Structural Determinants of the BK Channel γ-Subunits' Modulatory Potency

Abstract

BK channels consist of the pore-forming, voltage- and Ca2+-sensing α-subunits (BKα), either alone or together with the tissue-specific auxiliary β-subunits (β1-β4) or γ-subunits (γ1-γ4). The newly identified γ-subunits are a group of leucine-rich repeat (LRR)-containing membrane proteins, γ1 (LRRC26), γ2 (LRRC52), γ3 (LRRC55), and γ4 (LRRC38), that produce marked and graded shifts in the BK channel's voltage dependence of activation in the hyperpolarizing direction by ∼140 mV, 100 mV, 50 mV, and 20 mV, respectively, in the absence of calcium. The γ-subunits contain a single transmembrane segment (TM), a short intracellular C-terminal tail (C-tail), an N-terminal signal peptide, and a relatively large extracellular LRR domain, which comprises six tandem LRR structural units (LRRs1-6) and two cysteine-rich modules, LRRNT and LRRCT, capped on the N- and C-terminal sides. To identify structural determinants of their potent but different modulatory functions, we generated a series of chimeric constructs among these four γ-subunits by swapping various structural units and analyzed the resulting effects on their modulatory potency on BK channels. We found that: (1) the γ3- and γ4-subunits' relatively weaker potencies could not be improved by substitution with a γ1-subunit signal peptide, suggesting intrinsically weaker modulatory capabilities for these two subunits; (2) variation in amino acid sequences of the TM and C-tail regions determines the γ1, γ2, and γ3-subunits' differences in modulatory potency; (3) LRR domain also participates in the γ-subunits' modulatory function, as individual substitution of the γ1-subunit's LRR1, LRR2, LRR3, LRR4, LRR5, TM, and C-tail regions with those of γ4-subunit caused nearly full loss of γ1-subunit's effectiveness whereas replacement of LRRNT, LRR6, and LRRCT with the same regions from γ4-subunit were well tolerated.