Carbon Monoxide May Regulate BK slo1 Channel Activity by Partially Disrupting Heme Coordination

The putative gasotransmitter CO is reported to activate large-conductance K+ channels (BK); however, the mechanism remains controversial. We recently found evidence that M691, within the RCK1-RCK2 linker of the BK C-terminus, coordinates heme together with the distal, conserved heme regulatory motif (HRM), acting as a second axial ligand to the heme Fe atom. A similar binding configuration is observed in cytochrome c (CytC). Based on this and other lines of evidence, we have proposed that BK channels possess a CytC-like domain (BKCytCD). In CytC, CO associates with heme by cleaving the Met80 S-Fe2+ bond; in this work, we are testing the hypothesis that BK channels bind CO by a similar mechanism. We expressed and purified the intracellular C-terminal portion of the human BK channel (gating ring, GR). We first strengthened the evidence for the participation of M691 in heme coordination by detecting an absorption peak at 690nm, characteristic of the Met-Fe2+ bond, which was eliminated by either mutation M691A or HRM disruption (C615S/H616R). We then probed for CO-induced GR conformational rearrangements using the 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescent label in the presence of heme: the wild-type GR/heme complex exhibited strong ANS fluorescence reduction in response to CO, reporting structural transitions likely underlying CO regulation of BK conductance. The GR-M691A/heme complex was unresponsive up to 1.2 μM of CO. However, in the presence of CO-releasing CORM-2 ([RuCO3CI2]2), the GR-M691A/heme complex exhibited β/α absorption bands (540, 570nm) characteristic of CO binding. We propose that CO regulates BK conductance by inducing conformational rearrangements in its GR ligand-sensing domain. Specifically, CO substitutes M691 as the second heme axial ligand; cleavage of the M691-heme bond partially unfolds the BKCytCD, generating the optically-tracked conformational changes. This action may relieve heme-induced inhibition of BK opening.