Abstract
Abstract
In the presence of oxygen (O2), carbon monoxide (CO) is synthesised from heme by endogenous hemeoxygenases, and is a powerful activator of BKCa channels. This transduction pathway has been proposed to contribute to cellular O2 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 BKCa-αsubunit, have on channel CO sensitivity. Mutant BKCa-αsubunits 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 BKCa channels by the CO donor, tricarbonyldichlororuthenium (II) dimer, at 100μM. In the absence of KCN the CO donor increased wild-type channel activity in a concentration-dependent manner, with an EC50 of ca. 50μM. Single cysteine point mutations of residues C820, C995 and C1028 affected neither channel characteristics nor CO EC50 values. In contrast, the CO sensitivity of the C911G mutation was significantly decreased (EC50 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 EC50 values. These data highlight that a single cysteine residue is crucial to the activation of BKCa 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.
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Brazier, S., Telezhkin, V., Mears, R., Müller, C., Riccardi, D., Kemp, P. (2009). Cysteine Residues in the C-terminal Tail of the Human BKCaα Subunit Are Important for Channel Sensitivity to Carbon Monoxide. In: Gonzalez, C., Nurse, C.A., Peers, C. (eds) Arterial Chemoreceptors. Advances in Experimental Medicine and Biology, vol 648. Springer, Dordrecht. https://doi.org/10.1007/978-90-481-2259-2_5
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DOI: https://doi.org/10.1007/978-90-481-2259-2_5
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