On the role of Ca2+- and voltage-dependent inactivation in Cav1.2 sensitivity for the phenylalkylamine (-)gallopamil

Abstract

L-type calcium channels (Cav1.m) inactivate in response to elevation of intracellular Ca2+ (Ca2+-dependent inactivation) and additionally by conformational changes induced by membrane depolarization (fast and slow voltage-dependent inactivation). Molecular determinants of inactivation play an essential role in channel inhibition by phenylalkylamines (PAAs). The relative impacts, however, of Ca2+-dependent and voltage-dependent inactivation in Cav1.2 sensitivity for PAAs remain unknown. In order to analyze the role of the different inactivation processes, we expressed Cav1.2 constructs composed of different β-subunits (β1a-, β2a-, or β3-subunit) in Xenopus oocytes and estimated their (-)gallopamil sensitivity by means of the two-microelectrode voltage clamp with either Ba2+ or Ca2+ as charge carrier. Cav1.2 consisting of the β2a-subunit displayed the slowest inactivation and the lowest apparent sensitivity for the PAA (-)gallopamil. A significantly higher apparent (-)gallopamil-sensitivity with Ca2+ as charge carrier was observed for all 3 β-subunit compositions. The kinetics of Ca2+-dependent inactivation and slow voltage-dependent inactivation were not affected by drug. The higher sensitivity of the Cav1.2 channels for (-)gallopamil with Ca2+ as charge carrier results from slower recovery (τrec,Ca ≈ 15 seconds versus τrec,Ba ≈ 3 to 5 seconds) from a PAA-induced channel conformation. We propose a model where (-)gallopamil promotes a fast voltage-dependent component in Cav1.2 inactivation. The model reproduces the higher drug sensitivity in Ca2+ as well as the lower sensitivity of slowly inactivating Cav1.2 composed of the β2a-subunit.