Intramembrane charge movements in frog skeletal muscle in strongly hypertonic solutions

Intramembrane charge movements were studied in intact, voltage-clamped frog (Rana temporaria) skeletal muscle fibers in external solutions made increasingly hypertonic by addition of sucrose. The marked dependence of membrane capacitance on test potential persisted with increases in extracellular sucrose concentration between 350 and 500 mM. Charge movements continued to show distinguishable early monotonic (q beta) decays and the strongly voltage-dependent delayed (q gamma) charging phases reported on earlier occasions. In contrast, a further increase to 600 mM sucrose abolished the most steeply voltage-sensitive part of the membrane capacitance. It left a more gradual variation with potential that closely resembled the function that resulted when q gamma charge was abolished by tetracaine in the presence of 500 mM sucrose. Charging transients were now simple monotonic (q beta) decays and lacked delayed (q gamma) transients. Furthermore, tetracaine (2 mM) altered neither the kinetic nor the steady-state features of the charge left in 600 mM sucrose. However, Ca2+ current activation in the same fibers persisted through such tonicity increases under identical conditions of temperature, external solution, and holding voltage. Tonicity changes thus accomplish an independent separation of q gamma and q beta charge as defined hitherto through their tetracaine sensitivity. Their effects on q gamma charge correlate with earlier observations of osmotic conditions on delta[Ca2+] signals (1987. J. Physiol. (Lond.) 383:615-627.) and the parallel effects of other agents on excitation-contraction coupling and q gamma charge. In contrast, they suggest that Ca2+ current activation does not require q gamma charge transfer whether by itself or as part of the excitation-contraction coupling process.     

Differential effects of tetracaine on charge movements and Ca2+ signals in frog skeletal muscle

The effects of tetracaine on charge movements and on antipyrylazo III signals monitoring intracellular delta [Ca2+] were compared in cut frog semitendinosus muscle fibers in a single vaseline gap-voltage clamp. Low tetracaine concentrations (25-40 microM) markedly reduced delta [Ca2+] signals and shifted the rheobase. However, they neither influenced charge movement nor that peak delta [Ca2+] value associated with the contractile threshold. Higher tetracaine concentrations (100-200 microM) partly inhibited charge movements in cut fibers. They separated a steeply voltage-sensitive charge, some of whose features resembled 'q gamma' reported in intact fibers, and whose movement preceded delta [Ca2+] signals at threshold. These findings: (a) directly confirm an earlier suggestion that tetracaine acts on steps in excitation-contraction coupling rather than myofilament activation; (b) show that tetracaine at low concentrations can directly interfere with sarcoplasmic reticular calcium release without modifying charge movement; (c) show that the tetracaine-sensitive charge, first found in intact fibers, also exists in cut fibers; and (d) make it unlikely that tetracaine-sensitive charge transfer is a consequence of Ca2+ release as suggested on earlier occasions.     

Anatomical distribution of voltage-dependent membrane capacitance in frog skeletal muscle fibers

Components of nonlinear capacitance, or charge movement, were localized in the membranes of frog skeletal muscle fibers by studying the effect of 'detubulation' resulting from sudden withdrawal of glycerol from a glycerol-hypertonic solution in which the muscles had been immersed. Linear capacitance was evaluated from the integral of the transient current elicited by imposed voltage clamp steps near the holding potential using bathing solutions that minimized tubular voltage attenuation. The dependence of linear membrane capacitance on fiber diameter in intact fibers was consistent with surface and tubular capacitances and a term attributable to the capacitance of the fiber end. A reduction in this dependence in detubulated fibers suggested that sudden glycerol withdrawal isolated between 75 and 100% of the transverse tubules from the fiber surface. Glycerol withdrawal in two stages did not cause appreciable detubulation. Such glycerol-treated but not detubulated fibers were used as controls. Detubulation reduced delayed (q gamma) charging currents to an extent not explicable simply in terms of tubular conduction delays. Nonlinear membrane capacitance measured at different voltages was expressed normalized to accessible linear fiber membrane capacitance. In control fibers it was strongly voltage dependent. Both the magnitude and steepness of the function were markedly reduced by adding tetracaine, which removed a component in agreement with earlier reports for q gamma charge. In contrast, detubulated fibers had nonlinear capacitances resembling those of q beta charge, and were not affected by adding tetracaine. These findings are discussed in terms of a preferential localization of tetracaine-sensitive (q gamma) charge in transverse tubule membrane, in contrast to a more even distribution of the tetracaine-resistant (q beta) charge in both transverse tubule and surface membranes. These results suggest that q beta and q gamma are due to different molecules and that the movement of q gamma in the transverse tubule membrane is the voltage-sensing step in excitation-contraction coupling.     

