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Mutations of Nonconserved Residues within the Calcium Channel ₁-interaction Domain Inhibit β-Subunit Potentiation

¹ Centro de Neurociencia de Valparaíso and ² Programa de Doctorado en Ciencias mención Neurociencia, Universidad de Valparaíso, 2349400 Valparaíso, Chile
³ Institut für Neurophysiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany

Correspondence to Alan Neely: alan{at}cnv.cl

Voltage-dependent calcium channels consist of a pore-forming subunit (Ca_V1) that includes all the molecular determinants of a voltage-gated channel, and several accessory subunits. The ancillary β-subunit (Ca_Vβ) is a potent activator of voltage-dependent calcium channels, but the mechanisms and structural bases of this regulation remain elusive. Ca_Vβ binds reversibly to a conserved consensus sequence in Ca_V1, the ₁-interaction domain (AID), which forms an -helix when complexed with Ca_Vβ. Conserved aromatic residues face to one side of the helix and strongly interact with a hydrophobic pocket on Ca_Vβ. Here, we studied the effect of mutating residues located opposite to the AID-Ca_Vβ contact surface in Ca_V1.2. Substitution of AID-exposed residues by the corresponding amino acids present in other Ca_V1 subunits (E462R, K465N, D469S, and Q473K) hinders Ca_Vβ's ability to increase ionic-current to charge-movement ratio (I/Q) without changing the apparent affinity for Ca_Vβ. At the single channel level, these Ca_V1.2 mutants coexpressed with Ca_Vβ_2a visit high open probability mode less frequently than wild-type channels. On the other hand, Ca_V1.2 carrying either a mutation in the conserved tryptophan residue (W470S, which impairs Ca_Vβ binding), or a deletion of the whole AID sequence, does not exhibit Ca_Vβ-induced increase in I/Q. In addition, we observed a shift in the voltage dependence of activation by +12 mV in the AID-deleted channel in the absence of Ca_Vβ, suggesting a direct participation of these residues in the modulation of channel activation. Our results show that Ca_Vβ-dependent potentiation arises primarily from changes in the modal gating behavior. We envision that Ca_Vβ spatially reorients AID residues that influence the channel gate. These findings provide a new framework for understanding modulation of VDCC gating by Ca_Vβ.

Abbreviations used in this paper: AID, ₁-interaction domain; GV, conductance curve; I/Q, ionic-current to charge-movement ratio; Po, open probability; Q_on, charge movement; WT, wild-type.

© 2008 Gonzales-Gutierrez et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jgp.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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