Control of Resting Ca2+ Concentration in Skeletal Muscle

Chapter 56

Control of Resting Ca21 Concentration in Skeletal Muscle Jose R. Lopez and Paul D. Allen Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA

INTRODUCTION Ca21 is a critical signaling molecule within cells (1). Many cellular functions are directly or indirectly regulated by intracellular calcium concentration ([Ca21]i), and because cells extract specific information from changes in [Ca21]i over time, in space and in amplitude all three parameters must be very tightly regulated. Under resting conditions, skeletal muscle cells maintain [Ca21]i in a narrow range near 100 nM. This low cytosolic concentration has been described in both frog (2) and mammalian skeletal muscle (3 5). Because the gradient between resting myoplasmic free calcium and the extracellular space and internal stores is about 10,000-fold, to constantly keep [Ca21]i in the nM concentration range muscle cells use a complex dynamic equilibrium of Ca21 fluxes among pumps (plasma membrane Ca21 ATPase, and sarcoplasmic reticulum Ca21 ATPase) and an exchanger (Na1/Ca21exchanger) opposed by sarcoplasmic reticulum (SR) leak channels (ryanodine-insensitive “Ca21 leak”) as well as basal sarcolemmal Ca21 influx (6) which tunes myoplasmic Ca21 homeostasis at rest (see Figure 56.1).

MECHANISMS FOR Ca21 REMOVAL FROM THE MYOPLASM OF MUSCLE CELLS Sarcolemmal Outward Ca21 Transport Mechanisms It is generally accepted that the control of resting [Ca21]i in mammalian muscle cells is via plasmalemmal Ca21 extrusion via the sodium calcium exchanger (NCX) and the calmodulin-regulated plasma membrane Ca-ATPase (PMCA).

Sodium Calcium Exchanger (NCX) The NCX is an electrogenic (3Na1/1Ca21) and reversible counter transport system with a well-established role in

Muscle. DOI: http://dx.doi.org/10.1016/B978-0-12-381510-1.00056-9 © 2012 Elsevier Inc. All rights reserved.

Ca21 homeostasis in a variety of cells (7), including amphibian and mammalian skeletal muscle and mammalian myotubes (8 10). Three mammalian isoforms of the NCX protein that are products of three different genes have been cloned (11 13) and appear to have very similar properties (14). In its forward mode, NCX can operate as a high capacity and low affinity system for Ca21 transport, extruding Ca21 against its transmembrane electrochemical gradient, making use of the Na1 electrochemical gradient (7). Given the right balance between the respective electrochemical gradients, the NCX can also operate in the reverse mode, transporting Ca21 into cells and Na1 out (7). Although both NCX1 and NCX3 isoforms have been found in the transverse tubule (T-tubule) and the sarcolemma (10,15) in mammalian skeletal muscle, the physiological role for NCX in muscle has not yet been defined. Muscle fibers from amphibian skeletal muscle (8,16) and human myotubes (10) have been shown to exhibit Na1dependent Ca21 efflux (forward and reverse mode). Recently we have found that NCX in its reverse mode may play an important role in the regulation of resting [Ca21]i in muscle fibers which express RyR1s with mutations associated with malignant hyperthermia. A possible explanation for this is the high resting [Na1]i (15 mM) that we have found in these cells. In practical terms the elevation of [Ca21]i during contraction activates the NXC forward mode, and an elevation of [Na1]i above normal resting values will result in enhanced Ca21 influx via the NCX-reverse mode (Lopez et al., unpublished results).

Plasma Membrane Ca21-ATPase (PMCA) Plasma membrane calcium pumps, also known as the plasma membrane Ca21-ATPase or PMCA, are responsible for the expulsion of Ca21 from the cytosol of all eukaryotic cells. Together with NCX, they are the major plasma membrane transport system responsible for the long-term regulation of the resting [Ca21]i. Like the

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jSR Transverse tubule

Skeletal Muscle

nSR

SOC TRP Triadin Ora1

Ca2+

DHPR NCX

RyR1 SERCA1

PMCA

Junctin

ADP3− Matrix − space Inner membrane Pi Outer membrane

ADP4− ANT

Inter-membrane space

CASQ1 polymer

F1

CASQ1 monomer

Cytosol

FKBP12

+

FIGURE 56.1 Cartoon model of skeletal muscle showing all of the proposed involved proteins in the macromolecular CRU complex that play a significant role in controlling Ca21 homeostasis at rest.

