Voltage-dependent Na + channels are thought to sense membrane potential with fixed charges located within the membrane's electrical field. Measurement of open probability (Po) as a function of membrane potential gives a quantitative indication of the number of such charges that move through the field in opening the channel. We have used single-channel recording to measure skeletal muscle Na § channel open probability at its most negative extreme, where channels may open as seldom as once per minute. To prevent fast inactivation from masking the voltage dependence of Po, we have generated a clone of the rat skeletal muscle Na + channel that is lacking in fast inactivation (IFM1303QQQ). Using this mutant channel expressed in Xenopus oocytes, and the extra resolution afforded by single-channel analysis, we have extended the resolution of the hyperpolarized tail of the Po curve by four orders of magnitude. We show that previous measurements, which indicated a minimum of six effective gating charges, may have been made in a range of Po values that had not yet arrived at its limiting slope. In our preparation, a minimum of 12 charges must function in the activation gating of the channel. Our results will require reevaluation of kinetic models based on six charges, and they have major implications for the interpretation of $4 mutagenesis studies and structure/function models of the Na § channel.

Impedance studies were performed on small spherical clusters of embryonic chick heart cells grown in tissue culture. Each syncytial cluster was impaled with two microelectrodes; one injected low amplitude stochastic current and the other recorded the resulting perturbation of intracellular potential. The current and potential records were digitized, decomposed into their sinusoidal components, and the frequency domain impedance of the cluster was determined. The impedance data were compared with a theory for current flow in a spherical syncytium and values were derived for parameters describing the membranes and intercellular clefts of the tissue. The clusters were spontaneously active but usually became temporarily quiescent when impaled with two electrodes. The potential stabilized at a value close to -30 mV. At this depolarized potential, active slow currents, presumably present in the cardiac action potential, contributed noticeably to the linear impedance, producing a resonant peak in the magnitude of the impedance at a frequency of 1-3 Hz. The linearized impedance functions for these currents were characterized in the presence and absence of tetrodotoxin (TTX) and D-600. TTX had no noticeable effect on the impedance but D-600 essentially abolished the active currents. Although the ionic basis of these currents is not known, frequency domain analysis appears to be a viable technique for studying slow currents in heart muscle.

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