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-Ca Vβ contact surface in CaV1.2. Substitution of AID-exposed residues by the corresponding amino acids present in other Ca Vα1 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 Ca Vβ. At the single channel level, these CaV1.2 mutants coexpressed with CaVβ2a visit high open probability mode less frequently than wildtype channels. On the other hand, Ca V1.2 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 Ca Vβ spatially reorients AID residues that influence the channel gate. These findings provide a new framework for understanding modulation of VDCC gating by CaVβ. © 2008 Gonzales-Gutierrez et al.