Ca21 pump of the sarcoplasmic/endoplasmic reticulum (SERCA), which pumps Ca21 from the cytosol into the SR, PMCA belong to the family of P-type, named so because these calcium-transport proteins undergo autophosphorylation in which the γ-phosphate of ATP is transferred to a highly conserved aspartyl residue in the cytoplasmic portion of the protein (17). Mammalian PMCA are encoded by four separate genes and additional isoform variants are generated via alternative RNA splicing of the primary gene transcripts (18,19). The expression of different PMCA isoforms and splice variants is regulated in a developmental, tissue- and cell type-specific manner, suggesting that these pumps are functionally adapted to the physiological needs of particular cells and tissues. Different PMCA isoforms have different basal activities, affinities for calmodulin and rates of activation and

inactivation (20), suggesting they may have different physiological roles. The PMCA has low capacity but high affinity for Ca21 transport with a 1:1 Ca21/ATP stoichiometry. Work on purified protein reconstituted in liposomes had suggested electroneutrality (21) while other experiments have suggested that it is a partially electrogenic 1:1 Ca21:H1 transport process (22). The activity of the pump in native membranes has been found to be insensitive to the variation of the membrane potential, strongly supporting an electroneutral H1/Ca21 reaction (23). Under conditions found in the cell, the Kd of the pump for Ca21 is in the 200 500 nM range. Although the Ca21 affinity of the PMCA pump is lower than that of SERCA, it is high enough to enable it to operate with reasonable efficiency at Ca21 concentrations such as those prevailing in the

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Control of Resting Ca21 Concentration in Skeletal Muscle

myoplasm at rest. Accordingly, the PMCA has been conventionally defined as the fine tuner of cytosolic [Ca21]. However the physiological contribution of PMCA to the intracellular Ca21 homeostasis in skeletal muscle still is not fully defined. One significant reason for this is that there are no specific pharmacological inhibitors for any of the PMCA isoforms or splice variants and until muscle-specific genetic manipulation, i.e., selective “knock out or knock down” or overexpression of specific PMCA isoforms is done in skeletal muscle its regulation of contraction/relaxation in adult animals will be unknown.

SR Ca

21

Reuptake: the SERCA Pump

It is well established that active transport is required to import Ca21 into the lumen of SR after muscle activation (for review see 24). The SR of skeletal muscle is an extensively developed network of tubules and cisternae that is arranged in a precise geometric relationship to the contractile elements (25) and represents the primary calcium-storage organelle in striated muscle. The 100 kDa ATP-dependent Ca21 pump (SERCA) is an intrinsic component of the SR membrane in muscle cells, which makes up 50 80% of the total protein of endoplasmic reticulum (26). SERCA-mediated SR calcium reuptake controls the rate of muscle relaxation in skeletal muscle and is responsible for maintaining a 10,000-fold calcium gradient across the SR membrane. Like the PMCA, SERCA is a member of the family of P-type Ca21-ATPases (17). The ATP-dependent Ca21 pump is evenly distributed throughout the free SR and catalyzes the electrogenic transport of 2 Ca21 coupled to the hydrolysis of 1 mol of ATP, against a large electrochemical gradient of Ca21. Unlike the PMCA, SERCA is relatively insensitive to calmodulin (27,28). Brody’s disease is a rare recessive genetic disease associated with mutation in SERCA characterized by impaired relaxation, painless cramps, and stiffness following exercise (29). Three mutations in the SERCA1 gene have been identified in two affected families (30). These patients can still relax their muscles, although at a significantly reduced rate. This suggests that some compensation by ectopic expression of other SERCA isoforms (SERCA2 or SERCA3), and/or that Ca21 removal from the cytosol by the PMCA pump and/or the Na1/Ca21 exchanger could limit the defect in the Ca21-clearing activity of the SR. Although it has been generally thought that the only function of SERCA1 is the reuptake of Ca21 after muscle contraction and that the overall control of resting cytosolic [Ca21] is via the pumps and exchangers at the sarcolemma, recent studies suggest that this is not the case. Goonasakera et al. have shown that by overexpressing SERCA1 in skeletal muscle of mice with three severe

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myopathic conditions that the pathologic changes associated with these myopathies could be abrogated (31). Because some, and it is likely all, of these conditions are associated with increased resting [Ca21]i these data suggest that the control of resting [Ca21]i is not limited to the sarcolemma but may also be mediated by SR Ca21 uptake.

