Charge movement and the nature of signal transduction in skeletal muscle excitation-contraction coupling.

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CHARGE MOVEMENT AND THE NATURE OF Annu. Rev. Physiol. 1992.54:109-133. Downloaded from www.annualreviews.org by New York University - Bobst Library on 10/14/14. For personal use only.

SIGNAL TRANSDUCTION IN SKELETAL MUSCLE EXCITATION-CONTRACTION COUPLING Eduardo R{os Department of Physiology, Rush University School of Medicine, Chicago, Illinois

60612

Gonzalo Pizarro Departamento de Biofisica, Facultad de Medicina, Universidad de La Republica, Montevideo, Uruguay

Enrico Stefani Department of Physiology and Molecular Biophysics, Baylor College of Medicine, Houston, Texas 77006 KEY WORDS:

biomembranes, cell calcium. ionic channels. sarcoplasmic reticulum. voltage sensors

INTRODUCTION A fast signal transduction process takes place in skeletal muscle every time a depolarization of the surface membrane propagates down the transverse tubu lar (T) network and results in release of Ca2+ from the sarcoplasmic reticulum (SR). This transduction, which occurs in the millisecond to submillisecond 109

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RIOS, PIZARRO & STEFANI

time scale, is termed excitation-contraction (EC) coupling. Its mechanism is still unknown, but recent advances in measurement have led to a better definition of the processes involved and to the widespread hope that the mechanism will be clarified soon. Three processes can be conceptually distinguished in EC coupling: (a) voltage sensing-a process taking place in the T membrane, (b) transmission, which links voltage sensing to changes in the SR that allow Ca2+ release, and (c) the Ca release process. Recent advances in understanding processes (a) and (c) have occurred through the measurement of intramembrane charge movement, which is associated with the voltage-sensing process, and the development of methods to measure Ca release flux. These advances, together with biochemical and molecular biological studies, recently resulted in the identification of two molecular species, the dihydropyridine (DHP) receptor and the ryanodine receptor, that protagonize (a) and (c), respective ly. This review summarizes recent studies of charge movement and Ca release, pharmacological and biophysical interventions used to rationally modify the above, and relationships between charge movement and Ca release inferred from those studies. In light of these properties an� relationships, different mechanisms that have been proposed for the transmission step are critically considered. A more extensive review of these topics has recently appeared (118).

INTRAMEMBRANE CHARGE MOVEMENT The term refers to currents arising upon movement of charged molecules that are presumed to dwell in the cell membrane. All voltage-dependent processes that take place in membranes must have associated intramembranous charge movements (65). Skeletal muscle charge movement was initially described by Schneider & Chandler (122). A recorded current must satisfy some simple criteria to be interpreted as an intramembranous charge movement. (a) It must show saturation at both extremes of voltage, which corresponds to the adoption of two extreme positions or conformations by the carrier molecules. (b) The total charge moved must be a function of the initial and final states (voltages) only. This second law of charge movement implies that the amount of charge moved during an excursion in voltage (ON) must be equal to the charge that returns in the opposite excursion (OFF). This principle was used by Huang (70) to reject a model in which charge movement arises as a consequence of transient communication between T tubule and SR lumen (93). Violations of this law are interpreted in terms of additional phenomena, like inactivation during a pulse, which may lead to less charge movement in the OFF transient. (c) The charge movement current is expected to have the direction of the applied field. When a current is found going inward during a depolarizing pulse, it

SIGNAL TRANSDUCTION IN E-C COUPLING

111

should be ionic. An interesting exception to this rule has been recently describ ed (104). Thermodynamic considerations lead in the simplest hypothesis of a two

state system to the Boltzmann equation, which relates charge moved to membrane potential (122),


Q(V)

=

Qmax

1 I + e

_

(V

_

1.

V)/K

The slope factor K == KTlz' e, z' represents the apparent valence of the moving charge and V is the midpoint voltage (with K, T, and e representing Boltz mann's constant, temperature and electron charge, respectively). An apparent valence z, of 3 was obtained fitting Equation 1 to ex perimental Q(V) data; this, and the value of Qmax correspond to a density of =

moving particles of 500-600 per JJ.m2 (28a). The value is close to the number of feet structures determined by electron microscopy [700 (48)] and suggests

that there is one voltage-sensing molecule for every foot, an idea that was later revised (18).

Voltage Distribution and Kinetics Two different distributions of charge movement with voltage can be mea sured, depending on the holding potential (HP). If the membrane is held at or

negative to the normal resting potential, the bulk of the charge moves when the voltage is made more positive. This charge was termed charge 1 by Adrian & Almers (2) and is characteristic of a state in which the cell is ready to respond to electrical depolarization (a primed or available state). On the other hand, fibers held depolarized for several seconds enter an inactivated (or refractory) state, in which it is impossible to elicit contraction by electric stimulation. In these conditions the charge moves in a more negative range of potentials and is called charge 2 (3). In the following we summarize the measured properties of charges 1 and 2 in frog muscle. In most studies a Boltzmann function, Equation 1, is fit to the Q(V) data, which provides three descriptive parameters. CHARGE 1 In studies using Ca2+ as the main extracellular divalent cation, the value of Qmax varies between 20 and 40 nCIJ.LF, the midpoint transition

voltage (V) between -50 and -15 mY, and the slope factor between 7 and 21.5 nC!J.LF (tables and specific references in References 118, 76). Two of the reasons for the large variability have been identified; one is the extracellular dominant anion. According to Hu i & Chandler (76), substitution of S04 for Cl- results in the doubling of Qmax. It was reported (16) that high ex tracellular [CaH] or [CoH] results in an elevation of Qrnax in part because of the reduction in the amount of charge that moves with the control pulses. In -

