title: Identification of a thiol/disulfide redox switch in the human BK channel that controls its affinity for heme and CO.
abstract: Heme is a required prosthetic group in many electron transfer proteins and redox enzymes. The human BK channel, which is a large-conductance Ca(2+) and voltage-activated K(+) channel, is involved in the hypoxic response in the carotid body. The BK channel has been shown to bind and undergo inhibition by heme and activation by CO. Furthermore, evidence suggests that human heme oxygenase-2 (HO2) acts as an oxygen sensor and CO donor that can form a protein complex with the BK channel. Here we describe a thiol/disulfide redox switch in the human BK channel and biochemical experiments of heme, CO, and HO2 binding to a 134-residue region within the cytoplasmic domain of the channel. This region, called the heme binding domain (HBD) forms a linker segment between two Ca(2+)-sensing domains (called RCK1 and RCK2) of the BK channel. The HBD includes a CXXCH motif in which histidine serves as the axial heme ligand and the two cysteine residues can form a reversible thiol/disulfide redox switch that regulates affinity of the HBD for heme. The reduced dithiol state binds heme (K(d) = 210 nm) 14-fold more tightly than the oxidized disulfide state. Furthermore, the HBD is shown to tightly bind CO (K(d) = 50 nm) with the Cys residues in the CXXCH motif regulating affinity of the HBD for CO. This HBD is also shown to interact with heme oxygenase-2. We propose that the thiol/disulfide switch in the HBD is a mechanism by which activity of the BK channel can respond quickly and reversibly to changes in the redox state of the cell, especially as it switches between hypoxic and normoxic conditions.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Yi
authors.forename: Jeffrey T
authors.initials: JT
authors.lastname: Morgan
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Ragsdale
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002248:Carbon Monoxide / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005290:Ferric Compounds / Q000378:metabolism; D005296:Ferrous Compounds / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D004220:Disulfides; D005290:Ferric Compounds; D005296:Ferrous Compounds; C494841:KCNMA1 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D013438:Sulfhydryl Compounds; D006418:Heme; D002248:Carbon Monoxide; D006419:Heme Oxygenase (Decyclizing); C105245:heme oxygenase-2; D003545:Cysteine