Asymmetric membrane currents and fluxes of Ca 2+ release were determined in skeletal muscle fibers voltage clamped in a Vaseline-gap chamber. The conditioning pulse protocol 1 for suppressing Ca z+ release and the "hump" component of charge movement current (I~), described in the first paper of this series, was applied at different test pulse voltages. The amplitude of the current suppressed during the ON transient reached a maximum at slightly suprathreshold test voltages (-50 to -40 mV) and decayed at higher voltages. The component of charge movement current suppressed by 20 I~M tetracaine also went through a maximum at low pulse voltages. This anomalous voltage dependence is thus a property ofI,, defined by either the conditioning protocol or the tetracaine effect. A negative (inward-going) phase was often observed in the asymmetric current during the ON of depolarizing pulses. This inward phase was shown to be an intramembranous charge movement based on (a) its presence in the records of total membrane current, (b) its voltage dependence, with a maximum at slightly suprathreshold voltages, (c) its association with a "hump" in the asymmetric current, (d) its inhibition by interventions that reduce the "hump," (e) equality of ON and OFF areas in the records of asymmetric current presenting this inward phase, and (f) its kinetic relationship with the time derivative of Ca release flux. The nonmonotonic voltage dependence of the amplitude of the hump and the possibility of an inward phase of intramembranous charge movement are used as the main criteria in the quantitative testing of a specific model. According to this model, released Ca 2÷ binds to negatively charged sites on the myoplasmic face of the voltage sensor and increases the local transmembrane potential, thus driving additional charge movement (the hump). This model successfully predicts the anomalous voltage dependence and all the kinetic properties of I~ described in the previous papers. It also Address reprint requests to Dr.

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 [Ca 2+] transients, flux of calcium release from the sarcoplasmic reticulure (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 ( > 700 ms), which deplete Ca 2+ 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.

SUMMARY1. Intramembrane charge movements and changes in intracellular calcium concentration were recorded simultaneously in voltage clamped cut skeletal muscle fibres of the frog in the presence and absence of tetracaine.2. Extracellular application of 20 /IM tetracaine reduced the increase in myoplasmic [Ca2"]. The effect on the underlying calcium release flux from the sarcoplasmic reticulum was to suppress the peak of the release while sparing the steady level attained at the end of 100 ms clamp depolarizations.3. While the peak of the release flux at corresponding voltages was reduced by 62 o after the addition of tetracaine, the rate of inactivation was the same when the pulses elicited release fluxes of similar amplitude.4. Higher concentrations of tetracaine, 0-2 mm, abolished the calcium signal in stretched fibres whereas in slack fibres this concentration left a non-inactivating calcium release flux.5. Lowering the extracellular pH antagonized the effect of the drug both on charge movements and on calcium signals. The permanently charged analogue tetracaine methobromide lacked effects on excitation-contraction coupling.6. These results imply that the two kinetic components of calcium release flux have very different tetracaine sensitivities. They are also consistent with an intracellular site of action of the drug at low concentration. Taken together they strongly suggest that the inactivating and non-inactivating components of calcium release correspond to different pathways: one that inactivates, is sensitive to tetracaine and is controlled by calcium, and another that does not inactivate, is much less sensitive to tetracaine and is directly controlled by voltage.

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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 Ca r+ 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 > Sr > Mg > Ba for group IIa cations and Li > Na > K > Rb > Cs for group Ia. Ca 2 § 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.Address reprint requests to Dr.