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Voltage-dependent gating of sodium channels: correlating structure and function
TINS-January 1986
7
Voltage-dependent gating of sodium channels: correlating structure and function Voltage-dependent gating of ionic channels is a fundamental process underlying much of the information transfer and processing in the nervous system. The sodium channel is the first voltage-sensitive ionic channel whose subunit structure and partial amino acid sequence are known. Here, the unique structural features of this ionic channel are considered together with biophysical information about its voltage-dependent gating and a sliding helix model of voltage sensing is proposed. Previous articles in TINS 1,2 have chronicled the biochemical characterization, functional reconstitution, and primary structure determination of the protein components of voltage-sensiqve sodium channels. In all tissues examined to date, the principal protein component is a glycoprotein of 260 kDa which has a core polypeptide of approximately 210 kDa. In mammalian neurons, this ct subunit is associated by noncovalent forces with a [31 glycoprotein subunit of 36 kDa and by disulfide linkages with a 132 glycoprotein subunit of 33 kDa. In skeletal muscle, one or two small subunits of approximately 37 kDa are also associated with the principal sodium channel polypeptide, whereas only a single sodium channel polypeptide of 260 kDa has been observed in eel electroplax. These purified polypeptide preparations mediate selective ion transport and voltage-dependent gating when incorporated into pure phospholipid vesicles or planar hilayers. cDNAs encoding the amino acid sequence of the sodium channel glycoprotein from electroplax and the 0t subunit of the sodium channel from rat brain, have been cloned and their sequences determined, revealing the primary structure of the principal protein component of the sodium channel 3'4. Four internally homologous transmembrane domains of approximately 300 amino acids each are connected by shorter stretches of non-, homologous amino acids3,4. Noda etal. 4 have proposed that these four homologous domains surround a central aqueous transmembrane pore which serves as the ion conducting pathway of the sodium channel. This sequence of 1800-1900 amino acids also contains the key to understanding the molecular basis of voltage-dependent gating of the sodium channel. The classical voltage clamp studies of
Hodgkin and Huxleys established that sodium channels undergo changes in functional state as a function of voltage and time. Three functionally distinct states (resting, active, and inactivated) were defined in their work. It is now believed by most investigators that these changes of functional state are due to voltage-dependent conformational changes in protein component(s) of the sodium channel. On theoretical grounds, a membrane protein that responds to a change in membrane potential must have charged and/or dipolar amino acid residues located within the membrane electrical field. Changes in the membrane potential then exert a force on these proteinbound dipoles and charges. If the energy of the field-charge interactions is great enough, the protein may be induced to undergo a change in conformation to a new stable state in which the net charge or the location of charge within the membrane electrical field has been altered*. For such a voltage-driven change of state, the steepness of the relationship between protein state and membrane potential defines the number of charges that move, according to a Boitzmann distribution. On this basis, Hodgkin and Huxley5 predicted that activation of sodium channels would require charge displacement equivalent to the movement of six positive charges from the intracellular to the extracellular
Closed
side of the membrane. Such a movement of membrane-bound charge gives rise to a capacitive current that can, in principle, be detected using electrophysiological techniques. Capacitive currents associated with activation of sodium channels (gating currents) were first detected by Armstrong an.d Bezanflla6 in studies of the squid giant axon. They recorded gating currents that were approximately 0.3% of the sodium current during voltage clamp step and corresponded in size to the expected movement of about 6 charges per channel 6,7. The outward gating current reaches a maximum in 80 l~sec as sodium channel activation begins. The movement of gating charge is largely complete by the peak of the •sodium current, as expected if it were associated with the process of activation. More detailed analysis of the voltage and time dependence of gating currents supports the conclusion that they represent charge movements associated with the change of sodium channel state from resting to active7,a,9. In addition, inactivation of sodium channels during a depolarizing prepulse blocks gating currents with the same time and voltage dependence as sodium currents s,l°. These experiments leave little doubt that the small capacitive currents measured in these experiments are due to movements of charged groups on the sodium channel during activation. In all probability, these charged groups are amino acids whose position in the protein structure is altered in the conformational change leading to activation. What hints about the functional architecture of sodium channels can be *See article by A ld r ic h in F e b r u a r y issue.