1 12



fact we often obtain Qrnax values greater than 50nCiJLF in experiments using 10 mM [C02+] extracellularly (A. Gonzalez, G. Pizarro, E. Rfos, un published). CHARGE 2 The distribution of charge 2 has also been determined by several authors (2, 6, 21, 25, 43). Its Qrnax is about 45 nCIJLF, V is near -100 mY, and K varies from 19 to 39 mY. With a four Vaseline-gap clamp (115), smaller values of K are found (around 12 mV). Charge 2 has a Qrnax similar to that of charge 1, a steepness factor somewhat smaller, and the main difference is in the midpoint voltage, shifted some 70 mV to more hyperpolarized voltage. KINETICS Studies on frog (66) and rat fibers (129) show a bell-shaped voltage dependence of the time constants of decay of charge 1. The maximum time constant in frog muscle at 15°C is 10 ms, while for rat at 7, 15, and 25°C it is 3, 4, and 6 ms. Because of the temperature dependence and the bell shaped voltage dependence, Simon & Beam (130) interpreted these time constants as true molecular lags. Similarly Brum & Rfos (21) found bell shaped voltage dependence of the time constant of charge 2 (measured in depolarized fibers of the frog) and a time constant as long as 16 ms at the mid-point. Doubts remain, however, regarding the source of these kinetic delays; Lamb (83) showed that making the external solution hyperosmotic causes an acceleration of charge movement current (which on rabbit fibers is roughly exponentially decaying). This is interpreted as a consequence of more rapid charging of the tubular membranes. On the basis of this observation, Lamb estimated the characteristic times of the molecular transition to be less than 0.5 ms.

The Significance of Charge 2 The measurement of charge movement was followed by the finding of charge immobilization, with similar characteristics as the immobilization of gating current in nerve (28a). Maintained depolarization results in the approximately exponential disappearance of almost all the charge moved by a pulse from -80 to -20 mY, (time constant 18 s at 15°C). The ability to release Ca2+ disappears (inactivates) in parallel. Charge and EC coupling can be restored with a time constant of 40 s by repolarizing to -80 mV (repriming). A simple state model of the voltage sensor that accounts for inactivation and charge immmobilization (2, 28a, 108) includes a resting (R), an activat ing (A), and an inactivated or refractory (I) state. This simple scheme was later modified to account for the observation of reduction in the amount of charge 2 in parallel with recovery of charge 1 during repriming (21). Since charge 2 is present in amounts comparable to charge 1, with an apparent valence of the same order, a relationship between the two may be


113

suspected. In addition to the theoretical implications, there is an important technical reason to wonder about charge 2. The center voltage of its distribu tion (roughly -100 mY) is the same at which controls for linear capacitance


are recorded in polarized fibers; therefore the controls used conventionally

will be in error if charge 2 is present. Several groups have attempted to determine the fate of charge 2 when the HP is taken from 0 mV (at which charge 2 is defined) to -100 mV (the HP from which charge 1 movement measurements are made). The experiments gave inconclusive results. First Adrian & Almers (2), then Brum & Rios (21), found evidence of reduction of charge 2 by 50 to 60% at an HP of -100 mY. Lamb (85) did not find change in charge in the voltage range of ch arge 2 in rabbit fibers. Feldmeyer et al (41) found a small reduction that was not statistically significant. Caputo & Bolanos (25) had results consistent with interconversion. Rios et al (116) measured capacitance in depolarized and polarized fibers at various [Ca]o.

They found that upon polarization to -90 mV capacitance decreased in the range negative to -70 mV (range of charge 2) and increased positive to -70 mV. The decrease in charge 2 was especially large in extracellular solutions with large concentrations of Ca2+ or Co2+ (up to the disappearance of 90 to 100% of charge 2 in the presence of 100 mM Ca2+ or 215 mM cobalt sulfate). These conflicting results may arise from the difficulties of measuring charge movement in a very negative voltage range with the two or three

Vaseline-gap clamp techniques (27, 118). Recent experiments with the four Vaseline-gap clamp, in which current through inhomogeneously polarized membrane regions is excluded from the measurement, confirmed the finding of substantial reduction of charge 2 (30 to 60% reduction at HP = -90 mY, n = 7) (115). Based on the observation of reciprocal changes in charges 1 and 2 brought about by the many interventions just described, Brum & Rios (21) proposed that both types of charge movement originate at the same set of molecules (the voltage sensors of EC coupling). Charge 1 originates as the sensor moves between the resting state, R, and the activating state, A, whereas charge 2 originates in transitions between two (or more) inactivated states. The similar

ity in Qrnax and K values is accounted for assuming that only the transitions represented horizontally are voltage-dependent, generating charge move ments, whereas the vertical transitions are voltage-independent.

1*

charge 2 A 1

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&

STEFANI

This model, similar to one of Bezanilla et al (15) for the sodium channel of the squid giant axon, accounts for the voltage distribution and kinetics of charge 2 and accounts for voltage-dependent inactivation without additional assumptions (21, 118).


