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


191

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. 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

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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. L., WEDEN,L. and SALAMA,G. (1983) Heavy metals induce rapid calcium release from sarcoplasmic reticulum isolated from skeletal muscle. Proc. natn. Acad. Sci. U.S.A. 80, 1526-1530. ADAMS, B. A. and BEAM, K. G. (1990) Muscular dysgenesis in mice: a model system for studying excitation-contraction coupling. F A S E B J. 4, 2809-2816. ADAMS,D. J. and GAGE,P. W. (1976) Gating currents associated with sodium and calcium currents in an aplysia neuron. Science 192, 783-784. ADAMS,B. A., TANABE,T., MIKAMI,A., NUMA,S. and BEAM,K. G. (1990)Intermembrane charge movement restored in dysgenic skeletal muscle by injection of dihydropyridine receptor cDNAs. Nature 346, 569-572. ADRIAN,R. H. and ALMERS,W. (1976a) The voltage dependence of membrane capacity. J. Physiol. 254, 317-338. ADRIAN,R. H. and ALMERS,W. (1976b) Charge movement in the membrane of striated muscle. J. Physiol. 254, 339-360. ADRIAN,R. H. and HUANG,C. L.-H. (1984a)Charge movements near the mechanical threshold in skeletal muscleof RanD temporaria. J. Physiol. 349, 483-500. ADRIAN,R. H. and HUANG,C. L.-H. (1984b) Experimental analysis of the relationship between charge movement components in skeletal muscle of RanD temporaria. J. Physiol. 353, 419-434. ADRIAN,R. H. and PE~,S, A. (1979) Charge movement and membrane capacity in frog muscle. J. Physiol. 289, 83-97. ADRIAN,R. H. and PERES,A. R. (1977) A gating signal for the potassium channel. Nature 267, 800-804. ADRIAN,R. H., CHANDLER,W. K. and RAICOWSKI,R. F. (1976) Charge movement and mechanical repriming in skeletal muscle. J. Physiol, 254, 361-388. AIREY,J. A., BECK,C. F., MURAKAMI,K., TANKSLEY,S. J., DEERINCK,T. J., ELLISMAN,M. H. and SUTKO,J. L. (1990) Identification and localization of two triad junctionalfoot protein isoformsin mature avian fast twitch skeletal muscle. J. biol. Chem. 265, 14187-14194. ALDRICH,R. W., COREY,D. P. and STEVENS,C. F. (1983) A reinterpretation of mammalian sodium channel gating based on single channel recording. Nature 306, 436441. ALMERS,W. and BEST,P. M. (1976) Effects of tetracaine on displacement currents and contraction of frog skeletal muscle. J. Physiol. 262, 583-611. ALMERS,W., FINIC,R. and PALADE,P. T. (1981) Calcium depletion in frog muscle tubules: the decline of calcium current under maintained depolarization. J. Physiol. 312, 177-207. ALMERS,W., STANFIELD,P. R. and STUrIMER,W. (1983) Slow changes in currents through sodium channels in frog muscle membrane. J. Physiol. 339, 253-271. ANDERSON,K., LAI, F. A., LIU, Q.-Y., ROUSSEAU,E., ERIKSON,H. P. and MEISSNER,G. (1989) Structural and functional characterization of the purified cardiac ryanodine receptor-Ca2÷ release channel complex. J. biol. Chem. 264, 1329-1335. ARMSTRONG,C. M. (1969)Inactivation of the potassium conductance and related phenomena caused by quaternary ammonium ion injection in squid axons. J. gen. Physiol. 54, 553-575. ARMSTRONG,C. M. (1971) Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons. J. gen. Physiol. 58, 413-437. ARMSTRONG,C. M. and BEZANILLA,F. (1973) Currents related to movement of the gating particles of the sodium channels. Nature 242, 459-461. ARMSTRONG,C. M. and BEZANILLA,F. (1977) Inactivation of the sodium channel II. Gating current experiments.J. gen. Physiol. 70, 567-590. ARMSTRONG,C. M., BEZANILLA,F. and ROJAS,E. (1973) Destruction of sodium conductance inactivation in squid axons perfused with pronase. J. gen. Physiol. 62, 375-391. ARMSTRONG,C. M., BEZANILLA,F. M. and HOROWlCZ,P. (1972) Twitches in the presence of ethylene glycol bis(beta-aminoethyl ether)-N, N'-tetraacetic acid. Biochim. biophys. Acta 267, 60~608. ASHLEY,C. C., MULLIGAN,I. P. and LEA, T. J. (1991) Ca 2+ and activation mechanisms in skeletal muscle. Q. Rev, Biophys. 24, 1-73. ASOa'RA,K. and VERGARA,J. (1986) Levelsof inositol phosphates in stimulated and relaxed muscle. 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