v
Open
Fig. L Stepwise activation of the sodium channel by voltage-driven conformatio~l change in each domain. The sodium channel ~ subunit is illustrated as a square array of four homologous domains. Depolarization causes a sequential series of conformational changes in the individual domains. After each domain has changed conformation, an open ion channel is formed. Each conformational change i~ associated with a net transfer of protein-bound positive charge (AQ) outward across the membrane. © 1986, ElsevierScience Publishers B.V,, Amsterdam 0378- 5912/86/$02.00
TINS-January
X
derived from comparison of gating current measurements and sodium channel primary structure? The time course of voltage-dependent activation of sodium channels is sigmoid, as though individual channels must make transitions through multiple nonconducting states before activation. The time courses of gating current and sodium channel activation in squid giant axon are fit best by the assumption that the channel must pass through five closed states, although four or six such states are not excluded7. Since the principal polypeptide of the sodium channel has four homologous domains which are postulated to form its transmembrane pore, Noda et al. 4 have proposed that the multiple transitions leading to activation of the sodium channel represent conformational changes in each of the four channel domains. According to this hypothesis, depolarization must cause sequential voltage-dependent conformational changes in each of the four channel domains before an active ion permeability pathway is formed, as illustrated diagrammatically in Fig. 1. Each channel domain must have a voltage-sensitive gating element which transfers the equivalent of one or more net charges (AQ) across the membrane, contributes to the measured gating current, and triggers the protein conformational change necessary for activation. The requirement for large movements of protein-bound charges within each domain of the sodium channel structure focuses attention on regions of concentrated positive and negative charge in the homologous domains. The sequence of electroplax sodium channel contains a region of concentrated positive charge within each domain3'4. Every third amino acid residue for 18-21 residues is positively charged while the intervening residues are hydrophobic. These segments, named $4 by Noda et al. 4, are predicted to form 0¢helices. The corresponding helix formed by the $4 segment of domain IV of the electroplax sodium channel is illustrated in Fig. 2. It forms a cylinder with a hydrophobic surface and a spiral band of positively charged residues. Noda et al. 4 suggested that these regions might be involved in channel gating. However, their topological model placed these segments projecting into the cytoplasm on the intracellular face of the membrane where they would be little affected by the membrane electrical field and no
specific suggestions of gating mechanism were made. One might anticipate that components of the sodium channel involved in the unique voltage-dependent gating function would have been highly conserved during the course of evolution. For our present purposes, this is an important assumption since the most complete structural information is derived from the eel electroplax while the most detailed physiological information comes from neuronal studies. At a recent meeting of the Society of General Physiologists at the Marine Biological Laboratories in Woods Hole, USA, the primary structure of the tx subunit of the sodium channel from mammalian brain, deduced from the nucleotide sequence of the corresponding cDNA, was compared with that of the electroplax sodium channel3, and found to be highly homologous, with an average amino acid identity of approximately 60%. One of the most striking features of this comparison was the finding that A
1986
the $4 segments are essentially identical between these two proteins. No other region is as highly conserved. Positively-chargedhelices such as the one depicted in Fig. 2 may therefore play a central role in voltage-dependent gating. The requirement for transmembrane movement of 6 gating charges places critical restraints upon the underlying structural mechanisms. Mechanisms in which individual amino acid residues are translocated all the way across the permeability barrier seem excluded by the rigid a-helical nature of the transmembrane regions of channel proteins ill3. Two other classes of mechanisms should be considered. (1) Diffuse gating charge mechanisms, in which a large number of charges widely dispersed through the molecule move a fraction of the way across the membrane on activation, are plausible. For example, a movement of 60 gating charges 10% of the way across the membrane would produce the observed gating current. However, such mechanisms do not lead to specific B
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54 Fig. 2. The $4 helix o f domain IV in or-helical conformation. A. A ball-and-stick, three-dimensional representation of the $4 helix of domain IV. Darkened circles represent the or-carbon of each amino acid residue. Open circles show the direction o f projection o f the side chain of each residue away from the core of the helix. Nonpolar residues are illustrated in thin letters by their single letter code: F, Phe; A, Ala; I, lie; L, Leu; V, Val; G, Gly; T, Thr. Positively charged amino acids are illustrated in bold letters: R, arg. B. The spiral path made by the positively charged arginine residues is illustrated in the form of a ribbon wrapped around the central core of the helix.
