Cysteine Modification Alters Voltage- and Ca2+-dependent Gating of Large Conductance (BK) Potassium Channels

Abstract

The Ca²⁺-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 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 Ca²⁺-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.