MECHANISMS FOR Ca21 ENTRY INTO THE MYOPLASM IN SKELETAL MUSCLE Sarcolemmal Ca21 Entry Mechanisms It was established decades ago that excitation contraction (EC) coupling relies on the depolarization-dependent release of stored calcium in the SR for skeletal muscle contraction (32,33). More recently, growing evidence suggests that alternative calcium signaling pathways rely on sarcolemmal calcium entry (34 36). Two forms of Ca21 entry have been characterized in skeletal muscle fibers: excitation-coupled calcium entry (ECCE), which is activated in muscle cells during prolonged membrane depolarization and is independent of the filling state, and store-operated calcium entry (SOCE) (37), which occurs in response to depletion of the internal stores (for reviews see 38, 39).

Excitation-Coupled Ca21 Entry (ECCE) It has been reported that the interaction between the DHPR and RYR1 during activation promotes entry of extracellular Ca21 into the myoplasm in skeletal myotubes (5,37) and adult muscle fibers (40). This form of Ca21 entry is termed excitation-coupled Ca21 entry (ECCE). Although the molecular identity of the ECCE pore remains undefined, some authors have suggested it is the L-type calcium channel (41 43). ECCE requires the expression of both the DHPR and RYR1, as it is absent in both dysgenic (CaV1.1-null) and dyspedic (RYR1-null) myotubes (37). Interestingly, ECCE is enhanced by conformational changes in RYR1 such as those caused by ryanodine, certain cysteine mutations and mutations that are associated with malignant hyperthermia (5,40,44). Pharmacologically, ECCE can be blocked by 2-aminoethyl diphenylborate (2-APB), SKF 96356, La31, Gd31, and dantrolene (37,43,45). ECCE is independent of stores and can occur both without any store depletion (36,37,40,45) and in cells in which stores are fully depleted (37,46). Thus, it appears that ECCE may be important in normal skeletal muscle in helping to maintain force generation during tetanic stimulation, and accentuated ECCE may contribute to the pathophysiological increase in myoplasmic Ca21 in malignant hyperthermia.

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Store-Operated Ca21 Entry (SOCE) Capacitative Ca21 entry, or store-operated Ca21 entry (SOCE), was first described and characterized in nonexcitable cells, where it constitutes a major pathway for Ca21 influx (38). The electrical manifestation of this Ca21 entry in the continuous presence of extracellular Ca21 is a Ca21 current, Icrac, whose mechanism has recently been described as an interaction between Orai1 in the plasmalemma and STIM1 in the ER (47). Activation of SOCE is not restricted to nonexcitable cells, but is also observed in excitable cells including neurons, cardiac myocytes, and smooth muscle cells (48). This mode of Ca21 entry has also been described in myotubes and in adult skeletal muscle fibers, where release from SR Ca21 stores is operated by RyRs (6,35,49 51). The physiological role of SOCE in skeletal muscle is not well understood, but it is likely to be important for sustaining calcium stores to prevent muscle weakness and contribute calcium needed to modulate muscle-specific gene expression (52). Although other mechanisms have been proposed as the basis of SOCE in skeletal muscle, such as activation of SOCE channels by IP3 receptors (50) or RyR1 as the SR Ca21 sensor (53,54) and via TRPC proteins as the SOCE channel (53,55,56), it has recently been shown that SOCE in muscle is the result of highly Ca21-selective CRAC channel activation (57,58), resulting from store depletion causing STIM1 aggregation and reorganization in diverse regions of the SR that interact and activate Ca21 entry via Orai1 channels sarcolemma (57).

Transient Receptor Potential Channels (TRPs) TRP channels show a great diversity in activation and ion selectivity. Transcripts for TRPC subfamily members 1, 2, 3, 4, and 6 have been found in skeletal muscle and confirmed by Western blot. Immunohistochemical staining shows the presence of TRPC1, 3, 4, and 6 in the sarcolemma (55,59 61), however in other studies TRPC1, 4, and 6 showed a sarcolemmal localization while TRPC3 was preferentially found in the intracellular compartment, both in isolated fibers (61) and muscle cross-sections (59) but comparisons of TRPC channel expression between different skeletal muscles revealed significant variations (60). TRPC1, 2, 3, 4, and 6 mediate the transmembrane flux of cations down their electrochemical gradients, and because they are not selective for Ca21 their activation can increase both the intracellular Ca21 and Na1 concentrations. The mechanism by which TRPC channels are activated is unknown but possible mechanisms include (i) hydrolysis of phosphatidylinositol bis-phosphate (PIP2); (ii) diacylglycerol (DAG); (iii) by inositol (1,4,5)

