence of sarcoplasmic reticulum membrane. The exclusion of calcium ATPase antibody binding from circumscribed areas of the structure suggest the presence of non-sarcoplasmic reticulum membrane, perhaps from mitochondria and T-tubules. The sarcoball membrane contains calcium (Stein and Palade, 1988) and chloride (Hals et al., 1989; Lewis and Bretag, 1991) channels that are similar to channels in skeletal and cardiac sarcoplasmic reticulum vesicles incorporated into lipid bilayers (Smith et al., 1985, 1988; Miller, 1978; Rousseau, 1989). In contrast to isolated channels (Smith et al., 1985; Ma et al., 1988; Bezprozvanny et al., 1991), calcium channels in sarcoballs do not appear to be inactivated by cytoplasmic calcium in excess of 1 mM (Stein and Palade, 1988). A comparison

Contraction in skeletal muscle

199

between results obtained with sarcoball and lipid bilayer techniques may indicate whether the characteristics of the calcium release channel are altered by isolation and incorporation into an artificial environment. IV. ACTIVATION AND INACTIVATION OF EC C O U P L I N G Asymmetric charge movement, calcium release from isolated sarcoplasmic reticulum vesicles and skinned muscle fibres, and single calcium release channels provide essential information on the properties of individual protein molecules, but give limited information about the way in which the voltage sensor regulates calcium release in intact cells. On the other hand, calcium transients and contractions show the overall response of the system to depolarization, but not the contribution of the individual components of EC coupling. The properties of voltage-activated contraction during prolonged, steady-state, depolarization are described in this section. The changes in tension are sufficiently slow that intrafibril diffusion times and the kinetics of the contractile protein response (Section III.2.b, above) are not rate-limiting. Two approaches have been used to assess the relative importance of the voltage sensor, calcium release channels and the properties of the myofilaments in the time course and voltage-dependence of the final contractile response. The first is to examine the effects on voltage-activated contractions of experimental manipulations that specifically alter one component of EC coupling. The second is to compare the basic characteristics of charge movement, calcium release channels, calcium transients, and the calcium sensitivity of the contractile proteins in preparations that demonstrate differences in their contractile properties. The accumulated evidence derived from both of these approaches allows reasonable speculation about the contribution of each of the steps in EC coupling to the nature of the tension response during prolonged depolarization.

1. Depolarization-dependent Activation of Contraction

(a) The voltage-dependence of tension is set by the voltage sensor The close relationship between the voltage-dependence of tension and asymmetric charge movement in different types of normal mammalian muscle fibres, and with chronic changes in fibre-type (see b, below), supports the idea that charge movement gates EC coupling (III.2.a above). The observations also suggest that the voltage dependence of tension is set by asymmetric charge movement. Indeed, differences in the calcium sensitivity of the contractile proteins are not reflected in the relationship between tension and membrane potential. The higher calcium concentration for half maximal activation of tension in slow-twitch fibres (Stephenson and Williams, 1981) might be expected to shift the curve relating tension and membrane potential to more positive membrane potentials in slow-twitch fibres. However, both tension and charge movement occur at membrane potentials that are about 15 mV more negative in slow-twitch soleus fibres than in fast-twitch EDL fibres (Dulhunty and Gage, 1985). The differences of 1 to 5 mV between the membrane potential for 50% activation (Va) of tension and charge movement (Dulhunty and Gage, 1983, 1985) are very small considering the intervening steps of calcium release and activation of the contractile proteins. Tension at a given membrane potential can be altered by ligands such as caffeine that act primarily on the calcium release channel (Luttgau and Oetliker, 1968; Heistracher and Hunt, 1969a), or that might act on the contractile proteins. Thus experimental manipulations that change the voltage-dependence of tension do not necessarily imply that the voltage sensor is the primary target. However, the effects of depolarization, small changes in divalent cation concentrations or addition of lipid insoluble drugs--factors that act primarily on the surface membrane, can reasonably be interpreted in terms of an effect on the D H P receptor protein. (b) Tension-voltage curves: a labile property of muscle The voltage-sensitivity of EC coupling is normally assessed from tension-voltage curves JPB 57=3-F

200

A . F . DULHUNTY

that are constructed by plotting relative tension at the peak, or plateau, of a depolarizationinduced contracture (as in Fig. 3) against membrane potential (Fig. 5). The data is well described by a Boltzmann equation which, although not strictly applicable to the multi-step relationship between tension and membrane potential, provides a useful means of comparing data. The relationship between tension, Ta, and membrane potential, Vm, can be expressed as (Dulhunty and Gage, 1983):

(V~- V,~)/ka]

T. = Tm~x/[ + ex p

(1)

where Tmax is maximum tension and Va is the potential at which Ta = 0.5 Tmax a n d factor.

A Z

o

(~ Z w Inl > I-

J w M

S

EDL 1.00

.......

Z 0

~ e ' e "

"'" 0.80

k a is a

slope

SOLEUS 1.00 0.80

m

0.60

0.60

O.40

0.40

)

0.20 0.00 -60

-48

-36

-24

-12

MEMBRANE P O T E N T I A L

0 (mY)

0.20 0.00 -60

-48

-96

-24

-12

MEMBRANE P OTE N TI A L

0 (mY)

FIG. 5. Activation curves for tension in fast-twitch E D L (A) and slow-twitch soleus (B) fibres from rat.

K-contracture tension was recorded from small bundles of 5 to 15 fibres and plotted against membrane potential in high K ÷ solutions. In A, the filled circles are average normalized tension from control EDL fibres and the open circles show average data from fibres denervated for 3 to 5 weeks. In B, the filled circles are average normalized tension from control soleus fibres and the open circles are from soleus fibres 6 to 12 weeks after spinal cord transection. The standard errors are within the dimensions of the symbols in A and are shown as vertical capped bars in B. The curves through the data were generated using equation 1. In A, Va and k a are - 1 3 mV and 6.2 mV respectively for normal fibres (continuous line) and - 34 mV and 2.7 mV for denervated fibres (broken line). In B, 1,~ and ka are - 2 4 mV and 6.0 mV respectively for normal fibres (continuous line) and - 16 mV and 3.5 mV for fibres from spinal cord transected animals (broken line) (modified from Dulhunty and Gage, 1983, 1985).

