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

Voltage-dependent calcium channels consist of a pore-forming subunit (Ca(V)α(1)) that includes all the molecular determinants of a voltage-gated channel, and several accessory subunits. The ancillary β-subunit (Ca(V)β) is a potent activator of voltage-dependent calcium channels, but the mechanisms and structural bases of this regulation remain elusive. Ca(V)β binds reversibly to a conserved consensus sequence in Ca(V)α(1), the α(1)-interaction domain (AID), which forms an α-helix when complexed with Ca(V)β. Conserved aromatic residues face to one side of the helix and strongly interact with a hydrophobic pocket on Ca(V)β. Here, we studied the effect of mutating residues located opposite to the AID-Ca(V)β contact surface in Ca(V)1.2. Substitution of AID-exposed residues by the corresponding amino acids present in other Ca(V)α(1) subunits (E462R, K465N, D469S, and Q473K) hinders Ca(V)β's ability to increase ionic-current to charge-movement ratio (I/Q) without changing the apparent affinity for Ca(V)β. At the single channel level, these Ca(V)1.2 mutants coexpressed with Ca(V)β(2a) visit high open probability mode less frequently than wild-type channels. On the other hand, Ca(V)1.2 carrying either a mutation in the conserved tryptophan residue (W470S, which impairs Ca(V)β binding), or a deletion of the whole AID sequence, does not exhibit Ca(V)β-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(V)β, suggesting a direct participation of these residues in the modulation of channel activation. Our results show that Ca(V)β-dependent potentiation arises primarily from changes in the modal gating behavior. We envision that Ca(V)β spatially reorients AID residues that influence the channel gate. These findings provide a new framework for understanding modulation of VDCC gating by Ca(V)β.