Article,

Ca$^2+$-dependent inactivation of cardiac L-type Ca$^2+$ channels does not affect their voltage sensor.

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J. Gen. Physiol., 102 (6): 1005--1030 (December 1993)

Abstract

Inactivation of currents carried by Ba2+ and Ca$^2+$, as well as intramembrane charge movement from L-type Ca$^2+$ channels were studied in guinea pig ventricular myocytes using the whole-cell patch clamp technique. Prolonged (2 s) conditioning depolarization caused substantial reduction of charge movement between -70 and 10 mV (charge 1, or charge from noninactivated channels). In parallel, the charge mobile between -70 and -150 mV (charge 2, or charge from inactivated channels) was increased. The availability of charge 2 depended on the conditioning pulse voltage as the sum of two Boltzmann components. One component had a central voltage of -75 mV and a magnitude of 1.7 nC/microF. It presumably is the charge movement (charge 2) from Na$^+$ channels. The other component, with a central voltage of approximately -30 mV and a magnitude of 3.5 nC/microF, is the charge 2 of L-type Ca$^2+$ channels. The sum of charge 1 and charge 2 was conserved after different conditioning pulses. The difference between the voltage dependence of the activation of L-type Ca$^2+$ channels (half-activation voltage, V, of approximately -20 mV) and that of charge 2 (V of -100 mV) made it possible to record the ionic currents through Ca$^2+$ channels and charge 2 in the same solution. In an external solution with Ba2+ as sole metal the maximum available charge 2 of L-type Ca$^2+$ channels was 10-15\% greater than that in a Ca$^2+$-containing solution. External Cd2+ caused 20-30\% reduction of charge 2 both from Na$^+$ and L-type Ca$^2+$ channels. Voltage- and Ca$^2+$-dependent inactivation phenomena were compared with a double pulse protocol in cells perfused with an internal solution of low calcium buffering capacity. As the conditioning pulse voltage increased, inactivation monitored with the second pulse went through a minimum at about 0 mV, the voltage at which conditioning current had its maximum. Charge 2, recorded in parallel, did not show any increase associated with calcium entry. Two alternative interpretations of these observations are: (a) that Ca$^2+$-dependent inactivation does not alter the voltage sensor, and (b) that inactivation affects the voltage sensor, but only in the small fraction of channels that open, and the effect goes undetected. A model of channel gating that assumes the first possibility is shown to account fully for the experimental results. Thus, extracellular divalent cations modulate voltage-dependent inactivation of the Ca$^2+$ channel. Intracellular Ca$^2+$ instead, appears to cause inactivation of the channel without affecting its voltage sensor.

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