T I N S - January 1986
and testable structural models since voltage-dependent gating is proposed to be a holistic property encompassing a large fraction of the molecule. (2) Localized gating charge mechanisms, in which full charge transfer is postulated to result from simultaneous neutralization of one positive charge at the inner membrane surface and exposure of a different positive charge at the extracellular surface, are also plausible. For these mechanisms, a process by which simultaneous charge exposure and neutralization would be coupled to protein conformational change must be proposed. The structural features of the $4 segments of each homologous domain of the sodium channel seem well designed for such a charge transfer model. In considering how gating ch,rge might be transferred across the membrane, Armstrong7 suggested a hypothetical model in which each gating element contains a row of paired positive and negative charges that extends across the membrane and acts as a charge transfer mechanism. Changes in the transmembrane electric field cause translocation of the negative charges relative to the positive charges, resulting in loss of a positive charge on the intracellular side of the membrane and appearance of a positive charge on the extracellular side. Thus, full charge translocation is achieved without movement of individual charged species fully across the bilayer. The ¢x helices formed by $4 segments of the sodium channel are ideally suited for mediating such charge transfer. If the $4 segments are postulated to span the membrane, an 0~ helix forms a rigid structural element placing positive charges at regular intervals along a spiral path across the permeability barrier (Fig. 2). Alternatively, these segments may form less stable 310 helices* which would place the positive charges in a linear array across the membrane (not shown). In order to remain stable within the membrane, these positive charges must form ion pairs with negative charges located on adjacent transmembrane segments in the sodium channel protein. Transmembrane segments in integral membrane proteins are commonly in the c~helix conformation12'13. Since the positive charges are arranged in a spiral, extending around nearly the full circumference of the postulated $4 helix, multiple helical elements must con*An u n u s u a l a n d e n e r g e t i c a l l y less s t a b l e h e l i x with 3 rather than 3.5 residuesper turn.
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Fig. 3. Movement of the $4 helix in response to membrane depolarization. The proposed transmembrane $4 helix is illustrated as a cylinder with a spiral ribbon ofpositive charge as in Fig. 2B. At the resting membrane potential (left), all positively charged residues are paired with fixed negative charges on other transmembrane segments of the channel and the transmembrane segment is hem in that position by the negative internal membrane potential. Depolarization reduces the force holding the positive charges in their inward position. The $4 helix is then proposed to undergo a screw-like motion through a rotation of approximately 60~ and an outward displacement approximately 34. This movement leaves an unpaired negative charge on the inward surface of the membrane and reveals an unpaired positive charge on the outward surface to give a net charge transfer (AQ) of +1. Note that all intramembranous positive charges are paired in both conformations. If this helical segment adopts the relatively unstable 30 helix conformation, the positive and negative charges would form a linear array across the membrane and a simple outward translocation without rotation would give equivalent charge transfer.
tribute the neutralizing negative charges. If the $4 helix and its surrounding transmembrane segments are oriented perpendicular to the plane of the membrane, the negative charges neutralizing the $4 helix must be located on five or six neighboring transmembrane segments. If the transmembrahe helices which contribute the neutralizing negative charges are tilted up to 20°, as in bacteriorhodopsin12, three or four critically-oriented transmembrane segments may suffice. Determination of the full complement of transmembrane segments of the sodium channel will be necessary before these neutralizing negatively-charged residues can be definitely identified. Fig. 3 illustrates a hypothetical voltage-sensing element composed of one positively-charged $4 helix and a fixed array of negative charges from
surrounding transmembrane segments. The resting membrane potential (negative inside) exerts a force on each of the charges of the transmembrane voltagesensing elements. Positive charges are drawn in and negative charges are repelled outward. This force is proposed to hold each domain in the conformation characteristic of the resting or closed state. Upon depolarization, that force is relieved. Positive charges then move outward relative to negative charges and assume new ion pair partners, revealing a new unpaired positive charge at the extracellular surface and a new unpaired negative charge at the intracellular surface. The result is a net charge transfer of +1 associated with a conformational change in one of the four transmembrane d/omains of the sodium channel. This mbvement produces translocation
I Ii
