Regulation of BK Channel Auxiliary Gamma Subunits by N-Glycosylation

Abstract

BK channels consist of pore-forming, voltage- and Ca2+-sensing α subunits and auxiliary β and γ subunits. The auxiliary γ1-3 subunits potently modulate the BK channel's function by shifting voltage-dependence of channel activation by 50 - 150 mV toward the negative voltage direction. All BK channel γ subunits contain single or multiple consensus N-linked glycosylation sites in their extracellular LRR domains. We found that inhibition of protein N-glycosylation in HEK-293 cells by tunicamycin caused a great reduction (γ1) or full loss (γ2 and γ3) of the γ subunit's effectiveness in modulating BK channels. We identified the glycosylation sites of N147 in the γ1 subunit, N112, N148 and N211 in the γ2 subunit, and N82, N111 and N137 in the γ3 subunit to be all necessary for the γ subunits’ channel-modulation function. Blockade of glycosylation at single or multiple sites by mutation (N→Q) caused a decrease in protein size and a conversion of either all or a significant portion of BK channels to be unmodulated by the auxiliary γ subunit. Overexpression of the γ1-N147Q mutant can partially compensate its deficiency in BK channel modulation. We conclude that N-glycosylation plays a critical role in maintaining the modulatory function of the BK channel auxiliary γ subunits, presumably through facilitating the protein's surface expression.