Distinction between Steady-State Inactivation and Voltage-Dependent Facilitation in L-Type Ca^2＋ Channel α_1c and α_1c/β Subunits

Abstract

The L-type Ca²⁺ channel has a unique kinetic property known as voltage-dependent facilitation. Many researchers have repeatedly investigated the mechanism in response to the voltage-dependent facilitation since the first observation by Fenwick et al. in 1982. Electrophysiological evaluations of voltage-dependent facilitation, however, remain inconsistent, partially because of its unclear definition. Some scientists understand it as a current augmentation by a conditioning prepulse prior to the test pulse, and others understand it as a result of the U-shape steady-state inactivation curve. We therefore investigated to identify the distinction between the voltage-dependent facilitation and the steady-state inactivation, by use of Ba²⁺ as the charge in order to avoid the other inactivation mechanism or the Ca²⁺-dependent inactivation upon this analysis. Conventional whole-cell mode patch clamp technique was applied to Chinese hamster fibroblast (CHW) cells that express the α_1c subunit alone or the α_1c subunit with the β subunit (α_1c/β) derived from rabbit heart to investigate the voltage-dependent facilitation depending on the composition of the subunits. Coexpression of the β subunit augmented α₁ subunit channel current and shifted current-voltage relation towards hyperpolarized direction. In the experiment using conventional double pulse protocol to investigate steady-state inactivation, α_1c subunit channel current and α_1c/β subunit channel current were not fully inactivated. Subtraction of the steady-state inactivation component from whole recovered current enabled us to identify the voltage-dependent facilitation component of the L-type Ca²⁺ channel. The voltage-dependent facilitation of the α₁ subunit current and the α_1c/β subunit current were identical in kinetics, and could be generated at 0 mV or depolarized potentials partially overlapped with the potential range for the steady-state inactivation of the current. These results suggest that the voltage-dependent facilitation of the L-type Ca²⁺ channel could be formed by the α_1c subunit without interaction with the β subunit, and that the range for the voltage-dependent facilitation and the steady-state inactivation overlap each other at 0 mV or more depolarized potentials up to approximately+100 mV.