Key result
Cysteine modification of BK channels by MTSET, MTSES, or NEM alters voltage- and Ca2+-dependent gating, with effects on Ca2+ binding and channel opening strongly influenced by the C430 residue.
Cysteine modification alters the gating of BK channels, with residue C430 playing a critical role in RCK domain function and voltage/Ca2+-dependent gating.
C430 modulates BK gating in animal models; leaves open translation to human physiology or therapeutic targeting.
The Ca(2+)-activated K+ (BK) channel alpha-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 Ca2+ 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 Ca2+ and at negative potentials where voltage sensors are not activated. Combinations of [Ca2+] 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 Ca2+ binding and the relative stability of closed and open conformations as well as the coupling of voltage sensor activation and Ca2+ 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 Ca2+ affinity. However an additional mutation, C615S, in the Haem binding site reduced the effects of MTSET, consistent with a role for this region in Ca2+ binding.
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Zhang et al. (2005) studied BK channel function. Cysteine-modifying reagents (MTSET, MTSES, NEM) vs. Unmodified or mutant channels was evaluated on Voltage- and Ca2+-dependent gating of BK channels. Cysteine modification of BK channels by MTSET, MTSES, or NEM alters voltage- and Ca2+-dependent gating, with effects on Ca2+ binding and channel opening strongly influenced by the C430 residue.
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