On the Role of Ca ²⁺ - and Voltage-Dependent Inactivation in Ca _v 1.2 Sensitivity for the Phenylalkylamine (-)Gallopamil

Abstract

Abstract-L-type calcium channels (Ca v 1.m) inactivate in response to elevation of intracellular Ca 2ϩ (Ca 2ϩ -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 Ca 2ϩ -dependent and voltage-dependent inactivation in Ca v 1.2 sensitivity for PAAs remain unknown. In order to analyze the role of the different inactivation processes, we expressed Ca v 1.2 constructs composed of different ␤-subunits (␤ 1a -, ␤ 2a -, or ␤ 3 -subunit) in Xenopus oocytes and estimated their (-)gallopamil sensitivity by means of the two-microelectrode voltage clamp with either Ba 2ϩ or Ca 2ϩ as charge carrier. Ca v 1.2 consisting of the ␤ 2a -subunit displayed the slowest inactivation and the lowest apparent sensitivity for the PAA (-)gallopamil. A significantly higher apparent (-)gallopamil-sensitivity with Ca 2ϩ as charge carrier was observed for all 3 ␤-subunit compositions. The kinetics of Ca 2ϩ -dependent inactivation and slow voltage-dependent inactivation were not affected by drug. The higher sensitivity of the Ca v 1.2 channels for (-)gallopamil with Ca 2ϩ as charge carrier results from slower recovery ( rec,Ca Ϸ15 seconds versus rec,Ba Ϸ3 to 5 seconds) from a PAA-induced channel conformation. We propose a model where (-) antagonists, ie, phenylalkylamines (PAAs), benzothiazepines (ie, diltiazem) and 1,4-dihydropyridines (DHPs). [2][3][4][5] About 20 years ago, several groups demonstrated that Ca v 1.m inhibition by PAAs and diltiazem occurs predominately during membrane depolarizations ("use-dependent" action) when the channels are primarily in the open or inactivated conformation. [2][3][4]6 Since then, numerous studies in myocardial and smooth muscle cells have shown that Ca 2ϩ channel inhibition by these compounds, as well as by the new compound mibefradil, reflect a balance between channel shut-off during membrane depolarization and subsequent recovery at rest. [7][8][9][10][11] Over the last three years there has been substantial progress toward understanding the role of inactivation in Ca 2ϩ channel inhibition by Ca 2ϩ antagonists. Site directed mutagenesis enabled a detailed analysis of the impact of this process in PAA sensitivity. A comparison of the PAA sensitivities of Ca v 2.1 mutants and chimeric channel constructs inactivating at different rates revealed a strong correlation between the rates of fast voltage-dependent inactivation and apparent PAA sensitivities. 11-13 Similar findings were reported for Ca 2ϩ channel block by diltiazem. 11,14 Subsequently, the studies concentrated on the localization of inactivation determinants that are relevant for channel inhibition. Thus, inactivation determinants in the loop between domains I and II and ␤-subunit interaction have been shown to control PAA sensitivity of a Ca v 2.1 mutant 13 as well as the mibefradilsensitivity of wild ...

Molecular Determinants of Frequency Dependence and Ca2+ Potentiation of Verapamil Block in the Pore Region of Cav1.2

“…7B). Our finding that verapamil accelerates the inactivation of Ca v 1.2 channels and increases the fraction of channels inactivating is consistent with previous studies (Johnson et al, 1996;Sokolov et al, 2001). However, our results with EIIIQ are in contrast to observations with mutations in IVS6 which accelerated both verapamil block and recovery from verapamil block at ϩ10 mV (Johnson et al, 1996).…”

Structural Model for Phenylalkylamine Binding to L-type Calcium Channels

“…2ϩ potentiation of the LTCC block has been observed for different PAAs (8,55,56). In our models, a Ca 2ϩ ion bound to the side chains of E 3p50 and E 4p50 is coordinated by the nitrile nitrogen of PAAs.…”

Molecular Determinants of Ca2+ Potentiation of Diltiazem Block and Ca2+-Dependent Inactivation in the Pore Region of Cav1.2

“…C, change in peak current amplitude measured at ϩ10 mV from a holding potential of Ϫ60 mV when the extracellular solution is switched from 10 mM Ba 2ϩ to 10 mM Ca the modulation of (Ϫ)-gallopamil (a PAA) block of Ca v 1.2 by different ␤ subunits (Sokolov et al, 2001) concluded that channels with an accelerated voltage-dependent inactivation rate were also more sensitive to gallopamil block. Furthermore, Sokolov et al (2001) proposed that PAA block of Ca v 1.2 depends upon the fast voltage-dependent inactivation observed in Ba 2ϩ and not the further acceleration of inactivation observed in Ca 2ϩ . Thus, our data fit well with the model of Sokolov et al (2001), since the main effect of the I1627A mutation is to shift Ͼ80% of channels into the fast inactivating mode in Ba 2ϩ (i.e., the fast voltage-dependent inactivation of Sokolov et al, 2001; Fig.…”

“…Furthermore, Sokolov et al (2001) proposed that PAA block of Ca v 1.2 depends upon the fast voltage-dependent inactivation observed in Ba 2ϩ and not the further acceleration of inactivation observed in Ca 2ϩ . Thus, our data fit well with the model of Sokolov et al (2001), since the main effect of the I1627A mutation is to shift Ͼ80% of channels into the fast inactivating mode in Ba 2ϩ (i.e., the fast voltage-dependent inactivation of Sokolov et al, 2001; Fig. 8C).…”