A critical GxxxA motif in the gamma sub(6) calcium channel subunit mediates its inhibitory effect on Cav3.1 calcium current

The eight members of the calcium channel gamma subunit family are integral membrane proteins that regulate the expression and behaviour of voltage and ligand gated ion channels. While a subgroup consisting of gamma sub(2), gamma sub(3), gamma sub(4) and gamma sub(8) (the TARPs) modulate AMPA receptor localization and function, the gamma sub(1) and gamma sub(6) subunits conform to the original description of these proteins as regulators of voltage gated calcium channels. We have previously shown that the gamma sub(6) subunit is highly expressed in atrial myocytes and that it is capable of acting as a negative modulator of low voltage activated calcium current. In this study we extend our understanding of gamma sub(6) subunit modulation of low voltage activated calcium current. Using engineered chimeric constructs, we demonstrate that the first transmembrane domain (TM1) of gamma sub(6) is necessary for its inhibitory effect on Cav3.1 current. Mutational analysis is then used to identify a unique GxxxA motif within TM1 that is required for the function of the subunit strongly suggesting the involvement of helix-helix interactions in its effects. Results from co-immunoprecipitation experiments confirm a physical association of gamma sub(6) with the Cav3.1 channel in both HEK cells and atrial myocytes. Single channel analysis reveals that binding of gamma sub(6) reduces channel availability for activation. Taken together, the results of this study provide both a molecular and a mechanistic framework for understanding the unique ability of the gamma sub(6) calcium channel subunit to modulate low voltage activated (Cav3.1) calcium current density.