Heme signaling through the cytochrome c-like domain of the human BK channel

Large-conductance voltage- and Ca²⁺-activated potassium channels (BK channels) are key regulators of cellular excitability and are modulated by various signaling molecules, including heme. However, direct evidence for heme binding to BK channels is limited, and the mechanisms underlying heme-dependent signaling remain poorly understood. The cytochrome c-like (Cytcl) domain inside gating ring (GR) apparatus of the human BK channel is thought to mediate heme signaling. In addition to the heme-sensing motif ⁶¹²CKACH⁶¹⁶, residue Met691 may also facilitate heme recognition by the Cytcl domain. However, its precise role as a heme-sensing residue remains unclear. Analysis of the absorption spectra revealed that the Soret signal at 410 nm was significantly reduced in the C615S/H616R and M691A mutants compared to the wild type, and the characteristic β/α band peaks at 525/560 nm were absent in the mutant proteins. We found that the GR of the BK channel binds heme and undergoes conformational changes with high affinity (Kd ≈ 125 nM). In contrast, the Met691A mutant protein exhibited significantly reduced sensitivity to heme, with a higher Kd value of approximately 841 nM. Intracellular patch-clamp recordings on wild-type and Met691A channels expressed in Xenopus oocytes revealed that the Met691A mutation abolishes heme-induced inhibition of BK channel activity. This study demonstrates that heme functions as a native ligand for the GR machinery of the human BK channel, which can coordinate through residues ⁶¹²CKACH⁶¹⁶ and Met691 within the Cytcl domain. Formation of this interaction between heme and the Cytcl domain will lead to a substantial structural transition within the GR, which may represent heme-mediated signaling mechanisms of the human BK channel.