Mechanisms of Auxiliary α2δ-1 Subunit Regulation of Voltage-Gated Calcium Channels

Abstract

The voltage-gated calcium (CaV) channel α2δ-1 subunit is implicated in a variety of cardiovascular and neurological diseases and is the molecular target of gabapentin, a drug used for the treatment of epilepsy and neuropathic pain. The precise mechanisms by which α2δ-1 dysfunction causes disease and how its targeting by gabapentin is therapeutic are unclear, in part due to incomplete understanding of how α2δ-1 binds to and regulates CaV channels. Here, we investigated mechanisms underlying α2δ-1 regulation of neuronal CaV2.2 (α1B+β2a) channels reconstituted in HEK293 cells. Using whole-cell electrophysiology, α2δ-1 caused a dramatic four to five-fold increase in whole-cell current amplitude compared to channels reconstituted with α1B+β2a alone. The increase in current was wholly accounted for by an increase in channel surface density as reported by a quantum dot labeling technique. An optical pulse-chase assay showed that the increased surface density was achieved solely through reducing the rate of CaV2.2 endocytosis rather than by enhancing the rate of forward trafficking. To map the site of interaction between α2δ-1 and α1B, we inserted a small epitope tag into judiciously selected extracellular regions of the α1B subunit. We determined accessibility of the tag to quantum dot and modulation of whole-cell currents in the presence or absence of α2δ-1. From the pattern of this dual readout, we inferred an α2δ-1 binding site in the α1B domain III extracellular S5-S6 loop. Alanine scanning mutagenesis revealed discrete residues in this loop that was necessary for α2δ-1 regulation of α1B. These results provide novel insights into mechanisms underlying α2δ-1 regulation of CaV channels, focus the putative modes of gabapentin action, and offer a new target site for potential development of future therapeutic molecules.