Mutations of Nonconserved Residues within the Calcium Channel α1-interaction Domain Inhibit β-Subunit Potentiation

Abstract

Voltage-dependent calcium channels consist of a pore-forming subunit (CaVα1) that includes all the molecular determinants of a voltage-gated channel, and several accessory subunits. The ancillary β-subunit (CaVβ) is a potent activator of voltage-dependent calcium channels, but the mechanisms and structural bases of this regulation remain elusive. CaVβ binds reversibly to a conserved consensus sequence in CaVα1, the α1-interaction domain (AID), which forms an α-helix when complexed with CaVβ. Conserved aromatic residues face to one side of the helix and strongly interact with a hydrophobic pocket on CaVβ. Here, we studied the effect of mutating residues located opposite to the AID-CaVβ contact surface in CaV1.2. Substitution of AID-exposed residues by the corresponding amino acids present in other CaVα1 subunits (E462R, K465N, D469S, and Q473K) hinders CaVβ's ability to increase ionic-current to charge-movement ratio (I/Q) without changing the apparent affinity for CaVβ. At the single channel level, these CaV1.2 mutants coexpressed with CaVβ2a visit high open probability mode less frequently than wild-type channels. On the other hand, CaV1.2 carrying either a mutation in the conserved tryptophan residue (W470S, which impairs CaVβ binding), or a deletion of the whole AID sequence, does not exhibit CaVβ-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 CaVβ, suggesting a direct participation of these residues in the modulation of channel activation. Our results show that CaVβ-dependent potentiation arises primarily from changes in the modal gating behavior. We envision that CaVβ spatially reorients AID residues that influence the channel gate. These findings provide a new framework for understanding modulation of VDCC gating by CaVβ.