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

Big-conductance, Ca2+-activated K+ (BK) channels consist of Ca2+- and voltage-sensing, pore-forming (BK) subunits and regulatory auxiliary {beta} or {gamma} subunits. Concatenated subunit constructs are powerful tools for elucidating subunit stoichiometry in ion channel gating and regulation, allowing control over subunit arrangement, stoichiometry, and mutation. However, the additional S0 transmembrane segment in BK places its N- and C-termini on opposite sides of the membrane, preventing tandem BK channel subunit construction by conventional methods. To investigate the atypical "all-or-none" modulatory function of {gamma} subunits and the subunit stoichiometry of BK channel gating, we developed concatenated constructs containing 2 or 4 BK subunits by splicing them into modular forms that can be co-expressed to form functional channels. These constructs retained voltage and Ca2+ gating properties similar to intact BK channels. By fusing the LRRC26 ({gamma}1) subunit to the N-terminus of tandem BK constructs, we found that a single {gamma}1 subunit per subunit tetramer is sufficient to fully modulate the channel. Furthermore, the L312A mutation in the deep pore region exhibited a stoichiometrically graded effect on voltage-gated BK channel activation. In contrast, a V288A mutation at the selectivity filter induced channel inactivation only when present in all four BK subunits. Thus, by engineering concatenated BK constructs, we identified three distinct stoichiometric modes of BK channel gating control by LRRC26, the pore, and the selectivity filter. This study offers new molecular tools and advances our understanding of subunit stoichiometry in BK channel gating and modulation.