Components of Charge 1: Qf3 and Qy At least two kinetic components can be found in the current of charge I, 1f3 and I'Y, which carry corresponding charges Qf3 and Q'Y (6). 1f3 is fast, of an essentially exponential time course; I'Yis slow and is usually referred to as the hump, given its characteristic rising phase. Q'Y has a much steeper voltage dependence than Qf3 (2, 76), and I'Y is more susceptible to inactivation by prolonged depolarization (6). I'Y is specifically blocked by 2 mM tetracaine (69) and the muscle relaxant, dantrolene sodium (73, 74). Tetracaine blocks Ca signals together with I'Yin cut fibers (32, 142). These observations led to the hypothesis that Q'Yand Qf3 arise at different sets of voltage sensors, with different pharmacology, voltage sensitivity, and kinetics, which for discussion purposes will be termed 'Yand f3 sensors. This idea is supported by recent findings that some fibers have prominent Q'Y without an evident Qf3 (28b). Other experimental evidence (reviewed below) suggests that both Q'Yand most of Qf3 arise at the same set of voltage sensors, Q'Yoccurring as a consequence of calcium release. CALCIUM RELEASE AND CHARGE MOVEMENT In response to depolarization of the T tubules, Ca2+ is released to the myoplasm from the terminal cisternae of the SR. Advances in technique have allowed the determination of the flux of Ca release [R(t)] with adequate time resolution. This process starts with the measurement of intracellular Ca2+ transients, using optical techniques (9, 17). From the measured transients, the flux of Ca release can be inferred by various methods (reviewed in 118).

Transmission Delays Miledi and co-workers pioneered the recording of Ca transients in muscle, using the absorption dye arsenazo III (99), and continued with studies of voltage dependence and kinetics of activation. Miledi et al (98) showed that a subthreshold prepulse reduces the latency and increases the size of Ca2+ transients elicited by large brief pulses. This phenomenon is analogous to the effect of prepulses on the strength-duration curve, which was thought (4) to result from build-up of a hypothetical activator during the prepulses. Miledi et al (98, 100) established the correspondence of the activator with a process that builds up before Ca release and was termed coupler. Later work indicated that the activator/coupler is the intramembrane charge movement. The latency of


115

Ca release extrapolates to zero in the limit of high membrane potential, in other words, there seems to be no irreducible delay built into the transmission (147). Vergara & Delay (143) improved the study of delays by measuring simultaneously Ca2+ transients and T membrane potential with potentiometric dyes. They interpreted their results to support the existence of chemical steps in the transmission. This interpretation has been criticized on the basis that it does not properly take into account the intrinsic lags of charge movement (118).


Voltage-Dependence of Ca Release and Charge Movement Kovacs et al (81) simultaneously measured charge movements and Ca tran sients and found that most of the charge movement takes place at the onset of a depolarizing pulse before the release of calcium reaches its maximum level. Rakowski et al (109) demonstrated a close correspondence between maximum release flux and intramembrane charge moved. Melzer et al (96) showed that the waveform [R(t)], elicited by a voltage clamp pulse, first increases, reaches a maximum at 20 ms, then drops rapidly, reaching steady state at = 50 ms. Melzer et al (97) demonstrated a proportional relationship between peak release flux and the amount of suprathreshold charge moved by a variety of pulses of different amplitudes and durations. They interpreted their findings with a minimum model of activation of release by charge movement in which there are three states available to the voltage sensor =

that are linked by voltage-dependent transitions moving charges, Qab and Qbc The open probability of the release channel is determined proportionally by the occupancy of C. This model is sufficent to simulate the overall dependence of charge movement on voltage, assuming that the equilibrium constants of both reac tions are voltage-dependent through the Boltzmann function: 2. where Vab is the voltage at which KJ 1, and z' ab is the effective valence in the transition from A to B. A similar equation applies to the B-C step. This explicit formulation of a model for both charge and release resulted in Q(V) and R(V) curves. The model Q(V) was very similar to the experimental dependence, but the model R(V) did not reproduce the high steepness of the voltage dependence just above threshold. The simultaneous measurement of charge movement and either Ca tran sients or release flux also permitted kinetic comparisons and refinements of =


116

RIOS, PIZARRO '& STEFANI

the delay studies. Schneider et al (123) showed that the Ca transient elicited with 50-100 ms pulses was shifted to earlier in time by as much as 6 ms (At) when the same test voltage was reached in two steps, a first step to a subthreshold voltage, and a second to the final test pulse. In simultaneous measurements they found that the charge moved by the one-step pulse alone (Qref) was approximately equal to the sum of the charges moving during the first step (Qpre) and second step (Qtest). This was as expected of a capacitive charge. More interestingly, they demonstrated that the shift At was approx imately the time it took for a fraction of Qref equal to Qpre to move during the initial portion of the one-step pulse at the test voltage. This result suggests that the charge Qpre, close by definition to the threshold charge for Ca release, has to move before release can actually start. In terms of a state model, it is consistent with the threshold charge moving in sequential voltage-dependent transitions that precede the activating state; these transitions can proceed during the prepulse, which permits a faster activation with the two-step protocol. Schneider et al (125) showed that the decay of R(t) during a pulse is the result of Ca2+ -dependent inactivation. They then used the inactivating effect of a large Ca transient preceding the test to study the activation time course of the non-inactivatable component of release in isolation (131). This activation time course was reasonably parallel to the movement of charge 1, but the latter still preceded the activation of release (by about 2 ms at high voltages). This delay could reflect an irreducible time gap, due to an intervening chemical step (as proposed by Vergara & Delay, 143), but it should also have contributions from non-activating transitions involving charge movement. The delay in closure of the release channels upon membrane repolarization is shorter, close to the 0.5 ms resolution time of the method (110). These simultaneous measurements show a striking parallel between charge move ment and Ca release and establish the existence of a causal relationship between the two, but they neither prove nor rule out particular transmission mechanisms.