of the $4 ct helix one turn (5A) along a spiral path relative to the nearby interacting transmembrane helices. Movements of amino acid residues over similar distances have been observed in studies of conformational changes in much smaller proteins and therefore seem plausible. This movement of the $4 tx helix then initiates a conformational change in one domain as one step in channel activation. The driving force for conformational change in each domain is the electrically driven movement of its voltagesensing element. This sliding helix model of voltage sensing therefore provides a mechanism to connect the sigmoid time course of channel activation with the presence of four homologous domains4. Movement of six positive charges outward through the membrane permeability barrier in four to six separate transitions accompanies activation of the sodium channel, and analyses of gating currents suggest that the charge transfer associated with the final steps in channel activation is twice as large as that during the early steps7. However, four voltage-sensing elements like the one illustrated in Fig. 3 would mediate a charge transfer of +4 rather than +6 as predicted. Inspection of the four $4 segments of the amino acid sequence reveals that domains I and II would have four or five positive charges in the transmembrane region while domains III and IV would have six as illustrated in Fig~ 3. Moreover, the $4 helical segment of domain IV contains two additional positive charges that would be located outside the membrane electric field but available to move into the field upon depolarization. The observed gating charge movement of +6 may therefore be accommodated by the proposal that domains III and/or IV move through two steps of ion pair exchange (10A distance, 120° rotation) during activation. According to the proposed model, complete activation would require charge transfer of +1,
TINS-January
+1, +2, and +2 for a total charge transfer of +6 in close agreement with the expected value (Fig. 1). By this mechanism, modest changes in protein conformation can effect the major transmembrane movement of protein-bound charge required for sodium channel activation without violation of known principles of protein structure. Each of these unique structural elements is proposed to initiate a conformational change in its own domain. The structure of each individual domain defines the energetic barrier to its voltage-driven conformational change and therefore the sequence of conformational transitions that occurs during the sigmoid time course of activation. Additional structural information will be needed to construct a mechanism for coupling of the conformational changes in each domain to opening of a high conductance transmembrane pore and to subsequent closing by inactivation. Kosower j4 has presented a model of sodium channel structure having a total of 20 transmembrane segments including the four positively charged $4 segments. However, it is not clear whether this proposed structure could mediate selective ion conductance or voltage-depending gating. Subsequent to submission of this article, I received from colleagues two reports describing new three-dimensional models of the electroplax sodium channel derived from computer-assisted secondary structure analysis. In contrast to previous analyses of this amino acid sequence4'14, Greenblatt et al.~l and Guy and Seetharamulu ~5, using independent methods of analysis, arrive at the conclusion that each homologous domain contains eight transmembrane segments including the positively charged segment $4. As in the sliding helix model described here, Guy and Seetharamulu 15propose a helical screw model in which outward movement of the $4 segments underlies voltagedependent gating. Thus, analysis of
1980
predicted secondary structure leads to a suggested mechanism for voltagedependent gating involving an outward spiral motion of positively charged 0~ helices as does the consideration of gating current properties and sequence homology between brain and electroplax channels discussed here. The sliding helix or helical screw model provides a discrete, testable hypothesis for the structural basis of voltagedependent gating that may prove valuable in further probing of the structure and function of voltagesensitive ionic channels. Selected references I Catterall, W. A. (1982) Trends NeuroSci. 5, 303--306 2 Catterall, W. A. (1985) Trends NeuroSci. 8, 39-41 3 Auld, V., Marshall, J., Goldin, A., Dowsett, A., Catterall, W.A., Davidson, N. and Dunn, R. (1985) J. Gen. Physiol. 86, 10a 4 Noda, M., Shimizu, S., Tanabe, T., Takai, T., Kayano, T., Ikeda, T., Takahashi, H., Nakayama, Y., Kanaoka, Y., Minamino, N., Kangawa, K., Matsuo, H., Raflery, M. A., Hirose, T., lnayama, S., Hayashida, H., Miyata, T. and Numa, S. (1984) Nature (London) 312, 121-127 5 Hodgkin, A. L. and Huxley, A. F. (1952) J. Physiol. 117, 500-544 6 Armstrong, C. M. and Bezanilla, F. (1973) Nature (London) 242, 459--461 7 Armstrong, C. M. (1981) Physiol. Rev. 61, 644-683 8 BezaniUa, F. and Armstrong, C. M. (1974) Science 183,753-754 9 Keynes, R. D. and Rojas, E. (1974) J. Physiol. 239, 393-434 l0 Armstrong, C. M. and Bezanilla, F. (1977) J. Gen. Physiol. 70, 567-590 11 Greenblatt, R. E., Blatt, Y. and Montal, M. (1986) FEBS Lett, in press 12 Henderson, R. and Unwin, P. N. T. (1975) Nature (London) 275, 28-32 13 Kyte, J. and Doolinle, R. (1982) J. Mol. Biol. 157, 105-132 14 Kosower, E. M. (1985) FEBS Lett. 182,234242 15 Guy, H. R. and Seetharamulu, P. (1986) Proc. Natl Acad. Sci. USA, in press WILLIAM A CATTERALL The Department o f Pharmacology, University of Washington, Seattle, WA 98195, USA.