The voltage-dependence of tension varies between species, or between fibre types within one species, and can be shifted by chronic procedures that alter fibre-type, V, is between - 40 mV and - 50 mV for amphibian twitch fibres (Hodgkin and Horowicz, 1960), - 15 mV in snake twitch fibres (Heistracher and Hunt, 1969a), - 11 mV for mammalian fast-twitch fibres and - 2 7 m V for mammalian slow-twitch fibres (Dulhunty and Gage, 1985). Denervation converts fast-twitch fibres to slow-twitch fibres and produces a shift to more negative potentials in the activation of tension in EDL (Fig. 5A; Dulhunty and Gage, 1985). Spinal cord transection (Fig. 5B) and hyperthyroidism, on the other hand, transform mammalian slow-twitch fibres into fast-twitch fibres and produce a shift in the voltagedependence of tension in soleus to more positive potentials (Dulhunty and Gage, 1983; Dulhunty et al., 1987). In most cases the shifts in the voltage-dependence of tension follow parallel shifts in asymmetric charge movement and are therefore attributed to changes in the voltage sensor. Tension-voltage curves can be moved in either direction along the voltage axis by altering the surface charge on the T-tubule membrane: ions, particularly multivalent ions, can induce strong effects either by general screening of surface charge or by specifically binding to charged groups near the voltage sensor. Addition of millimolar concentrations of divalent cations to the external solution, neutralizes surface charge and shifts the tension-voltage curve to more positive membrane potentials (Caputo, 1981; Lorkovic and Rudel, 1983; Dorrscheidt-Kafer, 1976; Bolanos et al., 1986; Dulhunty and Gage, 1989). Perchlorate ions

Contraction in skeletal muscle

201

induce a negative shift in the voltage-dependence of both tension and charge movement (Luttgau et al., 1983; Csernoch et al., 1987). This shift is thought not to depend on a general addition of surface charge since neither the voltage-dependence of activation of sodium channels or potassium channels, or the voltage dependence of the inactivation of tension are similarly affected (Gomolla et al., 1983). However perchlorate may bind to specific sites on the D H P receptor and alter the membrane field seen by the voltage sensor as has been suggested for cobalt ions (Dulhunty and Gage, 1988). (c) Molecular regulation of voltage sensitivity In addition to surface charge effects, the amino-acid sequence of the D H P receptor and regulatory factors may contribute to the voltage-sensitivity of tension. The relationship between the amino acid sequence of the D H P receptor and the voltage-dependence of tension has not been directly investigated. However the results of studies with potassium channels may be relevant to the voltage sensor for EC coupling. The $4 segment of the D H P receptor (Section II.2.a, above) is homologous with the $4 segment of voltage-gated sodium and potassium channel proteins. The activation of Shaker potassium channels can be shifted along the voltage axis by mutations of charged and hydrophobic residues in the $4 segment of the protein (Papazian et al., 1991; McCormack et al., 1991). Mutations to many other parts of the potassium channel protein can also alter activation. The activation curve for delayed rectifier potassium channels from rat brain can be shifted by as much as + 40 mV by deletions to both the N- and C-terminal portions of the molecule, and the rate of activation can be altered by the same deletions (VanDongen et al., 1990). It would be interesting to know whether similar mutations of the D H P receptor would alter the voltage-dependence of EC coupling. Endogenous ligands for channel proteins, such as neurotransmitters, can also shift the activation curves of voltage-dependent processes along the membrane potential axis. Again, although this has not been specifically shown for EC coupling, it could well reflect a general property of voltage-activated molecules. V, for transient potassium currents in hippocampal neurons is shifted by 40 mV to more positive potentials by GABA, (gamma aminobutyric acid) agonists (Saint et al., 1990). Cyclic AMP may alter the voltage dependence of EC coupling in skeletal muscle, fl-adrenergic effectors (adrenaline and terbutaline) shift tension to more positive membrane potentials via an action of cyclic AMP (Cairns and Dulhunty, unpublished) and hence lead to an increase in the amplitude of twitch and tetanic contractions in mammalian skeletal muscle (Fig. 6).

5ran [ m lOs

!

J.

CONTROL

1

ADRENALINE

CONTROL

FIG. 6. The effects of the adrenaline (10 psi) on twitch and tetanic tension in small bundles of 5 to 15 rat soleus muscle fibres. The small vertical deflections are twitches and the large deflections are tetanic contractions. Similar effects on peak tension were observed after application of 10 #M terbutaline (a fl-adrenergic agonist) or 2 mM dibutyryl cyclic AMP to the bathing solution (Cairns and Dulhunty, unpublished).

202

A.F.

DULHUNTY

The site of action of cyclic AMP in EC coupling has not been clearly established. Adrenaline increases the amplitude of calcium transients in amphibian muscle (Brum et al., 1990). Cyclic AMP increase calcium influx through D H P sensitive calcium channels (see Hofmann et al., 1987 for review). However, asymmetric charge movement has not been found to be altered by cyclic AMP or adrenaline (Gamboa-Aldeco et al., 1989; Brum et al., 1990). The actions of fl-receptor agonists on tension may depend on stimulation of calcium release from the sarcoplasmic reticulum rather than being a direct effect on the voltage sensor: cyclic AMP phosphokinase has been shown to phosphorylate isolated calcium release channels (Timerman et al., 1990) and increase single channel activity (Hymel et al., 1989). The surface charge on the T-tubule membrane, the subtype of protein expressed by the cell (DHP receptors with slightly different amino acid sequences, for example), and regulatory factors, are all possible targets for modification by spinal cord transection, hyperthyroidism and denervation. Although denervation is well known to affect protein expression and turnover (Thesleff, 1974; Wallis amd Koenig, 1980), an additional effect on a general property of muscle fibre, like membrane surface charge or regulatory molecules, is suggested by the fact that two different voltage-dependent processes are altered in a similar manner. Sodium conductance, like EC coupling, is activated at more negative membrane potentials following denervation of fast-twitch fibres (Pappone, 1980). 2. Inactivation of Contraction

The decay of K-contracture tension and myoplasmic calcium concentrations during steady-state depolarization is thought to reflect the onset of the process that produces steadystate inactivation, seen as a reduction in the amplitude of test tensions evoked during sustained conditioning depolarizations (Fig. 7A). Steady-state inactivation is a voltage- and time-dependent process which is greater, and develops more rapidly, during conditioning depolarizations to more positive membrane potentials (Fig. 7B). The decline in test Kcontracture tension is biphasic (Fig. 7B): there is an initial rapid phase and then a slower phase of relaxation to a steady-state value reached after about 10 min.

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FIG. 7. Measurement of the development of steady-state inactivation in small bundles of rat soleus fibres. In A, the brief vertical deflections are tetanic contractions in response to stimulation at 50 Hz for 1 sec. The slower tension responses are K-contractures. Fibres were exposed to conditioning 40 mM K + (upper records) or 80 mM K + (lower records) solutions for various times, including 3 rain (horizontal lines, left hand panel) or 5 min (horizontal lines, right hand panel) and then exposed to a test 200 mM K + solution (double arrow). Test tension is normalized to mean contracture tension in 200 mM K + recorded before and after the conditioning/test sequence (the before response only is shown in A). The horizontal calibration is 3 min and the vertical calibration is 4 mN. In B, normalized test 200 mM K + contracture tension is plotted against time in the conditioning high potassium solution: filled circles, 20 mM K +; open circles, 40 mM K +; filled squares, 80 mM K +. The vertical bars show _+ 1 sem.

