Cysteine residue 911 in C-terminal tail of human BKCaα 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).

Heme Sensor Proteins

“…However, how heme binds and whether CO stimulates K ϩ efflux in a heme-dependent or heme-independent manner has still to be established. A cytochrome c-type CXXCH motif is present rather than the typical HRM (45)(46)(47)(48). Further studies are also required to establish the physiological relevance of the apparent heme-mediated inhibition of the arginine transferase activity of R-transferase and of putative heme binding to stanniocalcin glycoprotein hormones (52)(53)(54).…”

“…Studies on a peptide containing the CXXCH motif (which binds heme by His coordination) led to a conclusion that heme is the CO-binding site (45). However, mutating the CXXCH motif His did not diminish the effect of CO on channel permeability (despite heme loss), possibly because CO might also interact with an unidentified metal cluster (46,47). It was also proposed that BK channels do not bind heme but that CXXCH motif cysteines form a thiol/disulfide redox switch, similar to that proposed for Rev-erb␤ (48).…”

Relaxant Effect of a Water Soluble Carbon Monoxide-Releasing Molecule (CORM-3) on Spontaneously Hypertensive Rat Aortas

“…Moreover, a recent report suggests the involvement of ATP-dependent (K ATP ) potassium channels as part of the dilatatory mechanisms exerted by CORM-3 [13]. However, other authors report the central role of the KCa,α in CO effect [25,28,29].…”

T-Type Ca2+ Channel Regulation by CO: A Mechanism for Control of Cell Proliferation

“…A number of research groups, including our own, have demonstrated that specific ion channels are targets of regulation by CO, and that via ion channel modulation CO exerts many of its important physiological and pathological actions (Williams et al 2004;Jaggar et al 2005;Telezhkin et al 2011;Scragg et al 2008;Dallas et al 2012;Peers et al 2014). In the present study, we have examined whether T-type Ca 2+ channels represent another target for modulation by CO and, if so, how this might impact on VSMC proliferation.…”