Effect of the calcium buffer EGTA on the "hump" component of charge movement in skeletal muscle.

Three manifestations of excitation-contraction (E-C) coupling were measured in cut skeletal muscle fibers of the frog, voltage clamped in a double Vaseline gap: intramembrane charge movements, myoplasmic Ca2+ transients, and changes in optical transparency. Pulsing patterns in the presence of high [EGTA] intracellularly, shown by García et al. (1989. J. Gen. Physiol. 94:973-986) to deplete Ca2+ in the sarcoplasmic reticulum, were found to change the above manifestations. With an intracellular solution containing 15 mM EGTA and 0 Ca, 10-15 pulses (100 ms) to -20 mV at a frequency of 2 min-1 reduced the "hump" component of charge movement current. This effect was reversible by 5 min of rest. The same effect was obtained in 62.5 mM EGTA and 0 Ca by pulsing at 0.2 min-1. This effect was reversible by adding calcium to the EGTA solution, for a nominal [Ca2+]i of 200 nM, and was prevented by adding calcium to the EGTA solution before pulsing. The suppression of the hump was accompanied by elimination of the optical manifestations of E-C coupling. The current suppressed was found by subtraction and had the following properties: delayed onset, a peak at a variable interval (10-20 ms) into the pulse, a negative phase (inward current) after the peak, and a variable OFF transient that could be multi-phasic and carried less charge than the ON transient. In the previous paper (Csernoch et al., 1991. J. Gen. Physiol. 97:845-884) it was shown that several interventions suppress a similar component of charge movement current, identified with the "hump" or Q gamma current (I gamma). Based on the similarity to that component, the charge movement suppressed by the depletion protocols can also be identified with I gamma. The fact that I gamma is suppressed by Ca2+ depletion and the kinetic properties of the charge suppressed is inconsistent with the existence of separate sets of voltage sensors underlying the two components of charge movement, Q beta and Q gamma. This is explicable if Q gamma is a consequence of calcium release from the sarcoplasmic reticulum.     

Kinetic isoforms of intramembrane charge in intact amphibian striated muscle

The effects of the ryanodine receptor (RyR) antagonists ryanodine and daunorubicin on the kinetic and steady-state properties of intramembrane charge were investigated in intact voltage-clamped frog skeletal muscle fibers under conditions that minimized time-dependent ionic currents. A hypothesis that RyR gating is allosterically coupled to configurational changes in dihydropyridine receptors (DHPRs) would predict that such interactions are reciprocal and that RyR modification should influence intramembrane charge. Both agents indeed modified the time course of charging transients at 100-200-microM concentrations. They independently abolished the delayed charging phases shown by q gamma currents, even in fibers held at fully polarized, -90-mV holding potentials; such waveforms are especially prominent in extracellular solutions containing gluconate. Charge movements consistently became exponential decays to stable baselines in the absence of intervening inward or other time-dependent currents. The steady-state charge transfers nevertheless remained equal through the ON and the OFF parts of test voltage steps. The charge-voltage function, Q(VT), shifted by approximately +10 mV, particularly through those test potentials at which delayed q gamma currents normally took place but retained steepness factors (k approximately 8.0 to 10.6 mV) that indicated persistent, steeply voltage-dependent q gamma contributions. Furthermore, both RyR antagonists preserved the total charge, and its variation with holding potential, Qmax (VH), which also retained similarly high voltage sensitivities (k approximately 7.0 to 9.0 mV). RyR antagonists also preserved the separate identities of q gamma and q beta species, whether defined by their steady-state voltage dependence or inactivation or pharmacological properties. Thus, tetracaine (2 mM) reduced the available steady-state charge movement and gave shallow Q(VT) (k approximately 14 to 16 mV) and Qmax (VH) (k approximately 14 to 17 mV) curves characteristic of q beta charge. These features persisted with exposure to test agent. Finally, q gamma charge movements showed steep voltage dependences with both activation (k approximately 4.0 to 6.5 mV) and inactivation characteristics (k approximately 4.3 to 6.6 mV) distinct from those shown by the remaining q beta charge, whether isolated through differential tetracaine sensitivities, or the full approximation of charge-voltage data to the sum of two Boltzmann distributions. RyR modification thus specifically alters q gamma kinetics while preserving the separate identities of steady-state q beta and q gamma charge. These findings permit a mechanism by which transverse tubular voltage provides the primary driving force for configurational changes in DHPRs, which might produce q gamma charge movement. However, they attribute its kinetic complexities to the reciprocal allosteric coupling by which DHPR voltage sensors and RyR-Ca2+ release channels might interact even though these receptors reside in electrically distinct membranes. RyR modification then would still permit tubular voltage change to drive net q gamma charge transfer but would transform its complex waveforms into simple exponential decays.     

