RT Journal Article
SR Electronic
T1 Covalent Modification of a Volatile Anesthetic Regulatory Site Activates TASK-3 (KCNK9) Tandem Pore Potassium Channels
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP mol.111.076281
DO 10.1124/mol.111.076281
A1 Kevin E. Conway
A1 Joseph F. Cotten
YR 2011
UL http://molpharm.aspetjournals.org/content/early/2011/12/06/mol.111.076281.abstract
AB TASK-3 (KCNK9) tandem pore potassium channels provide a volatile anesthetic activated and Gαq 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 TASK-3's anesthetic binding site(s). We hypothesized 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. METHODS: TASK-3 channels were transiently expressed in Fischer rat thyroid cells, and their function studied by Ussing chamber. RESULTS: NEM irreversibly activated M159C TASK-3 with minimal effect on wild-type TASK-3. NEM-modified M159C channels were resistant to inhibition by both acidic pH and active Gαq protein. M159C channels, first inhibited by Gαq protein, were more slowly activated by NEM suggesting 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 Gαq protein or acidic pH. TASK-3 wild-type/M159C dimers expressed as a single polypeptide demonstrated modification of a single Cys-159 was sufficient for TASK-3 activation, and M159F/M159C and M159W/M159C dimers provided evidence for crosstalk between subunits. CONCLUSIONS: The data are consistent with residue 159 contributing to an anesthetic regulatory site(s) and suggest volatile anesthetics, through perturbations at a single site, increase TASK-3 channel activity and disrupt its regulation by active Gαq protein, a determinant of CNS arousal and consciousness.