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tris-phosphate (IP3); (iv) direct activation by changes in temperature, mechanical stimuli or inorganic ions (Ca21 and Mg21) (62). Co-immunoprecipitation and co-localization experiments suggested an association of TRPC1, TRPC4, and α-syntrophin in cultured myotubes (61,63). These data suggest that TRPC1 is associated via syntrophin to the complex of dystrophin-associated glycoproteins (DAG) and that TRPC1 may form a Ca21 channel or at least contributes to as a subunit of a Ca21 influx channel. In addition, connecting to the DAG complex, TRPC1 co-localizes with the scaffolding protein Homer 1 in skeletal muscle fibers (63). Knockdown of TRPC3 by small interference RNAs did not impair muscle differentiation and neither SOCE nor ECCE were impaired; however the association of TRPC3 with the calcineurinNFAT (nuclear factor of activated T cells) signal transduction pathway is thought to couple extracellular Ca21 influx with regulation of gene expression (64).

Resting Ca21 Entry and Resting [Ca21]i In quiescent muscle cells resting [Ca21]i is maintained, in part, by a continuous Ca21 influx that is not mediated by ECCE or SOCE. This resting Ca21 entry (RCaE) appears to play an important role in preserving the resting [Ca21]i in skeletal muscle (6). The cation channels responsible for RCaE, most probably TRPCs, show a variable Ca21/ Na1 ratio (65). Recent work demonstrated that RCaE could be blocked either by the Icrac blocker BTP2 or overexpression of a dominant negative form of Orai1 (E190Q) with a result being a reduction in resting [Ca21]i (6). In addition, we have recently found that Gd31 and GsMTx-4 (Chilean Rose Tarantula) can also block RCaE in muscle cells, and lower resting [Ca21]i and [Na1]i (Eltit et al., unpublished results). Together these data suggest a possible participation of both TRPCs and Orai in this novel Ca21 entry pathway. Additional investigation is required to completely identify the mechanisms that may be involved in the regulation of resting Ca21 entry in skeletal muscle.

SR Ca21 Leak A significant part of the Ca21 leak pathway from the SR into the cytoplasm arises from a ryanodine-insensitive constitutively open (POB1) low conductance conformation of RyR1 (6,66). Bastadin 5, a brominated macrocyclic derivative of dityrosine isolated from the marine sponge Iathella basta (67), modulates RyR1 gating behavior and can be used as a pharmacological tool to convert RyR1 from its leak conformation into a gating conformation (67). It is well known that the DHPR and the RyR1 engage in bidirectional signaling (68) and that physical coupling between them is essential for skeletal

Chapter | 56

Control of Resting Ca21 Concentration in Skeletal Muscle

excitation contraction coupling. Freeze-fracture images have shown that DHPRs are clustered in groups of four particles (tetrads), which due to steric hindrance are associated with alternate RyR1s in each Ca21 release unit (CRU). Thus alternate RyR1 channels are not physically coupled with DHPRs and the function of the uncoupled RyR1 population is unknown (69 71). Recently, Eltit and colleagues have identified a previously undiscovered orthograde signal from the DHPR to RyR1 that appears to dictate the ratio of actively gating RyR1 channels vs. those in the leak conformation (72). They speculate that since the RyR1 population not coupled to DHPRs in muscle should have a higher propensity to be in the leak conformation, this may be determinant in maintaining normal intracellular Ca21 homeostasis and is needed to keep myoplasmic [Ca21] and the SR Ca21 content in the physiological range (Figure 56.2).

METHODS FOR MEASUREMENTS RESTING [Ca21]I IN MUSCLE CELLS Measuring resting [Ca21]i in skeletal muscle has become an important issue for muscle physiologists. Two main approaches have been used for the determination of resting [Ca21]i in muscle: the introduction of indicators into muscle cells, by various means, which give luminescence, absorbance, or fluorescence signals that are proportional to the [Ca21]i and electrophysiologically using Ca21selective microelectrodes.