A quantitative description of the voltage-dependent capacitance in frog skeletal muscle in terms of equilibrium statistical mechanics

We present an analysis of experimental steady-state properties of charge movements in voltage-clamped frog skeletal muscle; by adopting an approach based on equilibrium statistical mechanics, detailed assumptions about the dynamics of the charge movement were avoided. Different components of the charge movements, as characterized by their sensitivities to local anaesthetics, were taken to correspond to different types of independent subsystem (integral membrane proteins). Quantitative agreement with the data for the q beta and q gamma components was obtained for the simplest subsystems, having two energy levels; however, q alpha required three (or more) energy levels. Each of the subsystems can be interpreted as having a charged group, minimum valency z, able to occupy two or more positions within the membrane. The tetracaine-sensitive q gamma charge can be described in terms of an ensemble of subsystems having z = 4.5, each occupying one of two levels whose free energies at zero applied voltage differ by 1.7 x 10(-1) eV. Likewise, the lidocaine-sensitive q beta has z = 1.0, free energy difference 1.9 x 10(-2) eV. However, the simplest model for the lidocaine-resistant q alpha involves a charge of valency 1.7 moving between three levels having relative free energies at zero applied voltage -9.7 x 10(-2), 0, and 2.1 x 10(-2) eV respectively, where the intermediate level 'sees' about 50% of the applied voltage.     

Effects of dantrolene on steps of excitation-contraction coupling in mammalian skeletal muscle fibers

The effects of the muscle relaxant dantrolene on steps of excitation-contraction coupling were studied on fast twitch muscles of rodents. To identify the site of action of the drug, single fibers for voltage-clamp measurements, heavy SR vesicles for calcium efflux studies and solubilized SR calcium release channels/RYRs for lipid bilayer studies were isolated. Using the double Vaseline-gap or the silicone-clamp technique, dantrolene was found to suppress the depolarization-induced elevation in intracellular calcium concentration ([Ca2+]i) by inhibiting the release of calcium from the SR. The suppression of [Ca2+]i was dose-dependent, with no effect at or below 1 microM and a 53 +/- 8% (mean +/- SEM, n = 9, cut fibers) attenuation at 0 mV with 25 microM of extracellularly applied dantrolene. The drug was not found to be more effective if injected than if applied extracellularly. Calculating the SR calcium release revealed an equal suppression of the steady (53 +/- 8%) and of the early peak component (46 +/- 6%). The drug did not interfere with the activation of the voltage sensor in as much as the voltage dependence of both intramembrane charge movements and the L-type calcium currents (I(Ca)) were left, essentially, unaltered. However, the inactivation of I(Ca) was slowed fourfold, and the conductance was reduced from 200 +/- 16 to 143 +/- 8 SF(-1) (n = 10). Dantrolene was found to inhibit thymol-stimulated calcium efflux from heavy SR vesicles by 44 +/- 10% (n = 3) at 12 microM. On the other hand, dantrolene failed to affect the isolated RYR incorporated into lipid bilayers. The channel displayed a constant open probability for as long as 30-50 min after the application of the drug. These data locate the binding site for dantrolene to be on the SR membrane, but be distinct from the purified RYR itself.     

The voltage sensor of excitation-contraction coupling in skeletal muscle. Ion dependence and selectivity

Manifestations of excitation-contraction (EC) coupling of skeletal muscle were studied in the presence of metal ions of the alkaline and alkaline-earth groups in the extracellular medium. Single cut fibers of frog skeletal muscle were voltage clamped in a double Vaseline gap apparatus, and intramembrane charge movement and myoplasmic Ca2+ transients were simultaneously measured. In metal-free extracellular media both charge movement of the charge 1 type and Ca transients were suppressed. Under metal-free conditions the nonlinear charge distribution was the same in depolarized (holding potential of 0 mV) and normally polarized fibers (holding potentials between -80 and -90 mV). The manifestations of EC coupling recovered when ions of groups Ia and IIa of the periodic table were included in the extracellular solution; the extent of recovery depended on the ion species. These results are consistent with the idea that the voltage sensor of EC coupling has a binding site for metal cations--the "priming" site--that is essential for function. A state model of the voltage sensor in which metal ligands bind preferentially to the priming site when the sensor is in noninactivated states accounts for the results. This theory was used to derive the relative affinities of the various ions for the priming site from the magnitude of the EC coupling response. The selectivity sequence thus constructed is: Ca greater than Sr greater than Mg greater than Ba for group IIa cations and Li greater than Na greater than K greater than Rb greater than Cs for group Ia. Ca2+, the most effective of all ions tested, was 1,500-fold more effective than Na+. This selectivity sequence is qualitatively and quantitatively similar to that of the intrapore binding sites of the L-type cardiac Ca channel. This provides further evidence of molecular similarity between the voltage sensor and Ca channels.     

