Concatenated tandem constructs of BK channels reveal three distinct types of subunit stoichiometry in channel modulation by the auxiliary γ subunit and mutations in the channel pore region

Abstract

AbstractThe large-conductance, Ca2+-activated K+(BK) channels consist of the Ca2+- and voltage-sensing and pore-forming α (BKα) subunits and regulatory auxiliary β or γ subunits. Concatenated tandem constructs have been proved to be necessary and powerful in understanding the subunit stoichiometry of K+channel gating and regulation by constraining the stoichiometry, organization, and mutation of individual subunits. However, because of its unique possession of an extra S0 transmembrane segment that puts the N- and C- termini on the opposite sides of the membrane, it is impossible to generate concatenated tandem subunit constructs of BK channels as those done with other K+channels. In order to investigate the molecular mechanisms of the atypical “all-or-none” modulatory function of the γ subunits and the subunit stoichiometry of BK channel gating, we have taken a novel strategy and generated functional concatenated tandem constructs containing 2 or 4 copies of the BKα subunits by splicing BKα into 2 protein constructs that can be co-expressed to form functional channels. We observed that the BK channels formed by concatenated tandem constructs are similar to intact channels in voltage and Ca2+gating properties. Interestingly, the BKγ1 subunit and mutations located at the S6 transmembrane segment and selectivity filter displayed 3 distinct types of subunit stoichiometry in BK channel modulation. By fusion of the BKγ1 subunit to the N-terminus of the concatenated tandem BKα constructs, we observed that a single molecule of BKγ1 subunit per tetrameric BKα channel is sufficient to fully modulate the channels. The Y294K, F303A, and L312A mutations at each BKα subunit has an incremental effect on BK channel gating. The V288A mutation at the selectivity filter produce an “all-or-none” effect but requires mutations at all 4 BKα subunits to have modulatory effect on channel gating, i.e., mutations at 1, 2, or 3 subunits have nearly no effect. Interestingly, the F307A mutation exhibited an atypical “all-or-none” modulatory function similar to that of the γ1 subunit. The diversity in subunit stoichiometry of BK channel modulation indicates distinct mechanisms in affecting BK channel gating. Overall, we have developed novel concatenated tandem constructs for BK channels and revealed three distinct types of subunit stoichiometry in BK channel modulation by the auxiliary γ subunit and mutations at the pore-lining S6 transmembrane segment and selectivity filter.