Inhibition of recombinant N-type Ca_V channels by the γ₂ subunit involves unfolded protein response (UPR)-dependent and UPR-independent mechanisms

Auxiliary γ subunits are an important component of high-voltage-activated calcium (Ca_V) channels, but their precise regulatory role remains to be determined. In the current report, we have used complementary approaches including molecular biology and electrophysiology to investigate the influence of the γ subunits on neuronal Ca_V channel activity and expression. We found that coexpression of γ₂ or γ₃ subunits drastically inhibited macroscopic currents through recombinant N-type channels (Ca_V2.2/ β₃/α₂δ) in HEK-293 cells. Using inhibitors of internalization, we found that removal of functional channels from the plasma membrane is an improbable mechanism of current regulation by γ. Instead, changes in current amplitude could be attributed to two distinct mechanisms. First, γ subunit expression altered the voltage dependence of channel activity. Second, γ subunit expression reduced N-type channel synthesis via activation of the endoplasmic reticulum unfolded protein response. Together, our findings (1) corroborate that neuronal γ subunits significantly downregulate Ca_V2.2 channel activity, (2) uncover a role for the γ₂ subunit in Ca_V2.2 channel expression through early components of the biosynthetic pathway, and (3) suggest that, under certain conditions, channel protein misfolding could be induced by interactions with the γ subunits, supporting the notion that Ca _V channels constitute a class of difficult-to-fold proteins.