12

Contraction in skeletal muscle

203

(a) Inactivation is related to a conformational state of the voltage sensor The hypothesis that the decline in tension with prolonged depolarization depends on inactivation of the voltage sensor is based on observations of immobilization of asymmetric charge movement (Section III.3.d, above) and other experiments which exclude effects of depolarization on the sarcoplasmic reticulum or contractile proteins. Caffeine has been employed to show that calcium stores are not depleted, and that the contractile proteins can generate normal tension, in mechanically refractory (inactivated) fbres. Low concentrations of caffeine (1-5 mM), release calcium from the sarcoplasmic reticulum (Delay et al., 1986; Fryer and Neering, 1989) without significantly increasing the calcium affinity of the myofilaments (Wendt and Stephenson, 1983). Two and a half mM caffeine produces nearly maximum tension during inactivation of tension by depolarization (Caputo, 1976) and contractures with 1 mM to 5 mM caffeine are enhanced by depolarization for 30 min to 96 hr(I) in 190 mM K + (Axelsson and Thesleff, 1958). Caffeine contractures in intact amphibian fibres maintain peak tension during a 40 sec exposure to 2.5 mM caffeine whereas K-contractures of the same peak amplitude decayed to zero tension within 20 sec (Caputo, 1976). This observation suggests that the calcium release channel does not inactivate. In addition, time-dependent kinetics that may reflect inactivation have not so far been described in studies of single calcium release channels. (b) Steady-state inactivation: shifts in voltage dependence Steady-state inactivation is usually measured with two pulses: a "conditioning" depolarization producing less than maximum tension is applied before a "test" depolarization that induces maximum tension (Fig. 7A). In K-contracture experiments in rat soleus fibres for example, solutions containing 20 mM tO 120mM K + produce conditioning depolarizations to potentials between - 50 mV and - 20 mV and 200 mM K ÷ produces a test depolarization to - 6 . 5 mV (Fig. 7A; Dulhunty, 1991).

A Z

o

SOLEUS

B

1.20

uJ I-

0.80

w > I-

0.40

J w rr

0.00

1.20

m

0,80

o

Z

EDL

Z

m

-

0.40

w 0.00

100

-60

-20

MEMBRANE POTENTIAL

20 (mY)

-

-100

-70

-40

~-

-10

MEMBRANE POTENTIAL

20 (mY)

FIG. 8. Steady-state inactivation curves for tension in small bundles of rat soleus (A) and E D L (B) fibres. The measurement of relative test 200 mM K + contracture tension is described in Fig. 7. In A, the filled circles show average test tension recorded in control solutions and the open symbols show average test tension recorded when 10 mM perchlorate was added to the bath prior to exposure to the conditioning K + solution (which also contained 10 mM perchlorate) or the 200 m~i K ÷ solution (with 10 mM perchlorate). In B, the filled symbols show the average test tension recorded in muscle fibres from normal rats and the open symbols show data recorded from EDL muscle from rats given daily injections of tri-iodothyronine (150/~g/kg body weight) for 10 to 14 days. The vertical capped bars show + 1 sere. The curves through the data were generated using equation 2. In A, Vj and k~ are - 3 7 . 5 mV and 2.2 mV respectively for normal fibres (continuous line) and - 4 3 mV and 3 mV in CIO2 (broken line). In B, Vi and k~ are - 5 0 mV and 10.0 mV respectively for normal fibres (continuous line) and - 3 8 mV and 9 mV for fibres from hyperthyroid rats (broken line) (modified from Chua and Dulhunty, 1989; Dulhunty e t al., 1992).

A plot of test K-contracture amplitude against conditioning membrane potential provides a steady-state inactivation curve (Fig. 8). The voltage-dependence of steady-state inactivation can be described by a Boltzmann equation of the form: Ti = Tmax/[1+ exp (V~- VO/k~]

(2)

204

A.F. DULHUNTY

where Ti is the test contracture amplitude at a conditioning membrane potential Vc. Vi is the potential at which T~=0.5 Tmax and kl is a slope factor. As with the voltage-dependence of activation of tension, steady-state inactivation differs between species and types of muscle fibre and can be altered by procedures that influence fibre-type. The membrane potential for 50% inactivation is - 35 mV in amphibian fibres (Luttgau and Spiecker, 1979), - 34 mV in mammalian slow-twitch fibres (Fig. 8A) and - 5 2 mV in mammalian fast-twitch fibres (Fig. 8B). V~in fast-twitch fibres is shifted by + 7 mV after denervation (Patterson and Dulhunty, unpublished) and by + 9 mV in hyperthyroidism (Fig. 8B); V~in slow-twitch fibres is shifted by - 4 mV in chronic hyperthyroidism (Chua and Dulhunty, 1989). Steady-state inactivation can also be shifted along the voltage axis, by - 20 mV or more, to more negative membrane potentials by reducing free extracellular calcium ions from millimolar to micromolar or nanomolar concentrations and by dihydropyridines (Luttgau and Spiecker, 1979; Luttgau et al., 1987) and by perchlorate ions (Fig. 8A). The effect of low external calcium concentrations on the steady-state inactivation curve has led to the suggestion that the transition to the inactive conformation of the voltage sensor depends on dissociation of calcium bound to the protein molecule (Section IV.3.c, below): dissociation occurs more rapidly if the free calcium concentration is lower (Luttgau et al., 1987). (c) Molecular mechanisms underlying inactivation The molecular changes associated with inactivation of EC coupling are not known. However, inactivation has been studied in other "simpler" voltage-dependent systems and the results may turn out to apply to the homologous DHP receptor protein also. Some voltage-dependent sodium and potassium channels inactivate. Potassium channels that do not normally inactivate can be blocked, once they are open, by quarternary ammonium ions (Armstrong, 1969, 1971). Inactivation of sodium channels is selectively destroyed when squid axons are perfused with the proteolytic enzyme pronase (Armstrong et al., 1973). These observations have led to the "ball and chain" hypothesis that inactivation occurs when a mobile part of the channel plugs the open pore (Armstrong and Bezanilla, 1977). The hypothesis is supported by observations on variants of Shaker potassium channels from Drosophila and on delayed rectifier and A-type potassium channels from rat brain: the voltage dependent potassium channel protein consists of one membrane spanning domain, which corresponds to one of the four domains of sodium and calcium channels; it is thought that four molecules cluster to form the potassium ion channel (see VanDongen et al., 1990 for summary). The different types of potassium channel are homologous in their membrane spanning domains, but differ in their cytoplasmic regions, including the terminal portions. Different kinetics of inactivation in each type of potassium channel can be related to variations in the N- and C-termini of the protein. A family of four rck proteins from rat brain, with similar transmembrane segments, but different N- and C-termini, form potassium channels with characteristics of either delayed rectifier (non-inactivating) channels or rapidly inactivating A channels: currents recorded from rck4, the protein with the longest Nterminus, shows the most rapid inactivation (Stuhmer et al., 1989b). There is a reduction in inactivation of Shaker potassium currents when the N-terminus of the protein is truncated (Hoshi et al., 1990). In addition, a peptide containing the first 20 amino acids in the Nterminus of the Shaker B (inactivating) channel can restore inactivation in a non-inactivating mutant of the channel (Zagotta et al., 1990). Mutations in the cytoplasmic region between repeats III and IV of the sodium channel protein slow inactivation and the "linker" region might contain the "ball" that blocks the Na ÷ channel (Moorman et al., 1990; Stuhmer, 1991). Inactivation is clearly more complex than the simple "ball and chain" model and can be influenced by mutations to many parts of the channel protein. Deletions to both the N- and C-termini of a delayed rectifier potassium channel from rat brain, encoded by the drkl gene, produce profound changes in the amount and rate of inactivation: more extensive deletions of both termini resulted in restoration of normal inactivation characteristics (VanDongen et al., 1990). Thus the "ball and chain" model may not apply to the slow inactivation of delayed rectifier channels. Mutations to both charged and hydrophobic residues of the $4 segment of