7
Voltage-dependent gating of sodium channels: correlating structure and function Voltage-dependent gating of ionic channels is a fundamental process underlying much of the information transfer and processing in the nervous system. The sodium channel is the first voltage-sensitive ionic channel whose subunit structure and partial amino acid sequence are known. Here, the unique structural features of this ionic channel are considered together with biophysical information about its voltage-dependent gating and a sliding helix model of voltage sensing is proposed. Previous articles in TINS 1,2 have chronicled the biochemical characterization, functional reconstitution, and primary structure determination of the protein components of voltage-sensiqve sodium channels. In all tissues examined to date, the principal protein component is a glycoprotein of 260 kDa which has a core polypeptide of approximately 210 kDa. In mammalian neurons, this ct subunit is associated by noncovalent forces with a [31 glycoprotein subunit of 36 kDa and by disulfide linkages with a 132 glycoprotein subunit of 33 kDa. In skeletal muscle, one or two small subunits of approximately 37 kDa are also associated with the principal sodium channel polypeptide, whereas only a single sodium channel polypeptide of 260 kDa has been observed in eel electroplax. These purified polypeptide preparations mediate selective ion transport and voltage-dependent gating when incorporated into pure phospholipid vesicles or planar hilayers. cDNAs encoding the amino acid sequence of the sodium channel glycoprotein from electroplax and the 0t subunit of the sodium channel from rat brain, have been cloned and their sequences determined, revealing the primary structure of the principal protein component of the sodium channel 3'4. Four internally homologous transmembrane domains of approximately 300 amino acids each are connected by shorter stretches of non-, homologous amino acids3,4. Noda etal. 4 have proposed that these four homologous domains surround a central aqueous transmembrane pore which serves as the ion conducting pathway of the sodium channel. This sequence of 1800-1900 amino acids also contains the key to understanding the molecular basis of voltage-dependent gating of the sodium channel. The classical voltage clamp studies of
Hodgkin and Huxleys established that sodium channels undergo changes in functional state as a function of voltage and time. Three functionally distinct states (resting, active, and inactivated) were defined in their work. It is now believed by most investigators that these changes of functional state are due to voltage-dependent conformational changes in protein component(s) of the sodium channel. On theoretical grounds, a membrane protein that responds to a change in membrane potential must have charged and/or dipolar amino acid residues located within the membrane electrical field. Changes in the membrane potential then exert a force on these proteinbound dipoles and charges. If the energy of the field-charge interactions is great enough, the protein may be induced to undergo a change in conformation to a new stable state in which the net charge or the location of charge within the membrane electrical field has been altered*. For such a voltage-driven change of state, the steepness of the relationship between protein state and membrane potential defines the number of charges that move, according to a Boitzmann distribution. On this basis, Hodgkin and Huxley5 predicted that activation of sodium channels would require charge displacement equivalent to the movement of six positive charges from the intracellular to the extracellular
Closed
side of the membrane. Such a movement of membrane-bound charge gives rise to a capacitive current that can, in principle, be detected using electrophysiological techniques. Capacitive currents associated with activation of sodium channels (gating currents) were first detected by Armstrong an.d Bezanflla6 in studies of the squid giant axon. They recorded gating currents that were approximately 0.3% of the sodium current during voltage clamp step and corresponded in size to the expected movement of about 6 charges per channel 6,7. The outward gating current reaches a maximum in 80 l~sec as sodium channel activation begins. The movement of gating charge is largely complete by the peak of the •sodium current, as expected if it were associated with the process of activation. More detailed analysis of the voltage and time dependence of gating currents supports the conclusion that they represent charge movements associated with the change of sodium channel state from resting to active7,a,9. In addition, inactivation of sodium channels during a depolarizing prepulse blocks gating currents with the same time and voltage dependence as sodium currents s,l°. These experiments leave little doubt that the small capacitive currents measured in these experiments are due to movements of charged groups on the sodium channel during activation. In all probability, these charged groups are amino acids whose position in the protein structure is altered in the conformational change leading to activation. What hints about the functional architecture of sodium channels can be *See article by A ld r ic h in F e b r u a r y issue.