Ca Release and Qy The voltage dependence of Ca release is much steeper than that of total charge movement; the steepness is closer to that reported for Q'Y ( 2.5 mV, Reference 76). In this and the following properties, Q'Y correlates better than Qf3 with Ca release: (a) the lowest test depolarizations that elicit Qy com ponents are just suprathreshold for Ca release (79); (b) minimum durations of depolarizations for visible contraction of cut voltage-clamped fibers observed at high magnification are the same as those that permit the development of an ON hump (68); (c) tetracaine affects Qy (69) at concentrations that block Ca release (142); (d) dantrolene sodium and hypertonic solutions suppress release =


117

and Q'Y (73); (e) high intracellular EGTA suppresses Q'Y and optical signals of Ca release at the same time (53). The observations on Q'Y and its close correla tion with EC coupling have led several authors, notably Huang (79) and Hui (73, 74), to propose that Q'Y arises at a chemically different set of voltage sensors, and that these I' sensors are the ones involved in controlling the Ca release process. These sensors would be specifically blocked by tetracaine and dantrolene and would be more sensitive to inactivation by depolarization. In the I' sen sor hypothesis, Q{3 and Qy are expected to activate and inactivate independently, without mutual interactions, and to have different roles (both or only one, presumably Q'Y, being involved in control of release). Huang & Peachey (72), measuring capacitance changes upon detubulation, demonstrated that Q'Y moves in the T tubule membrane. In contrast, Qf3 was found in equal proportion to the accessible linear capacitance in the T tubule and surface membranes; this was taken as evidence of the independence of Qf3 and Q'Y, and against the involvement of Qf3 in control of release. Additional ly, the peak of I'Y precedes the peak of the Ca transient by several ms in fibers clamped in a single Vaseline gap (32), in apparent contradiction with the hypothesis that I'Y is a consequence of Ca release.


THE "y SENSOR HYPOTHESIS

Many other observations, however, are not consistent with the existence of an independent set of sensors that control release, and stress instead the importance of Qf3. In most experiments with highly stretched frog fibers (19, 22, 41, 81, 97), and in mammalian m us cle (66, 67, 129), there is little kinetic evidence of Iy. Yet, the frog fiber experiments show large release fluxes, capable of raising the [Ca2+]i to sever al JLM, that are also very fast, reaching their peak in 10 to 20 ms. Therefore, if a kinetic definition of II' is used, its presence is not a requisite for large and fast fluxes of Ca release. A similar dissociation was revealed in recent studies on paralytic myotubes from mice with muscular dysgenesis (1) in which it is possible to evaluate the charge movement induced by expression of a DNA that encodes the voltage sensor as the difference between total charge movement before expression (presumably without EC coupling com ponents) and after expression. This charge movement does not have kinetic characteristics of I'Y even though the myotubes recover from paralysis after expression. Since the steepness factor in the stretched fiber experiments is typically greater than 12 mY and may be as high as 22 mV (97), the Q'Y component appears to be absent by the steepness criterion as well. In sum mary, there can be Ca release in the absence of Qy, but, in our experience, not Qy in the absence of release.

THE COMMON SENSOR HYPOTHESIS


118

Rios, PIZARRO & STEFANI

Another interpretation, first suggested by W. K. Chandler, (quoted in Reference 68) is that Qy is a consequence of Ca release and that it occurs because Ca2+ , locally increased upon release, binds to the myoplasmic side of the voltage sensors and increases the transmembrane potential difference, thus moving additional sensors belonging to a set common to Q/3 and Qy. Three laboratories recently reexamined this question and made several observations that are consistent with this idea. A number of interventions were applied on cut fibers, all of which were expected to suppress or block Ca release by a direct action on the SR channel. These included (a) a conditioning pulse protocol, shown to suppress the peak of Ca release by Ca-dependent in activation; (b) a pulsing protocol shown by Schneider et al (124) to deplete the SR of Ca (33); (c) a pulse protocol in the presence of high intracellularly EGTA, shown by Garcia et al (51) to deplete the SR (53); (d) the Ca release channel blocker ruthenium red (33); (e) I-heptanol and I-octanol, shown by Ma et al (91) to block the Ca release channel (56); (f) the Ca channel blocker octanoic acid (Gonzalez et aI, 56); (g) the Ca release channel blocker ryano dine (52; J. Ma, A. Gonzalez & E. Rios, unpublished). All these in terventions reduced Ca release as expected, and reduced or eliminated a component of charge movement that was identified as Iy based on its delayed onset. This current was identical in size and kinetics with the component blocked on the same fibers by 20 JLM tetracaine. (h) Szucs et al (138) showed that low concentrations of caffeine shift the threshold voltage for detectable Ca release some 10 mV to lower voltages, and simultaneously shift, on the same fibers and by the same amount, the voltage at which hump components of charge movement are first visible. It is conceivable that some of the above interventions (a to h), intended to alter primarily the release process, were instead affecting the hypothetical y sensor first and Ca release as a consequence. All interventions did accomplish their intended effect (depletion, inactivation, or simple block of release), however, and suppressed a component of charge with similar properties. This demonstrates that the effect on I y was secondary to suppression of release. Pizarro et al (104) formulated quantitatively the Ca2+ -binding model and found that it accounts for most of the properties of Iy, provided that the kinetic constants of charge movement are closer to the fast values proposed by Lamb (83) than to the values measured by Hollingworth & Marshall (66). To assume that the charged molecules involved in Q/3 and Qy belong to the same set of voltage sensors of EC coupling implies that Iy is both conse quence and cause of Ca release. This endows the mechanism with positive feedback: after Ca release begins, triggered by Q/3, the delayed movement of charge constituting Iy should activate more SR channels to open, more Ca2+ should be released, and the whole process should be self-reinforcing. This



119

positive feedback (107) could help to explain the high steepness of the relationship between Ca release and membrane potential near the threshold potential of release (94). Qf3 is defined as the monotonically decaying component of charge move ment, or as a tetracaine-resistant charge. With either definition it is a heterogenous set of components of diverse origin, including Na+ channel gating currents (141). It is possible to separate early components within I{3 by improving the time resolution of its measurement. Recent records of J. Garda & E. Stefani (unpublished), obtained with fast voltage clamp electronics, show three kinetic components in On charge movement current, I y and two early components, including one with a submillisecond time constant that is probably unrelated to EC coupling.