Altered inactivation of Ca2+ current and Ca2+ release in mouse muscle fibers deficient in the DHP receptor gamma1 subunit

Functional impacts of the skeletal muscle-specific Ca2+ channel subunit gamma1 have previously been studied using coexpression with the cardiac alpha1C polypeptide in nonmuscle cells and primary-cultured myotubes of gamma1-deficient mice. Data from single adult muscle fibers of gamma-/- mice are not yet available. In the present study, we performed voltage clamp experiments on enzymatically isolated mature muscle fibers of the m. interosseus obtained from gamma+/+ and gamma-/- mice. We measured L-type Ca2+ inward currents and intracellular Ca2+ transients during 100-ms step depolarizations from a holding potential of -80 mV. Ratiometric Ca2+ transients were analyzed with a removal model fit approach to calculate the flux of Ca2+ from the sarcoplasmic reticulum. Ca2+ current density, Ca2+ release flux, and the voltage dependence of activation of both Ca2+ current and Ca2+ release were not significantly different. By varying the holding potential and recording Ca2+ current and Ca2+ release flux induced by 100-ms test depolarizations to +20 mV, we studied quasi-steady-state properties of slow voltage-dependent inactivation. For the Ca2+ current, these experiments showed a right-shifted voltage dependence of inactivation. Importantly, we could demonstrate that a very similar shift occurred also in the inactivation curve of Ca2+ release. Voltages of half maximal inactivation were altered by 16 (current) and 14 mV (release), respectively. Muscle fiber bundles, activated by elevated potassium concentration (120 mM), developed about threefold larger contracture force in gamma-/- compared with gamma+/+. This difference was independent of the presence of extracellular Ca2+ and likely results from the lower sensitivity to voltage-dependent inactivation of Ca2+ release. These results demonstrate a specific alteration of voltage-dependent inactivation of both Ca2+ entry and Ca2+ release by the gamma1 subunit of the dihydropyridine receptor in mature muscle fibers of the mouse.     

Separation of Q beta and Q gamma charge components in frog cut twitch fibers with tetracaine. Critical comparison with other methods

Charge movement was measured in frog cut twitch fibers with the double Vaseline-gap technique. 25 microM tetracaine had very little effect on the maximum amounts of Q beta and Q gamma but slowed the kinetics of the I gamma humps in the ON segments of TEST-minus-CONTROL current traces, giving rise to biphasic transients in the difference traces. This concentration of tetracaine also shifted V gamma 3.7 (SEM 0.7) mV in the depolarizing direction, resulting in a difference Q-V plot that was bell-shaped with a peak at approximately -50 mV. 0.5-1.0 mM tetracaine suppressed the total amount of charge. The suppressed component had a sigmoidal voltage distribution with V = -56.6 (SEM 1.1) mV, k = 2.5 (SEM 0.5) mV, and qmax/cm = 9.2 (SEM 1.5) nC/microF, suggesting that the tetracaine-sensitive charge had a steep voltage dependence, a characteristic of the Q gamma component. An intermediate concentration (0.1-0.5 mM) of tetracaine shifted V gamma and partially suppressed the tetracaine-sensitive charge, resulting in a difference Q-V plot that rose to a peak and then decayed to a plateau level. Following a TEST pulse to greater than -60 mV, the slow inward current component during a post-pulse to approximately -60 mV was also tetracaine sensitive. The voltage distribution of the charge separated by tetracaine (method 1) was compared with those separated by three other existing methods: (a) the charge associated with the hump component separated by a sum of two kinetic functions from the ON segment of a TEST-minus-CONTROL current trace (method 2), (b) the steeply voltage-dependent component separated from a Q-V plot of the total charge by fitting with a sum of two Boltzmann distribution functions (method 3), and (c) the sigmoidal component separated from the Q-V plot of the final OFF charge obtained with a two-pulse protocol (method 4). The steeply voltage-dependent components separated by all four methods are consistent with each other, and are therefore concluded to be equivalent to the same Q gamma component. The shortcomings of each separation method are critically discussed. Since each method has its own advantages and disadvantages, it is recommended that, as much as possible, Q gamma should be separated by more than one method to obtain more reliable results.     

