The Cytochrome C-Like Domain of the Human BK Channel

Abstract

The voltage- and calcium-activated BK channel (Slo1) is modulated by small ligands that bind to its intracellular gating ring (GR) formed by four pairs of tandem homologous RCK1 and RCK2 domains. Heme binds reversibly to the GR at site 612CKACH616, a conserved heme regulatory motif (CXXCH) in the Cytochrome C (CytC) protein family, and it is located within the ∼120-residue linker connecting RCK1 and RCK2 domains. Most of this linker has thus far evaded structural definition. To gain structural insight on this functionally-significant region, we performed a sequence alignement of BK with CytC and CytC-like domains from different hemoproteins. We found that, in addition to the CXXCH motif, key structural and functional elements of CytC are conserved in the BK RCK1- RCK2 linker: firstly, the portion of the BK region resolved in the available atomic structures shares secondary structure elements with CytC proteins; secondly, CytC positively-charged residues critical for Apaf-1 and cardiolipin interaction align with BK residues K606, K623, R648, K684, K685 and K698; finally, CytC methionine-80, the second axial ligand to the heme iron, aligns with BK M691. These similarities support the premise that a CytC-like domain exists in the BK GR. To experimentally test this hypothesis, we expressed and purified this region (598IAS...LSG718) and probed its structural composition with Circular Dichroism spectroscopy. The α-helical composition of this protein increased following addition of heme (150 nM) from ≈35% to ≈51%, approaching the α-helical content of CytC (≈53%). Moreover, in the full GR, mutation M691A significantly attenuated heme-binding properties as shown by reduced Soret band formation compared to WT, suggesting that M691 is important for heme binding. These results demonstrate that BK channels possess four intracellular CytC-like domains, which may confer novel physiological functions to these ubiquitous ion channels.