Cysteine modification alters voltage- and Ca(2+)-dependent gating of large conductance (BK) potassium channels.
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.
Zhang
Guangping
G
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Horrigan
Frank T
FT
D000818:Animals; D002118:Calcium / Q000493:pharmacokinetics; D003545:Cysteine / Q000378:metabolism*; D004594:Electrophysiology; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D015640:Ion Channel Gating / Q000502:physiology*; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008698:Mesylates / Q000494:pharmacology; D051379:Mice; D024681:Potassium Channels, Calcium-Activated / Q000235:genetics / Q000502:physiology*
D007202:Indicators and Reagents; C465449:Kcnma1 protein, mouse; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008698:Mesylates; D024681:Potassium Channels, Calcium-Activated; C109153:(2-sulfonatoethyl)methanethiosulfonate; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D003545:Cysteine; D002118:Calcium