Tubular Optical Signals and Charge Movement Records from voltage-clamped fibers stained with membrane-impermeant voltage-sensitive dyes (61) give evidence of slower charging of T tubules when intramembrane charge moves, and in addition reveals a nonlinearity in the dependency of the signal amplitude on the applied voltage. This appears as an excess signal when depolarizing pulses are applied from a well polarized holding potential. The asymmetric dye signal has many of the properties of intramembrane charge movement, its voltage dependence is similar to that of Qy and it inactivates after prolonged depolarization. The signal seems to result from a change in internal surface potential, which changes the trans membrane potential. These results are relevant to the mechanism of Qy because the potential change measured has the polarity and many of the properties predicted by the Ca2+ -binding model of Qy. The kinetics of the potential change, however, seem to be faster and its amplitude greater than predicted by the binding model. More results from these techniques are eagerly expected.

PHARMACOLOGICAL INTERVENTIONS This section briefly summarizes a large body of work. A more complete survey can be found elsewhere ( 118).

Extracellular Cations The dependence of mechanical tension on external Ca2+ and other divalent cations is well established. K contractures fail in Ca2+ -free media and are shorter in low [Ca2+]e (24, 31, 60, 134). Uittgau & Spiecker (90) demon strated that low [Ca2+]e increases the tendency to inactivation. The simple explanation that external Ca2+ has to enter the fiber to trigger release was ruled out by the demonstration of mechanical activity in Ca2+ -free media (8,

12 0

Rios, PIZARRO

&

STEFANI

90). Furthermore, Ca2+ entering through the slow Ca channels of T mem branes (13) is not required to trigger release, as normal Ca transients and release can occur in the absence of detectable inward Ca2+ current (22, 23, 99). Eisenberg et al (37) found that a second contracture failed after a first one was carried out in the presence of D600, a phenylalkylamine with use dependent blocking effects on Ca channels. This paralysis was accomplished by inhibition of charge movement (41, 77, 106).


Brum et al (19, 22) measured simultaneous Ca release flux and charge movements and found that (a) the contribution of Ca2+ influx to the total

input of Ca2+ to the myoplasm was less than 5% and could not account for the

effect of reduction in [Ca2+Je;

(b)

low [Ca2+Je caused parallel shifts of the

steady state inactivation curve of Ca release and the steady state availability curve of charge 1 to lower voltages owing to an increased tendency to inactivation of the voltage sensor; (c) failure of release, reduction in charge 1,

and increase in charge 2 when [Ca2+Je was reduced below nM concentration with BGTA, and no other metal ions were present. This effect was rapid in onset (T "'" 2 min). When Ca2+ was readmitted, release recovered with similar

kinetics as repriming after a long depolarization (T = 1 min at 10°C; Refer

ence 105). The distribution of intramembrane charge with voltage became

equal in [Ca2+Je

=

0 to that of depolarized fibers (charge 2); thus [Ca2+]e

=

0

with no metal ion substitutes induces a situation analogous to voltage dependent inactivation. The need for Ca2+ or other metal ions can be explained by assuming that Ca2+ binding at a priming site on the voltage sensor stabilizes its non inactivated states and is essential for its function ( 1l3). When external Ca2+ is replaced by alkali ions or alkali-earths, release can still occur (19, 105). This implies that other cations may bind to the priming site. Relative affinities

Ax/Acesium

were calculated as the ratios of peak Ca

release flux in conditions that prevent saturation of the priming site. The sequence of affinities (Ca > Sr > Mg > Ba > Li > Na > K > Rb > Cs) and their values are surprisingly similar to the relative permeabilities of the L-type Ca channels of heart muscle, as calculated from reversal potentials in bi-ionic conditions (62). Thus even though membrane Ca currents do not appear to play any role in the function of the voltage sensor, the voltage sensor has a site with chemical properties similar to those of the intrapore Ca-binding site (or sites) of the Ca channels. This site on the sensor has to be occupied to prevent inactivation. An interesting question resulting from these studies is whether L-type Ca channels have this requirement for Ca2+ binding to a site accessible from the outside.


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Chaotropic Ions The best potentiators of contraction known are N03- , 1- , SCN- , and CI04 SCN- and CI04- are chaotropes, ions that at higher concentrations favor the partition of apolar groups into water and destablize folding of proteins, presumably by breaking long-range order in water (30). The effect of perchlorate on the voltage sensor is very different from its effect on Ca channels of the plasma and T membranes. It shifts the activation of charge movement and Ca release (89) to lower voltages, but does not shift the threshold potential of activation of slow Ca current in the frog (42). These pharmacological differences might correspond to structural differences be tween L channel and sensor, but the evidence is mounting that sensors and Ca channels are one and the same molecule. Therefore, a plausible interpretation is that the high sensitivity of the voltage sensor to CI04- is a consequence of specific interactions with neighboring molecules. This interpretation is sup ported by recent observations (117) of absence of effect of CI04- on the gating currents of Ca2+ channels of cardiac myocytes, on the currents through DHP-sensitive Ca channels from skeletal muscle reconstituted in bilayers, and on charge 2 of skeletal muscle. CI04- has its characteristic effects only on the skeletal muscle voltage sensor, and it requires the physiological interaction of EC coupling to be effective (117).