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

Potential-dependent inhibition of charge movement components by nifedipine was studied in intact, voltage-clamped, frog skeletal muscle fibers. Available charge was reduced by small shifts in holding potential (from -100 mV to -70 mV) in 2 microM nifedipine, without changes in the capacitance deduced from control (-120 mV to -100 mV) voltage steps made at a fully polarized (-100 mV) holding potential. These voltage-dependent effects did not occur in lower (0-0.5 microM) nifedipine concentrations. The voltage dependence of membrane capacitance at higher (10 microM) nifedipine concentrations was reduced even in fully polarized fibers, but shifting the holding voltage produced no further block. Voltage-dependent inhibition by nifedipine was associated with a fall in available charge, and a reduction in the charge and capacitance-voltage relationships and of late (q gamma) charging transients. It thus separated a membrane-capacitance with a distinct and steep steady-state voltage dependence. Tetracaine (2 mM) reduced voltage-dependent membrane capacitance and nonlinear charge more than did nifedipine. However, nifedipine did not exert voltage-dependent effects on charging currents, membrane capacitance, or inactivation of tetracaine-resistant (q beta) charge. This excludes participation of q beta, or the membrane charge as a whole, from the voltage-dependent effects of nifedipine. Rather, the findings suggest that the charge susceptible to potential-dependent block by nifedipine falls within the tetracaine-sensitive (q gamma) category of intramembrane charge.     

Excitation-contraction coupling in intact frog skeletal muscle fibers injected with mmolar concentrations of fura-2.

Experiments were carried out to test the hypothesis that mM concentrations of fura-2, a high-affinity Ca2+ buffer, inhibit the release of Ca2+ from the sarcoplasmic reticulum (SR) of skeletal muscle fibers. Intact twitch fibers from frog muscle, stretched to a long sarcomere length and pressure-injected with fura-2, were activated by an action potential. Fura-2's absorbance and fluorescence signals were measured at different distances from the site of fura-2 injection; thus, the myoplasmic free Ca2+ transient (delta [Ca2+]) and the amount and rate of SR Ca2+ release could be estimated at different myoplasmic concentrations of fura-2 ([fura-2T]). At [fura-2T] = 2-3 mM, the amplitude and half-width of delta [Ca2+] were reduced to approximately 25% of the values measured at [fura-2T] less than 0.15 mM, whereas the amount and rate of SR Ca2+ release were enhanced by approximately 50% (n = 5; 16 degrees C). Similar results were observed in experiments carried out at low temperature (n = 2; 8.5-10.5 degrees C). The finding of an enhanced rate of Ca2+ release at 2-3 mM [fura-2T] is opposite to that reported by Jacquemond et al. (Jacquemond, V., L. Csernoch, M. G. Klein, and M. F. Schneider. 1991. Biophys. J. 60:867-873) from analogous experiments carried out on cut fibers. In two experiments involving the injection of larger amounts of fura-2, reductions in SR Ca2+ release were observed; however, we were unable to decide whether these reductions were due to [fura-2T] or to some nonspecific effect of the injection itself. These experiments do, however, suggest that if large [fura-2T] inhibits SR Ca2+ release in intact fibers, [fura-2T] must exceed 6 mM to produce an effect comparable to that reported by Jacquemond et al. in cut fibers. Our clear experimental result that 2-3 mM [fura-2T] enhances SR Ca2+ release supports the proposal that delta [Ca2+] triggered by an action potential normally feeds back to inhibit further release of Ca2+ from the SR (Baylor, S.M., and S. Hollingworth. 1988. J. Physiol. [Lond.]. 403:151-192). Our results provide no support for the hypothesis that Ca(2+)-induced Ca2+ release plays a significant role in excitation-contraction coupling in amphibian skeletal muscle.     

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

Four manifestations of excitation-contraction (E-C) coupling were derived from measurements in cut skeletal muscle fibers of the frog, voltage clamped in a Vaseline-gap chamber: intramembranous charge movement currents, myoplasmic [Ca2+] transients, flux of calcium release from the sarcoplasmic reticulum (SR), and the intrinsic optical transparency change that accompanies calcium release. In attempts to suppress Ca release by direct effects on the SR, three interventions were applied: (a) a conditioning pulse that causes calcium release and inhibits release in subsequent pulses by Ca-dependent inactivation; (b) a series of brief, large pulses, separated by long intervals (greater than 700 ms), which deplete Ca2+ in the SR; and (c) intracellular application of the release channel blocker ruthenium red. All these reduced calcium release flux. None was expected to affect directly the voltage sensor of the T-tubule; however, all of them reduced or eliminated a component of charge movement current with the following characteristics: (a) delayed onset, peaking 10-20 ms into the pulse; (b) current reversal during the pulse, with an inward phase after the outward peak; and (c) OFF transient of smaller magnitude than the ON, of variable polarity, and sometimes biphasic. When the total charge movement current had a visible hump, the positive phase of the current eliminated by the interventions agreed with the hump in timing and size. The component of charge movement current blocked by the interventions was greater and had a greater inward phase in slack fibers with high [EGTA] inside than in stretched fibers with no EGTA. Its amplitude at -40 mV was on average 0.26 A/F (SEM 0.03) in slack fibers. The waveform of release flux determined from the Ca transients measured simultaneously with the membrane currents had, as described previously (Melzer, W., E. Ríos, and M. F. Schneider. 1984. Biophysical Journal. 45:637-641), an early peak followed by a descent to a steady level during the pulse. The time at which this peak occurred was highly correlated with the time to peak of the current suppressed, occurring on average 6.9 ms later (SEM 0.73 ms). The current suppressed by the above interventions in all cases had a time course similar to the time derivative of the release flux; specifically, the peak of the time derivative of release flux preceded the peak of the current suppressed by 0.7 ms (SEM 0.6 ms). The magnitude of the current blocked was highly correlated with the inhibitory effect of the interventions on Ca2+ release flux.(ABSTRACT TRUNCATED AT 400 WORDS)     

