D600 binding sites on voltage-sensors for excitation-contraction coupling in frog skeletal muscle are intracellular.

Charge movements were measured in frog cut twitch fibres mounted in a double Vaseline gap chamber at 14 degrees C with 30 microM D600 in the external solution. TEST-minus-CONTROL current traces appear normal with a hump current component (I gamma) embedded in the decay phase of the early current component (I beta) in the ON-segment and an exponentially decaying current transient in the OFF-segment. When a conditioning depolarization to 0 mV is applied at around 6 degrees C, charge movement is greatly reduced but not completely suppressed and no hump component can be visualized in the ON-segment. In addition, an extra capacitive component is generated having a time course slower than, and a polarity opposite to, that of the usual charge movement. This extra component makes the transients in both the ON- and OFF-segments appear bisphasic. When temperature is restored to 14 degrees C, the biphasic nature is greatly enhanced. After the application of a conditioning hyperpolarization, the shape of the TEST-minus-CONTROL current trace is converted back to that before paralysis, but the total amount of charge reprimed is less than 100% of control. In general, more Q beta is reprimed than Q gamma, and the amount of Q gamma reprimed varies over a wider range from fibre to fibre than that of Q beta. Extracellularly applied D890 cannot reproduce the blocking effect of D600 whereas intracellularly applied D890 can. As D890 is permanently charged and cannot permeate through the plasma membranes, it can be concluded that the binding sites for D600/D890 on the charge movement macromolecules must be on the myoplasmic side. This adds another parallelism between the charge movement entities and L-type calcium channels. However, the specific prerequisites for the blockage of the former not shared by the latter differentiates the two physiological units.