Contraction in skeletalmuscle

205

the Shaker potassium channel proteins shift the activation and inactivation curves along the voltage axis to either more positive or more negative membrane potentials (Papazian et al., 1991; McCormack et al., 1991). It is possible that changes in the conformation of the channel can influence the ability of the "ball" to block the pore. Although the voltage-dependence of inactivation of EC coupling is also likely to be an intrinsic property of the D H P receptor protein, it could be modified by external factors including membrane surface charge and modulatory molecules. The voltage-dependence of inactivation of A-currents in hippocampal neurons is shifted 40 mV in the positive direction by GABA B agonists (Saint et al., 1990). It is curious that Vi in two very different processes, EC coupling (Chua and Dulhunty, 1988) and sodium currents (Ruff et al., 1987, 1988), occurs at more negative membrane potentials in mammalian slow-twitch muscle than in fasttwitch fibres: a common factor may influence both phenomena. On a cautionary note, although we can speculate about the mechanisms of inactivation of EC coupling by drawing analogies between sodium or potassium channels and the D H P receptor protein, tension experiments with mutations and deletions in D H P receptors have yet to be performed. There is one important difference between the inactivation of K ÷ currents and inactivation of EC coupling. The rate of decay of A-currents is not voltagedependent (Zagotta et al., 1990), whereas the decay of K-contractures is strongly voltagedependent (Hodgkin and Horowicz, 1960; Dulhunty et al., 1992b). This difference could indicate that different molecular mechanisms are involved in the inactivation processes. The subunits of the D H P receptor may also play an important role in modulating inactivation of calcium channels. The ~1 subunit of the D H P receptor calcium channel expressed alone in Xenopus oocytes shows very slow inactivation kinetics: the rate of inactivation increases and the inactivation curve is shifted to more negative membrane potentials when various combinations of subunits are added, maximum effects are seen with the ~ and fl subunits (Catterall, 1991; Singer et al., 1991). 3. The Kinetics of Inactivation

The rate of inactivation can be assessed from the decay of K-contractures or from the rate of onset of steady-state inactivation. Although it is generally assumed that the decay of Kcontractures and the onset of steady-state inactivation reflect the same process, this may not necessarily by the case. Again, from analogies with ion channels, it has been shown that the decay of sodium currents in some mammalian neurons can be set by the kinetics of sodium channel opening (Aldrich et al., 1983). The rate of decay of K-contracture tension predicted by a sequential model for the activation and inactivation of the voltage sensor (Section IV. 3.c below) can also be strongly influenced by the activation rate constants (Gage and Dulhunty, unpublished observations). Thus any description of the kinetics of inactivation must be based on observations of steady-state inactivation as well as the decay of K-contracture tension. (a) Slow time course of inactivation in mammals The rate of decay of K-contracture tension and development of steady-state inactivation in phasic skeletal muscle fibres can vary over a 10-fold range. The decay of tension during steady depolarization takes 10-20 sec in amphibia (Hodgkin and Horowicz, 1960; Caputo, 1976), but requires 100--200 sec in mammals (Dulhunty, 1991). The slow decay of Kcontractures in mammals cannot be attributed to tension being maintained by an influx of external calcium: contractures are not altered by low external calcium concentrations (Dulhunty, 1991) or nifedipine (Dulhunty et al., 1992b) and are prolonged when external calcium is replaced by magnesium, cobalt or cadmium (Dulhunty and Gage, 1988). Kcontracture tension rapidly deactivates upon repolarization, suggesting that it is controlled by membrane potential and not maintained by a slow second messenger: the slow decay of tension in mammals is paralleled by the slow onset of steady-state inactivation (Dulhunty, 1991). Steady-state inactivation in mammals (Dulhunty, 1991) develops ten times more slowly than in amphibia (Nagai et al., 1979). The onset of steady-state inactivation is biphasic (see

206

A.F. DULHUNTY

Fig. 7B above; Nagai et al., 1979) and both phases are an order of magnitude slower in mammals. An "ultraslow" component of steady-state inactivation has been described in frog fibres that reaches completion after 100 sec (Caputo and Bolanos, 1990): it is not clear whether this is a part of the second phase of inactivation in amphibia described by Nagai et al. (1979), or whether it represents a third inactivated state. The slow phase of inactivation in rat soleus fibres continues for longer than 600 sec (Fig. 7B above). By analogy with ion channels, the different time course of inactivation in amphibia and mammals may reflect small differences in the amino acid sequence of the voltage sensitive molecule (VanDongen et al., 1990) or differences in the subunits of the DHP receptor expressed in different species (Catterall, 1991; Singer et al., 1991 ). (b) A biphasic time course of inactivation: two inactivated states That steady-state inactivation develops with a biphasic time course (Fig. 7; Nagai et al., 1979) could mean that there are two stages of inactivation in EC coupling. These have been referred to as "inactivation 1" and "inactivation 2" by Nagai et al. (1979), or "refractory" and "paralysed" states by Luttgau et al. (1986). The transition to the refractory state (inactivation 1) produces the rapid decay of K-contracture tension and is quickly reversed upon repolarization. The transition to the paralysed state (inactivation 2) occurs with longer periods of depolarization (Hodgkin and Horowicz, 1960), with deprivation of external calcium (Luttgau et al., 1986), and in the presence of the calcium channel agonists gallapomil (D600, Berwe et al., 1987) and nifedipine (Rios and Brum, 1987; Dulhunty and Gage, 1988). Recovery from the paralysed state is slow, with repriming times lasting many minutes. If the decay of K-contractures reflects the onset of steady-state inactivation it should, like the decay of test K-contracture tension, have a biphasic time course. Although the decay of K-contractures is normally monophasic (see Fig. 3 above), a slow phase of decay is unmasked in rat soleus fibres when perchlorate ions are added to the extracellular solution (Dulhunty et al., 1992b). K-contractures become amazingly slow, with large tensions that are generated for more than 10 rain and are independent of extracellular calcium concentration (Fig. 9). The slow decay is exponential and steeply voltage dependent. Tension during the slow K-contracture decays rapidly upon repolarization (Fig. 9A and B) and is thus under the direct control of the surface membrane potential. The time course of the slow decay of Kcontractures with perchlorate is similar to the time course of the slow component of steadystate inactivation and is thought to reflect the same mechanism. It has been suggested that the slow phase of inactivation is normally not seen in the decay of tension because it occurs when myoplasmic calcium concentrations are below threshold for contraction (Dulhunty et al., 1992b). The principal action of perchlorate is to shift the activation curve for asymmetric charge movement to more negative membrane potentials (Luttgau et al., 1983; Csernoch et al., 1987): the boosting effect of the anion on the voltage sensor may raise calcium concentrations to levels that remain above threshold for contraction during the slow phase of inactivation. The second inactivated state could reside in either the voltage sensor or the mechanism coupling the voltage sensor with the calcium release channel, but it cannot be attributed to changes in later stages of activation. The larger caffeine contractures after 30 min in 190 mM K ÷ and the lack of time-dependent kinetics in the isolated Ca 2 ÷ channel (Section IV.2.a, above) show that calcium stores are not depleted, the tension generating capacity of the myofilaments is normal and the calcium release channel is not inactivated. It is unlikely that fatigue processes, that reduce twitch and tetanic tension during repeated stimulation, contribute to paralysis since tension is close to zero during the development of this slow phase of inactivation. It is not easy to distinguish between the possibilities that slow inactivation arises in the voltage sensor, or in the coupling mechanism. However, it is interesting to note that a slow phase of inactivation is not unique to EC coupling. A similar process, that develops over a period of minutes, is seen in voltage dependent sodium channels (Brismar, 1977; Almers et al., 1983; Ruffet al., 1987, 1988). Given the sequence homology between the sodium channel protein and the DHP receptor (Section lI.2.a above), slow inactivation of EC coupling could well occur in the voltage sensor.

