Solution structure of extracellular loop of human β4 subunit of BK channel and its biological implication on ChTX sensitivity

Large-conductance Ca2+- and voltage-dependent K+ (BK) channels display diverse biological functions while their pore-forming a subunit is coded by a single Slo1 gene. The variety of BK channels is correlated with the effects of BK alpha coexpression with auxiliary beta (beta 1-beta 4) subunits, as well as newly defined. subunits. Charybdotoxin (ChTX) blocks BK channel through physically occluding the K+-conduction pore. Human brain enriched beta 4 subunit (h beta 4) alters the conductance-voltage curve, slows activation and deactivation time courses of BK channels. Its extracellular loop (h beta 4-loop) specifically impedes ChTX to bind BK channel pore. However, the structure of beta 4 subunit's extracellular loop and the molecular mechanism for gating kinetics, toxin sensitivity of BK channels regulated by beta 4 are still unclear. To address them, here, we first identified four disulfide bonds in h beta 4-loop by mass spectroscopy and NMR techniques. Then we determined its three-dimensional solution structure, performed NMR titration and electrophysiological analysis, and found that residue Asn123 of beta 4 subunit regulated the gating and pharmacological characteristics of BK channel. Finally, by constructing structure models of BK alpha/beta 4 and thermodynamic double-mutant cycle analysis, we proposed that BKa subunit might interact with beta 4 subunit through the conserved residue Glu264(BK alpha) coupling with residue Asn123(beta 4).