title: Cysteine residues in the C-terminal tail of the human BK(Ca)alpha subunit are important for channel sensitivity to carbon monoxide.
abstract: In the presence of oxygen (O(2)), carbon monoxide (CO) is synthesised from heme by endogenous hemeoxygenases, and is a powerful activator of BK(Ca) channels. This transduction pathway has been proposed to contribute to cellular O(2) sensing in rat carotid body. In the present study we have explored the role that four cysteine residues (C820, C911, C995 and C1028), located in the vicinity of the "calcium bowl" of C-terminal of human BK(Ca)-alphasubunit, have on channel CO sensitivity. Mutant BK(Ca)-alphasubunits were generated by site-directed mutagenesis (single, double and triple cysteine residue substitutions with glycine residues) and were transiently transfected into HEK 293 cells before subsequent analysis in inside-out membrane patches. Potassium cyanide (KCN) completely abolished activation of wild type BK(Ca) channels by the CO donor, tricarbonyldichlororuthenium (II) dimer, at 100microM. In the absence of KCN the CO donor increased wild-type channel activity in a concentration-dependent manner, with an EC(50) of ca. 50microM. Single cysteine point mutations of residues C820, C995 and C1028 affected neither channel characteristics nor CO EC(50) values. In contrast, the CO sensitivity of the C911G mutation was significantly decreased (EC(50) ca. 100 M). Furthermore, all double and triple mutants which contained the C911G substitution exhibited reduced CO sensitivity, whilst those which did not contain this mutation displayed essentially unaltered CO EC(50) values. These data highlight that a single cysteine residue is crucial to the activation of BK(Ca) by CO. We suggest that CO may bind to this channel subunit in a manner similar to the transition metal-dependent co-ordination which is characteristic of several enzymes, such as CO dehydrogenase.
authors.affiliation: School of Bioscience, Museum Avenue, Cardiff University, Cardiff CF11 9BX, UK. braziersp@cardiff.ac.uk
authors.forename: S P
authors.initials: SP
authors.lastname: Brazier
authors.forename: V
authors.initials: V
authors.lastname: Telezhkin
authors.forename: R
authors.initials: R
authors.lastname: Mears
authors.forename: C T
authors.initials: CT
authors.lastname: Müller
authors.forename: D
authors.initials: D
authors.lastname: Riccardi
authors.forename: P J
authors.initials: PJ
authors.lastname: Kemp
mesh_terms: D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism / Q000494:pharmacology*; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism*; D006801:Humans; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011190:Potassium Cyanide / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D011993:Recombinant Fusion Proteins; D002248:Carbon Monoxide; D003545:Cysteine; D011190:Potassium Cyanide