Ca2+ Ca2+ Ca2+

~2 mM

Ca2+

Ca2+ Ca2+

Ca2+

Ca2+

Ca2+ SR

~1 mM

Normal Ca2+ stores

~120 nM

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Optical Indicators Three major classes of indicators have been used: (i) bioluminescent calcium-activated photoproteins; (ii) metalochromic indicators; and (iii) tetracarboxylate Ca21 chelator dyes. The first two are mentioned largely for historical interest.

Photoproteins Aequorin (73) is the best known of the two photoproteins that have been used as intracellular Ca21 indicators and has to be either microinjected into the cell or loaded when the cell membrane is partially permeabilized and then allowed to reseal. Measurements of resting [Ca21]i with aequorin are problematic both because its signal is very small at calcium concentrations that resemble the normal myoplasmic resting free calcium in muscle fibers determined by other methods, calibration of the signal into absolute [Ca21] is not easy, because the signal is influenced by a variety of experimental variable (ionic strength, temperature, [Mg21] and its emission proportional roughly to the [Ca21] to the power 2.5 (74), and because it has a luminescence signal that is independent of [Ca21] which has been reported at very low [Ca21] (74). Resting [Ca21]i values in the range of 39 80 nM have been reported in skeletal muscle microinjected with aequorin (75,76).

Metalochromic Dyes Metallochromic dyes change color on binding Ca21 and several different metallochromic dyes have been used in living muscle cells (77 81) as intracellular Ca21 indicators, however it was found that the properties of these dyes made them unsuitable and no one has used them to determine resting [Ca21]i in muscle cells. Three important limitations of metallochromic Ca21 indicators are: (1); that a major fraction of the dye molecules are bound to both soluble and fixed proteins in the muscle cell, making them inaccessible to sense Ca21 ionic activity in the resting state (79,81); (2) it has been estimated that twothirds of the dye is not freely available in the cytoplasm (79); and (3) sulfhydryl groups reduce metallochromic Ca21 indicators to form azo anion radicals that inhibit SERCA function (82).

Ca2+ Orai1/Stim1

TRPC

RyR1 Gating state

NCX/PMCA

RyR1 Leak state DHPR

SERCA1

FIGURE 56.2 Cartoon showing DHPR control of RyR1 “leaks” and mechanisms for Ca21 entry into and extrusion from the cytoplasm.

Tetracarboxylate Fluorescent Ca21 Chelator Dyes Most currently used fluorescent Ca21 indicators (highaffinity or low-affinity) are members of a family of tetracarboxylate derivatives of BAPTA (Fura-2, Indo-1, Fluo-3, Fluo-4, Rhod-2, Calcium Green, etc.), which for the most part have a high selectivity for calcium over

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Mg21 and monovalent ions (83 85). One of the most attractive features of these Ca21 indicators is the ease with which they can be introduced into the muscle cells. Although as salts they are highly charged and alone cannot diffuse across the plasma membrane, in their acetomethyl ester (AM) salt form they become lipid-soluble and can penetrate the plasma membrane where the ester group is hydrolyzed by cytoplasmic esterases and the charged form of the tetracarboxylate derivative is trapped in the cytoplasm. Several studies have been conducted in diverse non-diseased muscle cells using these indicators to measure resting [Ca21]i (86 91). Unfortunately, a number of problems must be considered when tetracarboxylate indicators are used for the determination of resting [Ca21]i in muscle cells. (i) As BAPTA derivatives all have a chelator effect on Ca21 they reduce [Ca21]i. Thus in most cases the [Ca21]i that have been reported in muscle cells using this method have been significantly lower (range 20 80 nM) compared with those reported using Ca21 selective microelectrodes. (ii) The esterases capable of hydrolyzing the AM esters of these indicators are not located exclusively in the cytoplasm. They have been found in some cellular organelles such as SR and mitochondria, which can allow the indicator to be accumulated internally (92 94). (iii) Incomplete hydrolysis of the ester may occur in the interior of the muscle cell and different hydrolysis sub-products are generated whose properties are unknown (93). (iv) These indicators can leak out the cell in their unesterified or free acid form using an undefined active transport system, which is highly temperature-dependent (84,95,96). The leakage can be slowed by reducing the temperature or by treatment with probenecid. Unfortunately these interventions can also modify [Ca21]i. (v) All BAPTA-derivative fluorescent Ca21 indicators are subject to photobleaching and the product of the photobleaching is both fluorescent and relatively insensitive to Ca21(97). (vi) The fluorescence spectra or the calcium binding properties of these fluorescent Ca21 indicators are sufficiently altered in an intracellular environment to make the calibration curves done in aqueous buffers inappropriate (86,87,98,99). As an example, one indicator’s calculated Kd was smaller in aqueous solution alone than when it was measured with a myoplasmic protein, like aldolase, added to the buffer solution (100).