Contraction in skeletal muscle

A

lOmMperchlorate

lOmMperchlorate ine

.................................

40K

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

40K

5 rain

B

207

I mN

lOmMperchlorate

i

lOmMp rchlorate 10mMcobalt a

40K

.......................................

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

40K

FIG.9. Twitches (rapid verticaldeflections)and K-contractures in 40 mMK + and 10 mMperchlorate. A shows contractures in a bundle of fibresexposedfirst to solutions containing perchlorate alone and then to solutions containing perchlorate plus 15/aMnifedipine. B shows contractures in a different bundle of fibres exposed first to solutions containing perchlorate alone and then to solutions containing perchlorate plus 10 mMcobalt and lacking added calcium. The 40 mM K ÷ solution was added for the duration of the broken line below each record. The horizontal calibration is 5 min and the vertical calibration is 5 mN. (c) A sequential model with two inactivated states Although the site of slow inactivation of EC coupling is open to speculation, the refractory and paralysed states can be described by a simple "state" model (Luttgau et al., 1986; Dulhunty and Gage, 1988) which suggests that the D H P receptor voltage sensor exists in a number of conformations. Depolarization converts the molecule from a precursor, P, to an active state, A. The conversion to A requires a charge generating step with the formation of an intermediate state Q, followed by the rapid dissociation of calcium. The dissociation of calcium is included because activation is frequently enhanced when external calcium is reduced to micromolar or even lower concentrations (Graf and Schatzmann, 1984; Luttgau et al., 1986; Dulhunty and Gage, 1988). If depolarization is maintained, A is converted to the first inactivated state, I, and then to a second inactivated state, Y. It is proposed that conversion to Y involves a further dissociation of calcium since paralysis occurs more readily at micromolar external calcium concentrations (Luttgau et al., 1986; Dulhunty and Gage, 1988). This sequential model can be represented by Ca 2 +

Pc

~Q~---~-:-~.A,

Ca 2 +

'I~-----4Y.,

It is assumed that the calcium release channel opens when the voltage-sensitive molecule is in state A. K-contracture tension can be predicted by the model if it is assumed that myoplasmic calcium concentrations and tension are proportional to the concentration of A (Gage and Dulhunty, unpublished). The model predicts that the concentrations of A increases to a peak and then decays, rapidly initially as the molecule is converted to I and then slowly as it is converted to Y: a "pedestal" concentration of A remains once the steadystate equilibrium has been achieved (Fig. 10). That K-contractures (Fig. 3) and contractures induced by depolarizing voltage clamp steps (Luttgau et al., 1987) should decay monotonically to a baseline tension may at first seem inconsistent with the sequential model. However, tension would fall to zero when myoplasmic calcium concentrations drop below

208

A.F. DULHUNTY

IB

A 5 _<

5 _<

4

LU > I< ._1

4

3

3 F< J

2

2 1

1

threshold

0 0

threshold J

i

i

200

400

600

0 0

TIME (seconds)

i

i

200

400

m

600

TIME (seconds)

FiG. 10. Predicted changes in the relative concentration of voltage sensitive molecules in the active state, [A], followinga small depolarization in A and a larger depolarization in B. The relative [A] was calculated using the sequential model given in the text. The broken line shows a hypothetical [A] required to maintain myoplasmic calcium concentrations above threshold for contraction. Small changes in k~Acan shift the concentration of A above or below the threshold value. The rate constants used were'k 0 0 7 s e c - l i n A a n d 0 9 s e c - l i n B ; k e 0 7 s e c - ~ i n A a n d 0 0 5 s e c - ~ i n B ' k A 0.8 sec- ~in A and 0.18 sec- 1in B; kqA,0.001 sec- 1in A and B; k~t,0.10 sec- 1in A and 0.13 sec- 1in B; k~vand kzv,0.0001 in A and B. The concentration of activator fell below threshold when kQ^, was reduced from 0.012 sec-~ to 0.008 sec-~ in A, or from 0.018 sec-: to 0.012 sec-~ in B. •

PQ'

•

•

Q'

"

•

'

(2

,

threshold for c o n t r a c t i o n ( D u l h u n t y , 1991). "Pedestal" tensions (Section IV.4) would occur when calcium c o n c e n t r a t i o n s d u r i n g the slow decay of A, a n d in the steady-state, occasionally exceed the threshold for t e n s i o n ( C h u a a n d D u l h u n t y , 1988, 1989). The curves in Fig. 10 predicted by the m o d e l show that the relative c o n c e n t r a t i o n of A, like tension, rises to a lower peak a n d the rates of rise a n d decay are slower with small depolarizations (A) t h a n with large d e p o l a r i z a t i o n s (B). Small changes in the rate c o n s t a n t k~Acan move [A] a b o v e or below the level required to m a i n t a i n threshold c o n c e n t r a t i o n s of calcium ions.