- •

Ca Channel Antagonists At least five chemically different classes of Ca channel antagonists have been tested for EC coupling effects: dihydropyridines, phenylalkylamines, benzo thiazepines, diphenylbutylpiperidines, and benzolactams (59, 106, 119). The phenylalkylamine D600 induces paralysis (37) and eliminates charge 1 (77). The DHP nifedipine inhibits charge movement in mammalian (82, 84) and amphibian twitch muscles (111, 112 ). The block of charge movement is accompanied by simultaneous reduction of Ca release flux and is large only at depolarized holding potentials (111), as is the case for DHP block of Ca channels, in which the drug binds preferentially to the inactivated states (12). The proposal that the high affinity DHP receptors are in fact voltage sensors of EC coupling (111) is consistent with the voltage-dependence of DHP binding (126); it explains the effects of D600 because the phenylalkylamines (46) interact specifically with DHP receptors, and is roughly consistent with the density of DHP-binding sites (126), of feet structures in the T-SR junction (48), and of charge movement particles (2 8). The identity of voltage sensors and DHP receptors was proved by DNA expression (l 39). Feldmeyer et al (41) and Pizarro et al (106a ) showed that D600 paralysis does not eliminate charge movement, but changes the Q(V) dependence from that of charge 1 to that of charge 2, thus implying that the condition induced


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STEFANI

by the drug is similar to that nonnally reached through voltage-dependent inactivation. This is consistent with a preferential binding of Ca antagonists to inactivated states of the sensor (14, 44, 111). Feldmeyer et al (41) demonstrated recovery of Ca release and charge 1 upon hyperpolarization in the presence of D600; the strikingly parallel time course of both recovery processes constitutes evidence of involvement of charge 1 in EC coupling. As in other experiments with stretched fibers, these did not show clear humps, and the voltage dependence of charge movement was shallow, with charge increasing up to + 50 mV. Since all of the charge moved by a depolarizing pulse from -60 mV recovered in parallel with Ca release, and both release and charge continued to increase with voltage up to + 50 mV, the results indicate blockade of Qf3 by the drug and support the involvement of Qf3 in control of Ca release. Erdman & Liittgau (38) found similar effects with the phenylalkylamine ( -) D888 at concentrations consistent with a Kd of 1. 7 nM in binding to the inactivated states. The (+) enantiomer had a Kd of 12.9 nM; both KdS were consistent with the values found in [3H] binding studies (50, 55). This consistency is the strongest pharmacological evidence to date of the identity between voltage sensors and DHP receptors.

Local Anesthetics Tetracaine at 1 to 4 mM reduced charge 1 by 20 to 60% (69, 73, 142); the blocked charge was identified as Q'Y, and the Ca2+ transients were blocked as well (142). These observations suggest that tetracaine acts primarily on the voltage sensors (71, 73). In agreement with this, tetracaine blocked the tubular slow Ca2+ current at 400 /..IM (52). Other results are more consistent with a primary effect on the release channel. Low concentrations of extracellularly applied tetracaine (pK == 8.2 ; Reference 16) were ineffective at pH 5.5, both on K contractures (121) and on charge movement and Ca release (106b). Similarly 5 mM procaine (pK 8.95), virtually ineffective at pH 7.3, abolished K contractures at pH 9 (121). Benzocaine, which is neutral at pH 7, reduced K contractures at 1 mM and abolished them at 5 mM (121). The pennanently charged tetracaine methobromate had no effects ( l06b). This implies that the uncharged fonns are responsible for the effects, both on K contractures and on charge move ment and Ca release, and suggests that the site of action is intracellular, on the Ca channel of the SR. Sziics et al (138) showed that the effects on charge movement and Ca release of the SR release agonist caffeine and (25 fLM) tetracaine are opposite and antagonistic, which suggests that both act primari ly on the same process (presumably Ca release) and modify charge movement secondarily. The effects of tetracaine at concentrations under 100 J-LM are probably intracellular. ==

123


Ryanodine The specific Ca release channel ligand, ryanodine, induces tonic paralysis in skeletal muscle fibers and locks the release channel in a permanently open, low conductance state (133, 137). Baylor et al (10) sought effects of this drug on charge movement in the hope that locking the release channel in a partially open conformation would have backward repercussions in the chain of transmission, which would support the hypothesis of a direct (and rigid) mechanical link. Neither Baylor et al (10) nor Fryer et al (49) found obvious


effects, although the contracture induced by the drug hampered the ex periments. Garcia et al (52) and J. Ma, A. Gonzalez & E. Rios (unpublished) found that ryanodine (100 nM to 10 JLM) reducesCa release and a hump

in parallel, without major changes in

Q{3 or in the tubularCa current.

(Iy)

This is

consistent with the Ca2+-binding model of I 'Y.

Modulatory Effects on the Voltage Sensor Di Virgilio et al (35) first suggested the involvement of a G protein in EC coupling. Villaz et al (145) demonstrated the presence of

a

subunits of G

proteins in T membranes, and showed enhancement by GTP-'YS ofCa release induced in skinned fibers by caffeine. Garcia et al (54) reported that in

tracellular application of GTP-yS caused a 30% increase in charge 1 (in frog and rat) and increased ICa, but the application of protein kinase A, which

increased ICa, did not alter charge movement. Brum et al (20) found an increase in release flux by f3-adrenergic agonists, but no change in charge movement on extracellular application of epinephrine. These studies suggest possible roles of G proteins and protein phosphorylation in EC coupling.