Ca21-Selective Microelectrodes The basis for this technique is the use of a Ca21 selective membrane (ligand) that separates two aqueous solutions containing Ca21 and the readout is the electrical potential difference established between the solutions. In practice, the membrane in the Ca21-selective microelectrodes is a short column of Ca21-selective neutral carrier ligand,

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which separates two different solutions, a microelectrode filling solution (usually pCa7) and the solution outside the microelectrode tip. Neutral carriers are uncharged organic molecules that bind specific ions, generating an electrical potential (100,101). Current available neutral ligand sensors, ETH-1001 (102) and ETH-129 (103), have an adequate selectivity against Na1, K1, and H1 and have negligible interference from Mg2. Ca21-selective microelectrodes made with these ligands can give a well-calibrated measurement of [Ca21]i, over the full range of [Ca21]i of biological interest from pCa3 to below pCa8 in single muscle cells. Typically their response is Nernstian (29.5 mV/pCa unit at room temperature) from pCa3 7 and the signal down to pCa8 is useable. When Ca21-selective microelectrodes made with either ETH-1001 (103) or ETH-127 (104) have been used to measure [Ca21]i in skeletal muscle, the majority of values reported in normal muscle have been in the vicinity of 100 nM (2,4,5,6,85,105,106). However, Ca21-selective microelectrodes have some drawbacks. The most important is that they respond very slowly to changes in [Ca21]i due to the high electrical resistance of the neutral ligand. For measurements of resting [Ca21]i in quiescent muscle cells (steady-state), this slow response is not a limiting factor but it does make their use inappropriate to track rapid changes [Ca21]i, such as those observed during muscle activation. Another potential disadvantage of Ca21-selective microelectrodes lies in that they sample [Ca21]i from just one point which could under certain circumstances not represent the [Ca21]i in all parts of the cell. However, in skeletal muscle there is no evidence of intracellular Ca21 gradients at rest (107), so this problem is moot. Lastly this method is very demanding technically and requires special skills, and because with repeated use their sensitivity to Ca21 may change in the course of the experiments, a calibration curve must be carried out both before and after intracellular measurements. It has been suggested by some investigators that Ca21 measurements carried out using Ca21 selective microelectrodes do not represent the true intracellular Ca21 concentration due to membrane damage caused by electrode impalement (90,91). In general any muscle cell damage due to microelectrode impalements appears to be associated with either the use of microelectrodes with a tip size greater than 1 μm or inadequate care during cell impalement. In this regard we have monitored microelectrode impalements in muscle cells previously loaded with Indo1 AM or Fluo-4 AM and we have not observed any detectable increase in the fluorescence signal during or after the impalement was carried out, ruling out any potential membrane damage and leakage of Ca21 around the microelectrode (Yang et al., unpublished observations). In addition, measurements of [Ca21]i in muscle

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Control of Resting Ca21 Concentration in Skeletal Muscle

cells done after impalement in high extracellular [Ca21] solutions (12.5 mM) (105), are not different than those obtained when the impalement is done in normal Ringer solution (2 mM).

SUMMARY In summary, maintenance of the gradient between resting [Ca21]i inside muscle cells and the extracellular space and internal stores requires a complex dynamic equilibrium of Ca21 fluxes among pumps (PMCA, and SERCA) and an exchanger (NCX) which is opposed by sarcoplasmic reticulum (SR) leak channels (ryanodine insensitive “Ca21 leak”) as well as basal sarcolemmal Ca21 influx. Against what has been commonly thought and has been stated as the boundary theorem (108) which supposes that [Ca21]i is controlled solely by the sarcolemmal Ca21 fluxes, it is clear from recent data that this is not the case. Although sarcolemmal Ca21 fluxes do play an important role, the contribution of Ca21 fluxes in and out of the internal store must be considered in the overall equation.

ACKNOWLEDGMENTS The authors wish to express their thanks to Dr Jose M. Eltit for reading the manuscript and Francisco Altamirano for his help in preparation. Funding for the unpublished data was supported by grants to P.D.A. from NIH/NIAMS (AR43140, AR052354, and AR055104).

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