v
Open
Fig. L Stepwise activation of the sodium channel by voltage-driven conformatio~l change in each domain. The sodium channel ~ subunit is illustrated as a square array of four homologous domains. Depolarization causes a sequential series of conformational changes in the individual domains. After each domain has changed conformation, an open ion channel is formed. Each conformational change i~ associated with a net transfer of protein-bound positive charge (AQ) outward across the membrane. © 1986, ElsevierScience Publishers B.V,, Amsterdam 0378- 5912/86/$02.00
TINS-January
X
derived from comparison of gating current measurements and sodium channel primary structure? The time course of voltage-dependent activation of sodium channels is sigmoid, as though individual channels must make transitions through multiple nonconducting states before activation. The time courses of gating current and sodium channel activation in squid giant axon are fit best by the assumption that the channel must pass through five closed states, although four or six such states are not excluded7. Since the principal polypeptide of the sodium channel has four homologous domains which are postulated to form its transmembrane pore, Noda et al. 4 have proposed that the multiple transitions leading to activation of the sodium channel represent conformational changes in each of the four channel domains. According to this hypothesis, depolarization must cause sequential voltage-dependent conformational changes in each of the four channel domains before an active ion permeability pathway is formed, as illustrated diagrammatically in Fig. 1. Each channel domain must have a voltage-sensitive gating element which transfers the equivalent of one or more net charges (AQ) across the membrane, contributes to the measured gating current, and triggers the protein conformational change necessary for activation. The requirement for large movements of protein-bound charges within each domain of the sodium channel structure focuses attention on regions of concentrated positive and negative charge in the homologous domains. The sequence of electroplax sodium channel contains a region of concentrated positive charge within each domain3'4. Every third amino acid residue for 18-21 residues is positively charged while the intervening residues are hydrophobic. These segments, named $4 by Noda et al. 4, are predicted to form 0¢helices. The corresponding helix formed by the $4 segment of domain IV of the electroplax sodium channel is illustrated in Fig. 2. It forms a cylinder with a hydrophobic surface and a spiral band of positively charged residues. Noda et al. 4 suggested that these regions might be involved in channel gating. However, their topological model placed these segments projecting into the cytoplasm on the intracellular face of the membrane where they would be little affected by the membrane electrical field and no
specific suggestions of gating mechanism were made. One might anticipate that components of the sodium channel involved in the unique voltage-dependent gating function would have been highly conserved during the course of evolution. For our present purposes, this is an important assumption since the most complete structural information is derived from the eel electroplax while the most detailed physiological information comes from neuronal studies. At a recent meeting of the Society of General Physiologists at the Marine Biological Laboratories in Woods Hole, USA, the primary structure of the tx subunit of the sodium channel from mammalian brain, deduced from the nucleotide sequence of the corresponding cDNA, was compared with that of the electroplax sodium channel3, and found to be highly homologous, with an average amino acid identity of approximately 60%. One of the most striking features of this comparison was the finding that A
1986
the $4 segments are essentially identical between these two proteins. No other region is as highly conserved. Positively-chargedhelices such as the one depicted in Fig. 2 may therefore play a central role in voltage-dependent gating. The requirement for transmembrane movement of 6 gating charges places critical restraints upon the underlying structural mechanisms. Mechanisms in which individual amino acid residues are translocated all the way across the permeability barrier seem excluded by the rigid a-helical nature of the transmembrane regions of channel proteins ill3. Two other classes of mechanisms should be considered. (1) Diffuse gating charge mechanisms, in which a large number of charges widely dispersed through the molecule move a fraction of the way across the membrane on activation, are plausible. For example, a movement of 60 gating charges 10% of the way across the membrane would produce the observed gating current. However, such mechanisms do not lead to specific B
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i
/
/
/" i I
/ (~
/ x
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54 Fig. 2. The $4 helix o f domain IV in or-helical conformation. A. A ball-and-stick, three-dimensional representation of the $4 helix of domain IV. Darkened circles represent the or-carbon of each amino acid residue. Open circles show the direction o f projection o f the side chain of each residue away from the core of the helix. Nonpolar residues are illustrated in thin letters by their single letter code: F, Phe; A, Ala; I, lie; L, Leu; V, Val; G, Gly; T, Thr. Positively charged amino acids are illustrated in bold letters: R, arg. B. The spiral path made by the positively charged arginine residues is illustrated in the form of a ribbon wrapped around the central core of the helix.