MOLECULAR IDENTIFICATION OF THE VOLTAGE SENSOR Isolated skeletal muscle triads are enriched in DHP-binding activity (87), which suggests that the DHP receptors are located at the T-SRjunction. Block et al (18) described tetrads of intramembrane particles in freeze-fracture images of junctional T tubules and tentatively identified the tetrads with DHP receptors on the grounds that they were the only intramembrane (protein) particles of the junctional T membrane and that the isolated receptors and the particles had similar size. The density of DHP receptors is 230lJLm2 (126); that of T particles is 8001 JLm2 (18); both numbers are roughly consistent with the density of intramembrane-charged particles. A ratio of DHP to ryanodine

binding to rabbit triads of between 1.5 and 2 (7) is consistent with the

proposed stoichiometry of one T tetrad (four DHP sites) for every two feet

(18).


124

Rios, PIZARRO & STEFANI

Additional insight comes from work on myotubes from mice with muscular dysgenesis, a mutation that causes failure of EC coupling (12), lack of the slow Ca current (11), and of the al subunit (127). The absence of lea and the 0'1 protein are consistent with involvement of DHP receptors in EC coupling. This was confirmed by experiments in which paralytic myotubes were rescued by nuclear injection of cDNA encoding the 0'1 subunit (139). The dysgenic cells have much less charge movement than normal muscle. The expression of the DHP receptor is accompanied by an increase in charge movement ( 1). The difference between the charge movement in the myotubes after rescue by cDNA injection and the charge movement in the paralytic cell is presumably pure charge movement of DHP receptors. The V of a Boltz mann distribution that describes this difference is 5 mV, and its slope factor is 11 mV (1). The primary sequence of the (al) DHP-binding subunit was obtained by cloning and sequencing of its DNA (138), which was functionally expressed in dysgenic myotubes and cell lines 003, 139). This sequence includes four internal repeats (I to IV), each with six putative transmembrane segments. The highly conserved segments S4 are likely sites for the voltage sensing function, which would entail movement of its positively charged residues (three to five arginines and one or two lysines per S4 segment). -

Are the Voltage Sensors Functional Channels? A question posed since the identification of the voltage sensors is whether sensors are also functional Ca channels. When DNA encoding the DHP receptor is expressed in dysgenic myotubes, both EC coupling and slow Ca currents are restored (139); therefore, both sensors and Ca channels are products of the same gene. They could differ, however, because of altemati ve splicing or posttranslational modifications. Schwartz et al (126) estimated the fraction of DHP receptors actually functioning as Ca channels at fewer than 5%. This estimate would change, however, if a lower value of Popen was assumed for the functional channels (86). In agreement with this idea, bilayer studies show that DHP-sensitive channels have a very low Popen (92). Recent observations of biophysical and pharmacological differences between EC coupling and membrane Ca current (106, 119), as well as the finding of two forms of the DHP receptor protein, with molecular weights of 210 and 170 K (34), are more easily explained if sensors and channels are different. On the other hand, Feldmeyer et al (43) have shown that the slow Ca currents of skeletal muscle can activate almost instantaneously under special pulse protocols, thus indicating that the mole cule has the ability to respond at speeds adequate to participate in the (very fast) regulation of EC coupling. Therefore, whether voltage sensors of EC coupling actually function as channels remains a question.


125

MECHANISMS OF TRANSMISSION


Ca release from the SR is under control of potential changes across the T membrane. The mechanism of this communication between membranes, across a 12 to 17 nm gap, is still unknown. In view of the current advances, the question should be reformulated in terms of communication between the DHP receptor protein complex and the ryanodine receptor. At least six specific mechanisms have been proposed for this communication (118); three that have received the most attention are discussed below.

Calcium-Induced Calcium Release· A current form of this proposed mechanism (47) assumes that Ca2+ is released from a membrane-related intracellular compartment, but is difficult to sustain because muscle can release Caz+ for a long time in isotonic Caz+ -free intracellular EGTA (53). Calcium-induced calcium release (CICR) could also operate in skeletal muscle, amplifying an initial process of release elicited by other means (40, 135). It has been specifically proposed that the non-inactivating component of Ca release flows through a subset of release channels directly controlled by the voltage sensors, whereas the inactivating component flows through a ditTerent set of channels, secondarily activated by Ca2+ after the first set of channels have opened (114). Individual channels in bilayers and SR vesicles are opened by Ca2+. The sensitivity to Ca2+ is greater for vesicles than channels in bilayers, and the half-activating concentrations range from submicromolar to 100 p.M, depend ing on the preparation (120, 132; G. Meissner, personal communication). Fabiato (40) showed that calcium release can be elicited from subcellular bundles of myofibrils exposed to cytoplasmic Ca2+ as low as 0.2 p.M, provided that the change is made very rapidly. As with cardiac muscle (39), the observation is explained as a consequence of time- and Ca-dependent activation and inactivation of the release channel occurring in parallel. Jacquemond et al (79) found that the intracellular injection of several mM of the high affinity Ca buffer BAPT A selectively reduced the inactivating com ponent of Ca release flux. This observation suggests that the inactivating component is the result of CICR, whereas the non-inactivating component, which remains operative for the duration of the pulse, requires a different control mechanism. It does not clarify whether inactivating and non inactivating components of release flow through different channels.

Inositol Trisphosphate (lP3) Vergara et al (144) and Volpe et al (146) proposed that IP3 is the transmitter at the T-SR junction on the basis of releasing effects of IP3 in skinned fibers and fractionated SR. Regulatory actions of IP3 and PIPz on the Ca release channel

1 26


are now established (29, 88, 136); the precursors and enzymes necessary for IP3 metabolism are present in the T tubules (63 , 64) , and IP3 leveis have been

shown to increase in muscle after stimulation ( 10 1 , 144) .

