Cysteine Modification Alters Voltage- and Ca(2+)-dependent Gating of Large Conductance (BK) Potassium Channels

The Ca(2+)-activated K(+) (BK) channel α-subunit contains many cysteine residues within its large COOH-terminal tail domain. To probe the function of this domain, we examined effects of cysteine-modifying reagents on channel gating. Application of MTSET, MTSES, or NEM to mSlo1 or hSlo1 channels changed the voltage and Ca(2+) dependence of steady-state activation. These reagents appear to modify the same cysteines but have different effects on function. MTSET increases I(K) and shifts the G(K)–V relation to more negative voltages, whereas MTSES and NEM shift the G(K)–V in the opposite direction. Steady-state activation was altered in the presence or absence of Ca(2+) and at negative potentials where voltage sensors are not activated. Combinations of [Ca(2+)] and voltage were also identified where P(o) is not changed by cysteine modification. Interpretation of our results in terms of an allosteric model indicate that cysteine modification alters Ca(2+) binding and the relative stability of closed and open conformations as well as the coupling of voltage sensor activation and Ca(2+) binding and to channel opening. To identify modification-sensitive residues, we examined effects of MTS reagents on mutant channels lacking one or more cysteines. Surprisingly, the effects of MTSES on both voltage- and Ca(2+)-dependent gating were abolished by replacing a single cysteine (C430) with alanine. C430 lies in the RCK1 (regulator of K(+) conductance) domain within a series of eight residues that is unique to BK channels. Deletion of these residues shifted the G(K)–V relation by >−80 mV. Thus we have identified a region that appears to strongly influence RCK domain function, but is absent from RCK domains of known structure. C430A did not eliminate effects of MTSET on apparent Ca(2+) affinity. However an additional mutation, C615S, in the Haem binding site reduced the effects of MTSET, consistent with a role for this region in Ca(2+) binding.