title: Cysteine residue 911 in C-terminal tail of human BK(Ca)α channel subunit is crucial for its activation by carbon monoxide.
abstract: The large conductance, voltage- and calcium-activated potassium channel, BK(Ca), is a known target for the gasotransmitter, carbon monoxide (CO). Activation of BK(Ca) by CO modulates cellular excitability and contributes to the physiology of a diverse array of processes, including vascular tone and oxygen-sensing. Currently, there is no consensus regarding the molecular mechanisms underpinning reception of CO by the BK(Ca). Here, employing voltage-clamped, inside-out patches from HEK293 cells expressing single, double and triple cysteine mutations in the BK(Ca) α-subunit, we test the hypothesis that CO regulation is conferred upon the channel by interactions with cysteine residues within the RCK2 domain. In physiological [Ca(2+)](i), all mutants carrying a cysteine substitution at position 911 (C911G) demonstrated significantly reduced CO sensitivity; the C911G mutant did not express altered Ca(2+)-sensitivity. In contrast, histidine residues in RCK1 domain, previously shown to ablate CO activation in low [Ca(2+)](i), actually increased CO sensitivity when [Ca(2+)](i) was in the physiological range. Importantly, cyanide, employed here as a substituent for CO at potential metal centres, occluded activation by CO; this effect was freely reversible. Taken together, these data suggest that a specific cysteine residue in the C-terminal domain, which is close to the Ca(2+) bowl but which is not involved in Ca(2+) activation, confers significant CO sensitivity to BK(Ca) channels. The rapid reversibility of CO and cyanide binding, coupled to information garnered from other CO-binding proteins, suggests that C911 may be involved in formation of a transition metal cluster which can bind and, thereafter, activate BK(Ca).
authors.affiliation: Division of Pathophysiology & Repair, School of Biosciences, Museum Avenue, Cardiff University, UK.
authors.forename: Vsevolod
authors.initials: V
authors.lastname: Telezhkin
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Brazier
authors.forename: Ruth
authors.initials: R
authors.lastname: Mears
authors.forename: Carsten T
authors.initials: CT
authors.lastname: Müller
authors.forename: Daniela
authors.initials: D
authors.lastname: Riccardi
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Kemp
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002248:Carbon Monoxide / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011190:Potassium Cyanide / Q000494:pharmacology
chemical_list: C494841:KCNMA1 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D002248:Carbon Monoxide; D003545:Cysteine; D011190:Potassium Cyanide; D002118:Calcium