One problematic aspect of this hypothesis is that the density of precursor (PIP2) , estimated as at most 60 molecules per square micron of T membrane (64) , is tenfold lower than the density of the putative receptors (feet in the SR membrane, estimated at 700/J.t,M2 ; Reference 48). Additionally , the sensitiv ity of skinned fibers to extrinsic IP3 is maximal when the T membranes are


depolarized and the sensors are inactivated (36), which indicates that the putative receptor knows the state of the voltage sensor even before receiving the IP3 message, thus suggesting the existence of another pathway of com munication. Hidalgo & laimovich concluded after a thorough review (64) that a role of IP3 is highly likely, but a primary transmitter role has not been

demonstrated. Further studies are necessary to define modulatory actions of IP3 on EC coupling.

Mechanical Coupling According to this mechanism (28a), a charged molecule in the T membrane is mechanically linked to the SR channel. When the molecule moves under the changing electric field, it opens the channel by what was picturesquely envisioned as the pulling of a plug. There is not much evidence for or against this mechanism. Perhaps the strongest evidence in favor of mechanical coupling is the newly revealed structure of the release molecule, as well as the molecular nature of the sensor and its probable arrangement at the triad . The release channel has a large cytoplasmic moiety (45), sufficiently tall to reach the T membrane, thus giving structural base to the postulated mechanical link. The voltage sensor appears to be similar to a Ca channel , and there is a limited repertoire of functions that can be assigned to a channel-like molecule. In principle it should act by transporting calcium ions. Since the molecule does not seem to perform its function by transporting ions , it could be simply changing con formation and modifying a molecule(s) in close contact. It could be activating a phospholipase, or a G protein. It is more parsimonious , however, to think that it is acting on the release channel since the structural conditions for mechanical contact between both molecules seem to exist ( 1 8). The definitive evidence should include the demonstration of specific bind ing between the molecules. This evidence has been sought, with favorable but inconclusive results. Caswell collaborators ( 1 8a) found that labeled DHP receptors and ryanodine receptors bind to a 95-kd protein of the junctional SR. The 95-kd protein could be a link in a mechanical transmission chain. Yet another protein of the T tubules, of 28 kd (80), has been implicated in the transmission process because antibodies against this protein block the release


1 27

of Ca2+ from the SR compartment of suspensions of triads , which are induced by ionic substitutions that presumably depolarize the T membrane (78, 102). It is possible that the 95- and 28-kd proteins have structural roles, maintaining voltage sensor and Ca release channels in spatial positions , that allow their mechanical interaction.


SUMMARY The mechanism of transmission remains unclear. It is possible that release is reinforced by Ca2+ -induced activation secondary to opening of release chan nels by another primary mechanism. Multiple results favor some function of 1P3 in EC coupling; however, there are many arguments indicating that IP3 is not the primary transmitter. DHP receptors and ryanodine receptors are known to play essential roles in the triadic junction, but the biochemical make up of the junction has not been completely established, and there may be other essential proteins. The strongest argument in favor of mechanical coupling between voltage sensor and release channel is the close proximity of the channel protein to the T membrane and the fixed stoichiometry between sensors and channels .

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9. Baylor, S. M. 1982. Optical studies of excitation contraction coupling using voltage-sensitive and calcium-sensitive probes. In Handbook of Physiology: Sect. 10, Skeletal Muscle, ed. L. D . Peachey, R. H . Adrian, p p . 355-79. Be thesda, Md: Am. Physiol. Soc. 1 0 . Baylor, S. M . , Hollingworth, S . , Mar shall, M. W. 1989. Effects of intracellu lar ruthenium red on excitation contraction coupling in intact frog skeletal muscle fibres. J. Physiol. 408:617-35 1 1 . Beam, K. G . , Knudson, C. M . , Powell, J. A. 1986. A lethal mutation in mice eliminates the slow calcium current in skeletal muscle cells. Nature 320: 1 6870 1 2 . Bean, B. P. 1984. Nitrendipine block of cardiac calcium channels: High-af finity binding to the inactivated state . Proc. Nat/. Acad. Sci. USA 8 1 :638892 1 3 . Beaty, G. N . , Stefani, E. 1976. Inward calcium current in twitch muscle fibers of the frog. J. Physiol. 260:27P 14. Berwe, D . , Gottschalk, G . , Liittgau , H . Ch. 1987. The effects o f the Ca antagonist gallopamil (D600) upon ex citation-contraction coupling in the toe


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Charge movements in skeletal muscle.

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Reactivation of membrane charge movement and delayed potassium conductance in skeletal muscle fibres.

Effects of external calcium concentration and pH on charge movement in frog skeletal muscle.

Effect of sodium deprivation on contraction and charge movement in frog skeletal muscle fibres.

Acute acidosis attenuates leucine stimulated signal transduction and protein synthesis in rat skeletal muscle.

Calcium current activation and charge movement in denervated mammalian skeletal muscle fibres.

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Voltage-dependent block of charge movement components by nifedipine in frog skeletal muscle.

Kinetic properties of intramembrane charge movement under depolarized conditions in frog skeletal muscle fibers.

Intramembrane charge movement in developing skeletal muscle cells from fetal mice.

A non-linear voltage dependent charge movement in frog skeletal muscle.

Charge movement in the membrane of striated muscle.

The effects of lyotropic anions on charge movement, calcium currents and calcium signals in frog skeletal muscle fibres.

Charge movement and membrane capacity in frog muscle.

Interfering with calcium release suppresses I gamma, the "hump" component of intramembranous charge movement in skeletal muscle.