T I N S - January 1986
and testable structural models since voltage-dependent gating is proposed to be a holistic property encompassing a large fraction of the molecule. (2) Localized gating charge mechanisms, in which full charge transfer is postulated to result from simultaneous neutralization of one positive charge at the inner membrane surface and exposure of a different positive charge at the extracellular surface, are also plausible. For these mechanisms, a process by which simultaneous charge exposure and neutralization would be coupled to protein conformational change must be proposed. The structural features of the $4 segments of each homologous domain of the sodium channel seem well designed for such a charge transfer model. In considering how gating ch,rge might be transferred across the membrane, Armstrong7 suggested a hypothetical model in which each gating element contains a row of paired positive and negative charges that extends across the membrane and acts as a charge transfer mechanism. Changes in the transmembrane electric field cause translocation of the negative charges relative to the positive charges, resulting in loss of a positive charge on the intracellular side of the membrane and appearance of a positive charge on the extracellular side. Thus, full charge translocation is achieved without movement of individual charged species fully across the bilayer. The ¢x helices formed by $4 segments of the sodium channel are ideally suited for mediating such charge transfer. If the $4 segments are postulated to span the membrane, an 0~ helix forms a rigid structural element placing positive charges at regular intervals along a spiral path across the permeability barrier (Fig. 2). Alternatively, these segments may form less stable 310 helices* which would place the positive charges in a linear array across the membrane (not shown). In order to remain stable within the membrane, these positive charges must form ion pairs with negative charges located on adjacent transmembrane segments in the sodium channel protein. Transmembrane segments in integral membrane proteins are commonly in the c~helix conformation12'13. Since the positive charges are arranged in a spiral, extending around nearly the full circumference of the postulated $4 helix, multiple helical elements must con*An u n u s u a l a n d e n e r g e t i c a l l y less s t a b l e h e l i x with 3 rather than 3.5 residuesper turn.
9
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Fig. 3. Movement of the $4 helix in response to membrane depolarization. The proposed transmembrane $4 helix is illustrated as a cylinder with a spiral ribbon ofpositive charge as in Fig. 2B. At the resting membrane potential (left), all positively charged residues are paired with fixed negative charges on other transmembrane segments of the channel and the transmembrane segment is hem in that position by the negative internal membrane potential. Depolarization reduces the force holding the positive charges in their inward position. The $4 helix is then proposed to undergo a screw-like motion through a rotation of approximately 60~ and an outward displacement approximately 34. This movement leaves an unpaired negative charge on the inward surface of the membrane and reveals an unpaired positive charge on the outward surface to give a net charge transfer (AQ) of +1. Note that all intramembranous positive charges are paired in both conformations. If this helical segment adopts the relatively unstable 30 helix conformation, the positive and negative charges would form a linear array across the membrane and a simple outward translocation without rotation would give equivalent charge transfer.
tribute the neutralizing negative charges. If the $4 helix and its surrounding transmembrane segments are oriented perpendicular to the plane of the membrane, the negative charges neutralizing the $4 helix must be located on five or six neighboring transmembrane segments. If the transmembrahe helices which contribute the neutralizing negative charges are tilted up to 20°, as in bacteriorhodopsin12, three or four critically-oriented transmembrane segments may suffice. Determination of the full complement of transmembrane segments of the sodium channel will be necessary before these neutralizing negatively-charged residues can be definitely identified. Fig. 3 illustrates a hypothetical voltage-sensing element composed of one positively-charged $4 helix and a fixed array of negative charges from
surrounding transmembrane segments. The resting membrane potential (negative inside) exerts a force on each of the charges of the transmembrane voltagesensing elements. Positive charges are drawn in and negative charges are repelled outward. This force is proposed to hold each domain in the conformation characteristic of the resting or closed state. Upon depolarization, that force is relieved. Positive charges then move outward relative to negative charges and assume new ion pair partners, revealing a new unpaired positive charge at the extracellular surface and a new unpaired negative charge at the intracellular surface. The result is a net charge transfer of +1 associated with a conformational change in one of the four transmembrane d/omains of the sodium channel. This mbvement produces translocation
I Ii
