Interaction via a Key Tryptophan in the I-II Linker of N-Type Calcium Channels Is Required for β1 But Not for Palmitoylated β2, Implicating an Additional Binding Site in the Regulation of Channel Voltage-Dependent Properties

The Ca V β subunits of voltage-gated calcium channels regulate these channels in several ways. Here we investigate the role of these auxiliary subunits in the expression of functional N-type channels at the plasma membrane and in the modulation by G-protein-coupled receptors of this neuronal channel. To do so, we mutated tryptophan 391 to an alanine within the α-interacting domain (AID) in the I-II linker of Ca V 2.2. We showed that the mutation W391 virtually abolishes the binding of Ca V β1b and Ca V β2a to the Ca V 2.2 I-II linker and strongly reduced current density and cell surface expression of both Ca V 2.2/α2δ-2/β1b and/β2a channels. When associated with Ca V β1b, the W391A mutation also prevented the Ca V β1b-mediated hyperpolarization of Ca V 2.2 channel activation and steady-state inactivation. However, the mutated Ca V 2.2W391A/β1b channels were still inhibited to a similar extent by activation of the D 2 dopamine receptor with the agonist quinpirole. Nevertheless, key hallmarks of G-protein modulation of N-type currents, such as slowed activation kinetics and prepulse facilitation, were not observed for the mutated channel. In contrast, Ca V β2a was still able to completely modulate the biophysical properties of Ca V 2.2W391A channel and allow voltage-dependent G-protein modulation of Ca V 2.2W391A. Additional data suggest that the concentration of Ca V β2a in the proximity of the channel is enhanced independently of its binding to the AID by its palmitoylation. This is essentially sufficient for all of the functional effects of Ca V β2a, which may occur via a second lower-affinity binding site, except trafficking the channel to the plasma membrane, which requires interaction with the AID region.