Involvement of the carboxy-terminus region of the dihydropyridine receptor beta1a subunit in excitation-contraction coupling of skeletal muscle

Skeletal muscle knockout cells lacking the beta subunit of the dihydropyridine receptor (DHPR) are devoid of slow L-type Ca(2+) current, charge movements, and excitation-contraction coupling, despite having a normal Ca(2+) storage capacity and Ca(2+) spark activity. In this study we identified a specific region of the missing beta1a subunit critical for the recovery of excitation-contraction. Experiments were performed in beta1-null myotubes expressing deletion mutants of the skeletal muscle-specific beta1a, the cardiac/brain-specific beta2a, or beta2a/beta1a chimeras. Immunostaining was used to determine that all beta constructs were expressed in these cells. We examined the Ca(2+) conductance, charge movements, and Ca(2+) transients measured by confocal fluo-3 fluorescence of transfected myotubes under whole-cell voltage-clamp. All constructs recovered an L-type Ca(2+) current with a density, voltage-dependence, and kinetics of activation similar to that recovered by full-length beta1a. In addition, all constructs except beta2a mutants recovered charge movements with a density similar to full-length beta1a. Thus, all beta constructs became integrated into a skeletal-type DHPR and, except for beta2a mutants, all restored functional DHPRs to the cell surface at a high density. The maximum amplitude of the Ca(2+) transient was not affected by separate deletions of the N-terminus of beta1a or the central linker region of beta1a connecting two highly conserved domains. Also, replacement of the N-terminus half of beta1a with that of beta2a had no effect. However, deletion of 35 residues of beta1a at the C-terminus produced a fivefold reduction in the maximum amplitude of the Ca(2+) transients. A similar observation was made by deletion of the C-terminus of a chimera in which the C-terminus half was from beta1a. The identified domain at the C-terminus of beta1a may be responsible for colocalization of DHPRs and ryanodine receptors (RyRs), or may be required for the signal that opens the RyRs during excitation-contraction coupling. This new role of DHPR beta in excitation-contraction coupling represents a cell-specific function that could not be predicted on the basis of functional expression studies in heterologous cells.     

Effect of sarcoplasmic reticulum calcium depletion on intramembranous charge movement in frog cut muscle fibers

Cut muscle fibers from Rana temporaria (sarcomere length, 3.3-3.5 microns; temperature, 13-16 degrees C) were mounted in a double Vaseline-gap chamber and equilibrated for at least an hour with an internal solution that contained 20 mM EGTA and phenol red and an external solution that contained predominantly TEA-gluconate; both solutions were nominally Ca-free. The increase in total myoplasmic concentration of Ca (delta[CaT]) produced by sarcoplasmic reticulum (SR) Ca release was estimated from the change in pH produced when the released Ca was complexed by EGTA (Pape, P.C., D.-S. Jong, and W.K. Chandler. 1995. Journal of General Physiology. 106:259-336). The resting value of SR Ca content, [CaSR]R (expressed as myoplasmic concentration), was taken to be equal to the value of delta[CaT] obtained during a step depolarization (usually to -50 to -40 mV) that was sufficiently long (200-750 ms) to release all of the readily releasable Ca from the SR. In ten fibers, the first depolarization gave [CaSR]R = 839-1,698 microM. Progressively smaller values were obtained with subsequent depolarizations until, after 30-40 depolarizations, the value of [CaSR]R had usually been reduced to < 10 microM. Measurements of intramembranous charge movement, Icm, showed that, as the value of [CaSR]R decreased, ON-OFF charge equality held and the amount of charge moved remained constant. ON Icm showed brief initial I beta components and prominent I gamma "humps", even after the value of [CaSR]R was < 10 microM. Although the amplitude of the hump component decreased during depletion, its duration increased in a manner that preserved the constancy of ON charge. In the depleted state, charge movement was steeply voltage dependent, with a mean value of 7.2 mV for the Boltzmann factor k. These and other results are not consistent with the idea that there is one type of charge, Q beta, and that I gamma is a movement of Q beta caused by SR Ca release, as proposed by Pizarro, Csernoch, Uribe, Rodriguez, and Rios (1991. Journal of General Physiology. 97:913-947). Rather, our results imply that Q beta and Q gamma represent either two distinct species of charge or two transitions with different properties of a single species of charge, and that SR Ca content or release or some related event alters the kinetics, but not the amount of Q gamma. Many of the properties of Q gamma, as well as the voltage dependence of the rate of SR Ca release for small depolarizations, are consistent with predictions from a simple model in which the voltage sensor for SR Ca release consists of four interacting charge movement particles.     

