Positions of the cytoplasmic end of BK α S0 helix relative to S1-S6 and of β1 TM1 and TM2 relative to S0-S6.

The large-conductance, voltage- and Ca(2+)-gated K(+) (BK) channel consists of four α subunits, which form a voltage- and Ca(2+)-gated channel, and up to four modulatory β subunits. The β1 subunit is expressed in smooth muscle, where it slows BK channel kinetics and shifts the conductance-voltage (G-V) curve to the left at [Ca(2+)] > 2 µM. In addition to the six transmembrane (TM) helices, S1-S6, conserved in all voltage-dependent K(+) channels, BK α has a unique seventh TM helix, S0, which may contribute to the unusual rightward shift in the G-V curve of BK α in the absence of β1 and to a leftward shift in its presence. Such a role is supported by the close proximity of S0 to S3 and S4 in the voltage-sensing domain. Furthermore, on the extracellular side of the membrane, one of the two TM helices of β1, TM2, is adjacent to S0. We have now analyzed induced disulfide bond formation between substituted Cys residues on the cytoplasmic side of the membrane. There, in contrast, S0 is closest to the S2-S3 loop, from which position it is displaced on the addition of β1. The cytoplasmic ends of β1 TM1 and TM2 are adjacent and are located between the S2-S3 loop of one α subunit and S1 of a neighboring α subunit and are not adjacent to S0; i.e., S0 and TM2 have different trajectories through the membrane. In the absence of β1, 70% of disulfide bonding of W43C (S0) and L175C (S2-S3) has no effect on V50 for activation, implying that the cytoplasmic end of S0 and the S2-S3 loop move in concert, if at all, during activation. Otherwise, linking them together in one state would obstruct the transition to the other state, which would certainly change V50.