title: Covalent modification of a volatile anesthetic regulatory site activates TASK-3 (KCNK9) tandem-pore potassium channels.
abstract: TASK-3 (KCNK9) tandem-pore potassium channels provide a volatile anesthetic-activated and Gα(q) protein- and acidic pH-inhibited potassium conductance important in neuronal excitability. Met-159 of TASK-3 is essential for anesthetic activation and may contribute to the TASK-3 anesthetic binding site(s). We hypothesized that covalent occupancy of an anesthetic binding site would irreversibly activate TASK-3. We introduced a cysteine at residue 159 (M159C) and studied the rate and effect of Cys-159 modification by N-ethylmaleimide (NEM), a cysteine-selective alkylating agent. TASK-3 channels were transiently expressed in Fischer rat thyroid cells, and their function was studied in an Ussing chamber. NEM irreversibly activated M159C TASK-3, with minimal effects on wild-type TASK-3. NEM-modified M159C channels were resistant to inhibition by both acidic pH and active Gα(q) protein. M159C channels that were first inhibited by Gα(q) protein were more-slowly activated by NEM, which suggests protection of Cys-159, and similar results were observed with isoflurane activation of wild-type TASK-3. M159W and M159F TASK-3 mutants behaved like NEM-modified M159C channels, with increased basal currents and resistance to inhibition by active Gα(q) protein or acidic pH. TASK-3 wild-type/M159C dimers expressed as a single polypeptide demonstrated that modification of a single Cys-159 was sufficient for TASK-3 activation, and M159F/M159C and M159W/M159C dimers provided evidence for cross-talk between subunits. The data are consistent with residue 159 contributing to an anesthetic regulatory site or sites, and they suggest that volatile anesthetics, through perturbations at a single site, increase TASK-3 channel activity and disrupt its regulation by active Gα(q) protein, a determinant of central nervous system arousal and consciousness.
authors.affiliation: Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
authors.forename: Kevin E
authors.initials: KE
authors.lastname: Conway
authors.forename: Joseph F
authors.initials: JF
authors.lastname: Cotten
mesh_terms: D018685:Anesthetics, Inhalation / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D005033:Ethylmaleimide / Q000494:pharmacology; D024683:Potassium Channels, Tandem Pore Domain / Q000502:physiology*; D051381:Rats; D011916:Rats, Inbred F344; D013961:Thyroid Gland / Q000187:drug effects
chemical_list: D018685:Anesthetics, Inhalation; C407082:Kcnk9 protein, rat; D024683:Potassium Channels, Tandem Pore Domain; D005033:Ethylmaleimide