Positions of beta 2 and beta 3 subunits in the large-conductance calcium- and voltage-activated BK potassium channel

Large-conductance voltage-and Ca2+-gated K+ channels are negative-feedback regulators of excitability in many cell types. They are complexes of alpha subunits and of one of four types of modulatory beta subunits. These have intracellular N- and C-terminal tails and two transmembrane (TM) helices, TM1 and TM2, connected by an similar to 100-residue extracellular loop. Based on endogenous disulfide formation between engineered cysteines (Cys), we found that in beta 2 and beta 3, as in beta 1 and beta 4, TM1 is closest to alpha S1 and alpha S2 and TM2 is closest to alpha S0. Mouse beta 3 (m beta 3) has seven Cys in its loop, one of which is free, and this Cys readily forms disulfides with Cys substituted in the extracellular flanks of each of alpha S0-alpha S6. We identified by elimination m beta 3-loop Cys152 as the only free Cys. We inferred the disulfide-bonding pattern of the other six Cys. Using directed proteolysis and fragment sizing, we determined this pattern first among the four loop Cys in beta 1. These are conserved in beta 2-beta 4, which have four additional Cys (eight in total), except that m beta 3 has one fewer. In beta 1, disulfides form between Cys at aligned positions 1 and 8 and between Cys at aligned positions 5 and 6. In m. 3, the free Cys is at position 7; position 2 lacks a Cys present in all other. 2-. 4; and the disulfide pattern is 1-8, 3-4, and 5-6. Presumably, Cys 2 cross-links to Cys 7 in all other beta 2-beta 4. Cross-linking of m beta 3 Cys152 to Cys substituted in the flanks of alpha S0-S5 attenuated the protection against iberiotoxin (IbTX); cross-linking of Cys152 to K296C in the alpha S6 flank and close to the pore enhanced protection against IbTX. In no case was N-type inactivation by the N-terminal tail of m beta 3 perturbed. Although the m beta 3 loop can move, its position with Cys152 near alpha K296, in which it blocks IbTX binding, is likely favored.