Effects of D-600 on intramembrane charge movement of polarized and depolarized frog muscle fibers

Intramembrane charge movement has been measured in frog cut skeletal muscle fibers using the triple vaseline gap voltage-clamp technique. Ionic currents were reduced using an external solution prepared with tetraethylammonium to block potassium currents, and O sodium + tetrodotoxin to abolish sodium currents. The internal solution contained 10 mM EGTA to prevent contractions. Both the internal and external solutions were prepared with impermeant anions. Linear capacitive currents were subtracted using the P-P/4 procedure, with the control pulses being subtracted either at very negative potentials, for the case of polarized fibers, or at positive potentials, for the case of depolarized fibers. In 63 polarized fibers dissected from Rana pipiens or Leptodactylus insularis frogs the following values were obtained for charge movement parameters: Qmax = 39 nC/microF, V = 36 mV, k = 18.5 mV. After depolarization we found that the total amount of movable charge was not appreciably reduced, while the voltage sensitivity was much changed. For 10 fibers, in which charge movement was measured at -100 and at 0 mV, Qmax changed from 46 to 41 nC/microF, while V changed from -41 to -103 mV and k changed from 20.5 to 30 mV. Thus membrane depolarization to 0 mV produces a shift of greater than 50 mV in the Q-V relationship and a decrease of the slope. Membrane depolarization to -20 and -30 mV, caused a smaller shift of the Q-V relationship. In normally polarized fibers addition of D-600 at concentrations of 50-100 microM, does not cause important changes in charge movement parameters. However, the drug appears to have a use-dependent effect after depolarization. Thus in depolarized fibers, total charge is reduced by approximately 20%. D-600 causes no further changes in the voltage sensitivity of charge movement in fibers depolarized to 0 mV, while in fibers depolarized to -20 and -30 mV it causes the same effects as that obtained with depolarization to 0 mV. These results are compatible with the idea that after depolarization charge 1 is transformed into charge 2. D-600 appears to favor the conversion of charge 1 into charge 2. Since D-600 also favors contractile inactivation, charge 2 could represent the state of the voltage sensor for excitation-contraction coupling in the inactivated state.     

Perchlorate enhances transmission in skeletal muscle excitation- contraction coupling

The effects of the anion perchlorate (present extracellularly at 8 mM) were studied on functional skeletal muscle fibers from Rana pipiens, voltage-clamped in a Vaseline gap chamber. Established methods were used to monitor intramembranous charge movement and flux of Ca release from the sarcoplasmic reticulum (SR) during pulse depolarization. Saponin permeabilization of the end portions of the fiber segment (Irving, M., J. Maylie, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:1-41) substantially reduced the amount of charge moving during conventional control pulses, thus minimizing a technical error that plagued our previous studies. Perchlorate prolonged the ON time course of charge movement, especially at low and intermediate voltages. The OFFs were also made slower, the time constant increasing twofold. The hump kinetic component was exaggerated by ClO4- or was made to appear in fibers that did not have it in reference conditions. ClO4- had essentially no kinetic ON effects at high voltages (> or = 10 mV). ClO4- changed the voltage distribution of mobile charge. In single Boltzmann fits, the midpoint potential V was shifted -20 mV and the steepness parameter K was reduced by 4.7 mV (or 1.78-fold), but the maximum charge was unchanged (n = 9). Total Ca content in the SR, estimated using the method of Schneider et al. (Schneider, M. F., B. J. Simon, and G. Szucs. 1987. Journal of Physiology. 392:167-192) for correcting for depletion, stayed constant over tens of minutes in reference conditions but decayed in ClO4- at an average rate of 0.3 mumol/liter myoplasmic water per s. ClO4- changed the kinetics of release flux, reducing the fractional inactivation of release after the peak. ClO4- shifted the voltage dependence of Ca release flux. In particular, the threshold voltage for Ca release was shifted by about -20 mV, and the activation of the steady component of release flux was shifted by > 20 mV in the negative direction. The shift of release activation was greater than that of mobile charge. Thus the threshold charge, defined as the minimum charge moved for eliciting a detectable Ca transient, was reduced from 6 nC/microF (0.55, n = 7) to 3.4 (0.53). The average of the paired differences was 2.8 (0.33, P < 0.01). The effects of ClO4- were then studied in fibers in modified functional situations. Depletion of Ca in the SR, achieved by high frequency pulsing in the presence of intracellular BAPTA and EGTA, simplified but did not eliminate the effects of ClO4-.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of membrane processes in controlling skeletal muscle function