A

B

£

£6 F4 < m C

2 0

0

200

400

TIME (seconds)

600

A

0

150

i

300

i

450

t

600

TIME (seconds)

FIG. 1I. Changes in the relative concentration of voltage sensitivemoleculesin the active state, [AJ, predicted by the sequential model for control conditions and with 10 mMperchlorate. The broken line shows a hypothetical [A] required to maintain myoplasmic calcium concentrations above threshold for contraction. The rate constants in the model were adjusted so that the decay of I-A] follows the same time course as the decay of K-contractures and the initial decay is similar in control and perchlorate-containing solutions (Dulhunty et al., 1992). Graph A shows the decay of [A] to a maintained high pedestal in perchlorate, similar to the decay in tension in Fig. 9A. Graph B shows a more rapid fallin I-A]in perchlorate, similar to the decay of tension in Fig. 9A. The rate constants used in graph A for the control curve were: k~, 0.09sec-1; kov, 0.03 sec-1; kQA, 0.4sec-1; kAQ, 0.001 sec- ~;kay,0.15 sec- t; ktA,0.015; kv~,0.0005; kw, 0.0001. The rate constants for CtO2 in A were: kr~, 0.6 sec- I ; kov, 0.1 s e c -1; koA, 0.25 sec- I ; kAo,0.001 sec- 1 ; kay,0.04 sec- I ; k~A,0.015; kw, 0.0005; k=v,0.0001. All rate constants used in graph B, except ktv, were the same as those in graph A: kw was increased to 0.005 sec-~ in graph B. The biphasic K - c o n t r a c t u r e s with perchlorate (Fig. 9 above) can also be predicted by the sequential m o d e l (Fig. 11) if is a s s u m e d that the rate c o n s t a n t kpo is increased by the a n i o n (since a s y m m e t r i c charge m o v e m e n t is e n h a n c e d with perchlorate, L u t t g a u et al., 1983; C s e r n o c h et al., 1987) a n d thus the c o n c e n t r a t i o n of A formed at a p a r t i c u l a r m e m b r a n e

Contraction in skeletal muscle

209

potential is greater than in control solutions. It was necessary to assume that perchlorate also affects three other rate constants (kQv, kQAand kAi) in order to reproduce the similar initial decay times seen with K-contractures in control solutions and with perchlorate (Dulhunty et al., 1992b). With these changes in rate constants, the concentration of A remains above threshold for contraction during the slow decay. The different rates of decay of the slow phase shown in Fig. 11A and B, also seen with perchlorate (Fig. 9), were achieved by varying k~v. Unlike the first phase of the decay, the slow phase is unaffected by changes in the activation rate constants. The biphasic profile of the decay of K-contracture tension in perchlorate provides evidence to support the sequential model with two inactivated states of the voltage sensor. (d) Pedestal tensions: independent or sequential activation and inactivation processes An alternative model for the voltage sensor suggests that activation and inactivation are two independent voltage-dependent processes that act separately to open or close the calcium release channel (Luttgau and Spiecker, 1979; Caputo, 1981; Graf and Schatzmann, 1984). Pedestal tensions recorded during prolonged depolarization were thought to provide evidence for this model. K-contractures often decay to a maintained pedestal tension that is slightly greater than the base-line tension (see insert in Fig. 12; Chua and Dulhunty, 1988, 1989; Lamb and Stephenson, 1990b). It has been suggested that, as might be the case with sodium currents (AttweU et al., 1979; Colatsky, 1982), the pedestal tension is a steady-state tension that arises from incomplete inactivation of the voltage sensor. Indeed, the amplitude of the pedestal tension is voltage-dependent (Fig. 12B) and tensions are largest at membrane potentials near the area of maximum overlap between the activation (tension-membrane potential) and steady-state inactivation curves (Fig. 12). Steady-state tensions can be predicted using a model which assumes that (a) the activation and inactivation curves reflect activation and inactivation of the voltage sensor, (b) activation and inactivation of the voltage sensor are independent processes and (c) tension at the peak of a K-contracture reflects a steady-state level of activator that would be maintained if the independent inactivation did not reduce calcium release (Chua and Dulhunty, 1988, 1989). However, when pedestal tensions were measured at several membrane potentials within the area of overlap, it became clear that tensions predicted by this model (continuous curve, Fig. 12B) were generally larger than the observed pedestal tension. A poor fit to pedestal tension data by the independent model for activation and inactivation was also seen with perchlorate (Dulhunty and Zhu, unpublished). The anion was used in this study because it produces a large negative shift in the voltage-dependence of tension (Luttgau et al., 1983) and a much smaller shift in inactivation (Dulhunty et al., 1992b), thus increasing the area of overlap between the curves (Fig. 13A). It was expected that the amplitude of the pedestal tension would be increased by perchlorate. The pedestal tension did increase (Fig. 13B), but it remained significantly less than the predicted tension and was displaced along the voltage axis (continuous line, Fig. 13B). The measured pedestal tension in control solutions and with perchlorate could be predicted by the independent model if it was assumed that the steady-state activation curve for the voltage sensor was shifted towards more positive membrane potentials than the voltage-dependence of peak Kcontracture tension (broken lines, Fig. 12 and Fig. 13). Pedestal tensions in control solutions (Fig. 14A) and with perchlorate (Fig. 14B) can be predicted by the sequential "state" model (Section IV.3.c, above), if it is assumed that klv and kyi increase with voltage and that kw initially increases more rapidly than kvl (Gage and Dulhunty, unpublished observations). The same changes in rate constants also accurately predict the increased rate of decay of K-contracture tension at more positive membrane potentials. The ability of the sequential model to fit both the decay of K-contractures and the pedestal tension suggests that it provides a more appropriate description of the response of the voltage sensor than the independent model. Although the sequential model accounts for the complex changes in tension during depolarization, it gives no information about the actual molecular changes that transform the molecule from the precursor to the active or inactive states, or about the amino acid

210

A.F. DULI-IUNTY

A

E3 z 0

1.20

z w F iii > I--

0.80 0.40

l

uJ rr

0.00 -60

.

Z

-40

MEMBRANE

0.08

>

i

I

40K

w

2

4

n'f4

rnin

0.04

<

o

-20

POTENTIAL

0.12

,

_J W ~

0

0.00 -60

(mV)

-40

-20

MEMBRANE POTENTIAL

0 (mY)

FIG. 12. Overlap of activation and inactivation curves for tension in rat soleus fibres in control solutions (A) and pedestal tensions recorded in the same fibres (B). Pedestal tension is expressed relative to maximum tension recorded in 200 mM K +. The insert in B shows a conditioning contracture in 40 mM K* with a typical small pedestal tension. Test 200 mM K + solutions were added after I0 rain conditioning depolarization and the pedestal tension was measured between 8 and 10 rain in the conditioning solution. In A, the open circles are average test 200 mM K ÷ contracture tension, the filled circles are average peak conditioning K-contracture tension, and the vertical bars show __+1 sem. The broken line is an inactivation curve fitted to the open symbols using equation 2 above: V~= - 37.2 mV and ki = 2.2 mV. The continuous line is an activation curve fitted to the filled symbols using equation 1 above: v,= -28.7 mV and k,=2.8 inV. In B, the filled circles are the average pedestal tensions and the vertical bars show _+1 sem. The continuous line shows pedestal tensions predicted from the overlap of the activation and inactivation curves in A (Chua and Dulhunty, 1988, 1989). The broken line shows pedestal tensions predicted from the inactivation curve in A and a hypothetical activation curve in which V, was - 2 5 mV and ka was 3 mV.

