Abstract
The large conductance, voltage- and calcium-activated potassium channel, BKCa, is a known target for the gasotransmitter, carbon monoxide (CO). Activation of BKCa 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 BKCa. Here, employing voltage-clamped, inside-out patches from HEK293 cells expressing single, double and triple cysteine mutations in the BKCa 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 [Ca2+]i, all mutants carrying a cysteine substitution at position 911 (C911G) demonstrated significantly reduced CO sensitivity; the C911G mutant did not express altered Ca2+-sensitivity. In contrast, histidine residues in RCK1 domain, previously shown to ablate CO activation in low [Ca2+]i, actually increased CO sensitivity when [Ca2+]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 Ca2+ bowl but which is not involved in Ca2+ activation, confers significant CO sensitivity to BKCa 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 BKCa.
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The authors would like to thank The Medical Research Council and The British Heart Foundation for financial support.
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Telezhkin, V., Brazier, S.P., Mears, R. et al. Cysteine residue 911 in C-terminal tail of human BKCaα channel subunit is crucial for its activation by carbon monoxide. Pflugers Arch - Eur J Physiol 461, 665–675 (2011). https://doi.org/10.1007/s00424-011-0924-7
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DOI: https://doi.org/10.1007/s00424-011-0924-7