The role of membrane processes in the activation of skeletal muscle fibers has been investigated in details recently. It seems very probable that the intramembrane charge movement is the potential sensitive step of the excitation-contraction coupling. Considerable efforts were made to monitor the subsequent steps (SR function, Ca release) using optical methods. Experimental data were presented about the relations existing between charge movement process, Ca release and contraction threshold.     

Differential regulation of skeletal muscle L-type Ca2+ current and excitation-contraction coupling by the dihydropyridine receptor beta subunit

The dihydropyridine receptor (DHPR) of skeletal muscle functions as a Ca2+ channel and is required for excitation-contraction (EC) coupling. Here we show that the DHPR beta subunit is involved in the regulation of these two functions. Experiments were performed in skeletal mouse myotubes selectively lacking a functional DHPR beta subunit. These beta-null cells have a low-density L-type current, a low density of charge movements, and lack EC coupling. Transfection of beta-null cells with cDNAs encoding for either the homologous beta1a subunit or the cardiac- and brain-specific beta2a subunit fully restored the L-type Ca2+ current (161 +/- 17 pS/pF and 139 +/- 9 pS/pF, respectively, in 10 mM Ca2+). We compared the Boltzmann parameters of the Ca2+ conductance restored by beta1a and beta2a, the kinetics of activation of the Ca2+ current, and the single channel parameters estimated by ensemble variance analysis and found them to be indistinguishable. In contrast, the maximum density of charge movements in cells expressing beta2a was significantly lower than in cells expressing beta1a (2.7 +/- 0.2 nC/microF and 6.7 +/- 0. 4 nC/microF, respectively). Furthermore, the amplitude of Ca2+ transient measured by confocal line-scans of fluo-3 fluorescence in voltage-clamped cells were 3- to 5-fold lower in myotubes expressing beta2a. In summary, DHPR complexes that included beta2a or beta1a restored L-type Ca2+ channels. However, a DHPR complex with beta1a was required for complete restoration of charge movements and skeletal-type EC coupling. These results suggest that the beta1a subunit participates in key regulatory events required for the EC coupling function of the DHPR.     

Mechanisms of stimulated 45Ca efflux in skinned skeletal muscle fibers

Excitation-contraction (E-C) coupling in skeletal muscle can be studied in skinned fibers by direct assay of 45Ca efflux and simultaneous isometric force, under controlled conditions. Recent work provides evidence that such studies can address major current questions about the mechanisms of signal transmission between transverse tubules and sarcoplasmic reticulum and sarcoplasmic reticulum calcium release, as well as operation of the sarcoplasmic reticulum active Ca transport system in situ. Stimulation by imposed ion gradients at constant [K+][Cl-] product results in 45Ca release with two components: a large Ca2+-dependent efflux, responsible for contractile activation, and a small Ca2+-insensitive efflux. The Ca2+-insensitive stimulation is sustained, consistent with sustained depolarization, and appears to gradate the Ca2+-dependent stimulation; this component is likely to reflect intermediate steps in E-C coupling. Several lines of evidence suggest that the depolarizing stimulus acts on the transverse tubules. It is inhibited by the impermeant glycoside ouabain applied before skinning, which should specifically inhibit polarization of subsequently sealed transverse tubules. Sealed polarized transverse tubules also are the only plausible target for stimulation of 45Ca release by monensin and gramicidin D, which can rapidly dissipate Na+ and K+ gradients; a protonophore and the K+-specific ionophore valinomycin are ineffective. Ionophore stimulation is prevented by the permeant glycoside digitoxin; it is also highly Ca2+ dependent. Stimulation of 45Ca release by imposed ion gradients is potentiated by perchlorate, which potentiates charge movements and activation in intact fibers, and is inhibited selectively in highly stretched fibers, presumably by transverse tubule-sarcoplasmic reticulum uncoupling. These results relate the Ca2+-dependent sarcoplasmic reticulum efflux channel to the physiological transverse tubule-sarcoplasmic reticulum coupling pathway, which also could involve Ca2+.