,&

E3

z 0

1.20

z 0

0.28

z w F

0.80

w t.-

0.19

(~

w > h< ._J w rr

w

_>

0.4O

I< -,

0.00 -60

-

-40

MEMBRANE

-

-20 POTENTIAL

0

i

0 (mY)

Ill IT"

0.09

0.00 -60

-40

MEMBRANE

-20 POTENTIAL

(mY)

FIG. 13. Overlap of activation and inactivation curves for tension in rat soleus fibres in solutions containing 10 mu perchlorate (A) and pedestal tensions recorded in the same fibres (B). The symbols and curves in A and B were obtained in the same way as previously described for Fig. 12. Parameters used for the curves in A were - 4 3 mV and 3 mV for V~ and k t (inactivation curve) respectively (broken line) and - 4 3 mV and 3 mV for Va and k, (activation curve, continuous line). In B, the continuous line is predicted from the overlap between the activation and inactivation curves in A, and the broken line is predicted from the overlap of the inactivation curve and a hypothetical activation curve in which Va was -40.5 mV and ka was 1.5 mV.

r e s i d u e s o r s u b u n i t s o f t h e p r o t e i n i n v o l v e d i n e a c h o f t h e t r a n s i t i o n s . It r e m a i n s f o r m o l e c u l a r biology to solve these p r o b l e m s before we can finally u n d e r s t a n d the c o m p l e x c o n f o r m a t i o n a l c h a n g e s w h i c h a l l o w t h e v o l t a g e s e n s o r in t h e T - t u b u l e t o o p e n a n d c l o s e t h e c a l c i u m r e l e a s e c h a n n e l in t h e s a r c o p l a s m i c r e t i c u l u m . V. T H E

MECHANISM

OF EC COUPLING

A number of possible mechanisms for coupling between the voltage sensor and calcium release channels have been discussed throughout this article. It may be useful to finally summarise these mechanisms and look briefly at their relative merits. The structural,

Contraction in skeletal muscle

211

,& z ©

0.02

S

0.16

z w l-

0.01

Z Ill t.--

0.11

w > i,<

0.01

Ill :> I-

0.05

d

J

w n-

0.00 -60

!11 £c -40

-20

MEk, E~RANE P O T E N T I A L

0 (mY)

0.00 -60

-40

MEMBRANE

-20 POTENTIAL

(mY)

FIG. 14. Pedestal tensions predicted by the sequential model for the voltage sensor. Pedestal tension recorded in control solutions (A) and C102 (B) (also shown in Figs 12B and 13B respectively) are plotted against membrane potential. The curves through the data were predicted using the sequential model for the formation of active voltage sensor molecules as described for Figs 10 and 11, with the additional assumption that the rate constants kAl, klA and klv are voltage dependent and that k A, initially increases more rapidly with voltage than k~A or klv. In A, kAt increased from 0.07 sec- t at - 56 m V to 0.39 sec- ~ at 0 mV, k~A increased from 0.0016 sec- ~ at - 5 6 mV to 0.009 sec- ~ at 0 mV and ktv increased from 0.0001 sec -~ at - 5 6 mV to 0.0005 sec -~ at 0 mV. In B, kAt increased from 0.03 sec-~ at --50 mV to 0.35 sec-~ at - 2 2 mV, k~A increased from 0.004 sec-~ at - 5 0 mV to 0.046 s e c - t at --22 mV and k~v increased from 0.0001 sec-~ at - 5 0 mV to 0.0014 see-~ at - 2 2 mV.

physiological and pharmacological complexity of the calcium release channel raises the possibility that several of the postulated mechanisms may co-operate in releasing calcium during normal voltage-activated contractions. The currently most popular hypothesis for EC coupling is that the voltage sensor and calcium release channel are mechanically coupled (Chandler et al., 1976a), either directly or through intermediate molecules. It has been established that just a few amino acids in the cytoplasmic loop of the D H P receptor, between the second and third transmembrane segment, allow the protein to function as a voltage sensor for skeletal muscle EC coupling, rather than a "simple" calcium ion channel (Tanabe et al., 1990). The way in which this cytoplasmic segment of the protein communicates with calcium release channels is the subject of investigation and speculation. (1) Direct mechanical coupling. A direct coupling hypothesis in which the voltage sensor and calcium release channel are continuous across the triad junction is attractive because of its simplicity. A conformational change in the voltage sensor induces a conformational change in the ryanodine receptor macromolecule that opens the calcium release channel. Evidence for the hypothesis is the close apposition between particles (most likely voltage sensor proteins) in the j unctional T-tubule membrane and the feet that span the j unction and are thought to contain the calcium release channel in junctional terminal cisternae membranes (Block et al., 1988). The most compelling argument against the hypothesis is that the isolated DHP receptor protein does not bind to isolated ryanodine receptor calcium release channels (Brandt et al,, 1990). However it could be argued that the conformation of a high affinity binding site is distorted in the cell free system. (2) Mechanical coupling through intermediate proteins. The voltage sensor could be coupled to the calcium release channel by one or more intermediate molecules. Three of the lower molecular weight proteins that have so far been isolated from triad junctions have high affinities for both the DHP receptor and the ryanodine receptor and could, therefore, provide a physical bridge between the two proteins (Brandt et al., 1990; Kim et al., 1990b). The hypothesis of mechanical coupling in this case would suggest that conformational changes are transmitted across several molecules. A precedent for this type of transmission of conformational changes can be found in the troponin/tropomyosin interactions following calcium binding to troponin C (see Zot and Potter, 1987 for review). One major drawback of a hypothesis in which the only trigger for calcium release during voltage activated contraction depends on a mechanical coupling across the triad junction is that all calcium is released into the junctional gap and could be accumulated in the confined