of the $4 ct helix one turn (5A) along a spiral path relative to the nearby interacting transmembrane helices. Movements of amino acid residues over similar distances have been observed in studies of conformational changes in much smaller proteins and therefore seem plausible. This movement of the $4 tx helix then initiates a conformational change in one domain as one step in channel activation. The driving force for conformational change in each domain is the electrically driven movement of its voltagesensing element. This sliding helix model of voltage sensing therefore provides a mechanism to connect the sigmoid time course of channel activation with the presence of four homologous domains4. Movement of six positive charges outward through the membrane permeability barrier in four to six separate transitions accompanies activation of the sodium channel, and analyses of gating currents suggest that the charge transfer associated with the final steps in channel activation is twice as large as that during the early steps7. However, four voltage-sensing elements like the one illustrated in Fig. 3 would mediate a charge transfer of +4 rather than +6 as predicted. Inspection of the four $4 segments of the amino acid sequence reveals that domains I and II would have four or five positive charges in the transmembrane region while domains III and IV would have six as illustrated in Fig~ 3. Moreover, the $4 helical segment of domain IV contains two additional positive charges that would be located outside the membrane electric field but available to move into the field upon depolarization. The observed gating charge movement of +6 may therefore be accommodated by the proposal that domains III and/or IV move through two steps of ion pair exchange (10A distance, 120° rotation) during activation. According to the proposed model, complete activation would require charge transfer of +1,
TINS-January
+1, +2, and +2 for a total charge transfer of +6 in close agreement with the expected value (Fig. 1). By this mechanism, modest changes in protein conformation can effect the major transmembrane movement of protein-bound charge required for sodium channel activation without violation of known principles of protein structure. Each of these unique structural elements is proposed to initiate a conformational change in its own domain. The structure of each individual domain defines the energetic barrier to its voltage-driven conformational change and therefore the sequence of conformational transitions that occurs during the sigmoid time course of activation. Additional structural information will be needed to construct a mechanism for coupling of the conformational changes in each domain to opening of a high conductance transmembrane pore and to subsequent closing by inactivation. Kosower j4 has presented a model of sodium channel structure having a total of 20 transmembrane segments including the four positively charged $4 segments. However, it is not clear whether this proposed structure could mediate selective ion conductance or voltage-depending gating. Subsequent to submission of this article, I received from colleagues two reports describing new three-dimensional models of the electroplax sodium channel derived from computer-assisted secondary structure analysis. In contrast to previous analyses of this amino acid sequence4'14, Greenblatt et al.~l and Guy and Seetharamulu ~5, using independent methods of analysis, arrive at the conclusion that each homologous domain contains eight transmembrane segments including the positively charged segment $4. As in the sliding helix model described here, Guy and Seetharamulu 15propose a helical screw model in which outward movement of the $4 segments underlies voltagedependent gating. Thus, analysis of
1980
predicted secondary structure leads to a suggested mechanism for voltagedependent gating involving an outward spiral motion of positively charged 0~ helices as does the consideration of gating current properties and sequence homology between brain and electroplax channels discussed here. The sliding helix or helical screw model provides a discrete, testable hypothesis for the structural basis of voltagedependent gating that may prove valuable in further probing of the structure and function of voltagesensitive ionic channels. Selected references I Catterall, W. A. (1982) Trends NeuroSci. 5, 303--306 2 Catterall, W. A. (1985) Trends NeuroSci. 8, 39-41 3 Auld, V., Marshall, J., Goldin, A., Dowsett, A., Catterall, W.A., Davidson, N. and Dunn, R. (1985) J. Gen. Physiol. 86, 10a 4 Noda, M., Shimizu, S., Tanabe, T., Takai, T., Kayano, T., Ikeda, T., Takahashi, H., Nakayama, Y., Kanaoka, Y., Minamino, N., Kangawa, K., Matsuo, H., Raflery, M. A., Hirose, T., lnayama, S., Hayashida, H., Miyata, T. and Numa, S. (1984) Nature (London) 312, 121-127 5 Hodgkin, A. L. and Huxley, A. F. (1952) J. Physiol. 117, 500-544 6 Armstrong, C. M. and Bezanilla, F. (1973) Nature (London) 242, 459--461 7 Armstrong, C. M. (1981) Physiol. Rev. 61, 644-683 8 BezaniUa, F. and Armstrong, C. M. (1974) Science 183,753-754 9 Keynes, R. D. and Rojas, E. (1974) J. Physiol. 239, 393-434 l0 Armstrong, C. M. and Bezanilla, F. (1977) J. Gen. Physiol. 70, 567-590 11 Greenblatt, R. E., Blatt, Y. and Montal, M. (1986) FEBS Lett, in press 12 Henderson, R. and Unwin, P. N. T. (1975) Nature (London) 275, 28-32 13 Kyte, J. and Doolinle, R. (1982) J. Mol. Biol. 157, 105-132 14 Kosower, E. M. (1985) FEBS Lett. 182,234242 15 Guy, H. R. and Seetharamulu, P. (1986) Proc. Natl Acad. Sci. USA, in press WILLIAM A CATTERALL The Department o f Pharmacology, University of Washington, Seattle, WA 98195, USA.