212

A.F. DULHUNTY

space of that structure (Section II.3.c). The presence of extra-junctional calcium release channels would reduce problems with calcium accumulation. Extra-junctional ryanodine receptors have been identified (Dulhunty et al., 1992a). If the extra-junctional ryanodine receptors are functional calcium channels they cannot be gated by a direct mechanical link to the voltage sensor. Either the mechanical link extends to extra-junctional regions of the terminal cisternae, or two separate gating mechanisms exist for junctional and extrajunctional calcium release channels. (3) Coupling through calsequestrin. The calcium binding protein, calsequestrin, has been implicated in EC coupling (Ikemoto et al., 1989; Collins et al., 1990). It has the appropriate geometry (Franzini-Armstrong et al., 1987; Dulhunty, 1989) to physically couple junctional calcium release channels to extra-junctional calcium release channels and could also provide a physical link to the voltage sensor, either through parts of junctional ryanodine receptor protein not associated with the ion channel or through some of the other proteins that have been isolated with triad junctions. Calsequestrin in this scheme could provide the basis for rapid regulation of all calcium release channels by the voltage sensor. If calsequestrin forms a mechanical link between the voltage sensor and junctional and extra-junctional calcium channels, conformational changes in the protein would have to be transmitted over distances as great as 100nm in order to activate extra-junctional receptors. Once again the troponin/tropomyosin system provides a precedent for transmission of conformational changes over long distances. (4) Calcium-induced calcium release. Calcium-activated calcium release provides an alternative mechanism for activation of extra-junctional calcium channels in the terminal cisternae. Calcium release, activated by an influx ofextracellular calcium does not contribute to EC coupling in skeletal muscle (see Luttgau and Stephenson, 1986 for review and Section II.2.a). However some calcium channels may be activated by calcium released from the sarcoplasmic reticulum. It is unlikely that a large fraction of calcium channels are normally calcium-activated since the calcium-induced release is a regenerative process, while voltageactivated calcium release decays rapidly when muscle fibres are repolarized and the voltage sensor is deactivated (Simon et al., 1989; Klein et al., 1990; Lamb and Stephenson, 1991). However, calcium concentrations remain high for several milliseconds after the cessation of tetanic stimulation (Blinks et al., 1978; Miledi et al., 1982) and calcium release is maintained for several milliseconds after repolarization in muscles fibres under voltage clamp conditions (Simon et al., 1989; Garcia and Stefani, 1990). The delay between repolarization and the decay of the calcium transient is accentuated when calcium-induced calcium release is enhanced with caffeine (Simon et al., 1989; Klein et al., 1990). Thus calcium-induced calcium release may contribute to the elevation of myoplasmic calcium concentrations during voltage activated contraction. The fraction of calcium released by this mechanism, and by extrajunctional calcium release channels, remains to be determined. (5) E C coupling based on regulation by magnesium ions. If EC coupling depends on mechanical coupling with the voltage sensor, the question still remains of how this coupling gates the calcium release channel. One mechanism that has been suggested is that the conformational change transmitted from the voltage sensor lowers the affinity of the calcium release channel for magnesium, thus removing resting inhibition of the channels (Lamb and Stephenson, 1991 ). The suggestion was based on the old observation that the calcium release channel is blocked when magnesium is increased to millimolar concentrations (Section III.4.a) and the new observation that a reduction in magnesium concentration to 0.05 mM causes spontaneous opening of calcium release channels in skinned muscle fibres. Magnesium and calcium are two of many ligands that are important modulators of the calcium release channel. Other intrinsic compounds that modulate--but probably do not gate the channel during voltage-activated contraction--include ATP, cyclic AMP, calmodulin and IP 3. One question that is yet to be solved is whether all calcium release channels contain receptors for each of these compounds or whether there are different populations of channel protein with different distributions of receptors. The second possibility seems most likely in amphibian muscle where only 50% of calcium release channels show IP 3 sensitivity (Suarez-isla et al., 1991) and two isoforms of the ryanodine

Contractionin skeletalmuscle

213

receptor are found (Airey et al., 1990; Olivares et al., 1991). The distribution of these different subtypes of channel over junctional and extra-junctional membranes remains to be determined. VI. C O N C L U D I N G COMMENTS Voltage-activated contraction in skeletal muscle fibres depends on a chain of complex events which allow depolarization of the surface membrane of the largest cells in the body (3-5 cm in length and 50-100 #m in diameter) to generate maximum tension within a few milliseconds. Most of the important molecules in each event in the chain have now been identified, isolated and sequenced. However the details of the functional interactions between the molecules, particularly the communication between the voltage sensor and calcium release channel, have yet to be discovered. The functional characteristics of many of the events differ in fast- and slow-twitch mammalian skeletal muscle fibres and combine to determine the final contractile response of the muscle fibre. The available evidence suggests that the time course of isometric tension development in fast-twitch fibres at 37°C depends on the time course of calcium release and of EC coupling. At lower temperatures, and in slowtwitch fibres, the response of the contractile proteins may become rate-limiting. The voltage-dependence of tension during steady-state depolarization depends exclusively on the response of the voltage sensor in the T-tubule membrane. Similarly, the slow decay in tension during prolonged depolarization depends only on inactivation of EC coupling and is not influenced by rates of calcium dissociation from troponin, cross-bridge detachment, calcium dissociation from parvalbumin or calcium uptake by the sarcoplasmic reticulum. The activation and inactivation characteristics of tension, reflecting the voltage-dependent properties of the DHP receptor in the T-system, have much in common with the activation and inactivation characteristrics of voltage-dependent sodium, potassium and calcium ion channels. Molecular biology is being used extensively to investigate which parts of ion channel proteins determine the activation and inactivation properties of ion currents. Even though these techniques have not yet been applied to the voltage sensor for EC coupling, the results of experiments with ion channels can be extrapolated to the activation of EC coupling because of the homology between the membrane spanning portions of sodium and potassium channels and the DHP receptor. Although our knowledge of the processes involved in the complex mechanism of EC coupling has expanded enormously over the past few years, many challenging questions remain to be answered. Some of these questions relate to the basic mechanisms of voltagesensitive molecules and ion permeation through proteins, while others are specific to the problem of activation of skeletal muscle. Which part of the DHP receptor is the voltage sensor for EC coupling? Is this also the voltage sensor for the calcium channel in the same molecule? Do Qa and Qv reflect the movement of the same or different species of intramembrane charge? How does the voltage sensor for EC coupling communicate with and gate calcium release channels in the sarcoplasmic reticulum? What do the subconductance states in the ryanodine receptor calcium release channel and the DHP receptor calcium channel mean in terms of (a) the way ions move through channels and (b) the gating of EC coupling? Are there junctional and extra-junctional calcium release channels in the terminal cisternae? Are all calcium release channels gated by the voltage sensor? Does calciumactivated calcium release operate in normal EC coupling? What is the role of calsequestrin? What is the mechanism of inactivation of EC coupling? Are both components of inactivation located in the voltage sensor? The questions are legion and the answers to many are difficult to predict. The nature of the coupling between the DHP receptor and the calcium release channel is perhaps the greatest mystery. The development of a preparation in which single calcium release channel activity can be recorded following activation of the voltage sensor may be the only route to finally solving this problem. The application of molecular biology, and its use with conventional biochemistry and electrophysiology,has provided the answer to many questions that seemed as daunting 10 years ago and could provide the answer to many of the remaining questions. Other questions may have to await the discovery and development of new techniques.

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A.F. DULHUNTY ACKNOWLEDGEMENTS

The a u t h o r is grateful to Peter G a g e for e n c o u r a g e m e n t a n d s t i m u l a t i n g discussion a n d to P a u l i n e J u n a n k a r a n d Peter G a g e for c o m m e n t s o n the m a n u s c r i p t . Several previously u n p u b l i s h e d o b s e r v a t i o n s (manuscripts in p r e p a r a t i o n ) that are cited in the text were o b t a i n e d in c o l l a b o r a t i o n with P. R. J u n a n k a r , S. P. Cairns, P. W. Gage, P. H. Z h u , M. P a t t e r s o n , T. M. Lewis a n d C. Stanhope. I a m i n d e b t e d to Ms S. Curtis for expert assistance in c o m p i l i n g the reference list, to the electron microscope (particularly Ms L. Maxwell a n d Ms S. Bell) a n d p h o t o g r a p h i c services of the J C S M R for their assistance. REFERENCES AARON,B.-M. B., OIKAWA,K., REITHMEIER,R. A. F. and SYKES,B. D. (1984)Characterization of skeletal muscle calsequestrin by 1H NMR. J. biol. Chem, 259, 11876-11881. ABRAMSON,J. J., TRIMM,J. 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