Pharmacological and functional regulation of two-pore domain potassium channels

Abstract

Two pore domain potassium (K2P) channels underlie the background potassium leak currents of excitable cells. In this study, the whole cell patch clamp technique was used with transiently transfected human embryonic kidney cells, and cerebellar granule neurones (CGNs) in primary culture, to compare the pharmacological properties of acid sensitive K2P channels. Zn2+, La3+, Cu2+, ruthenium red and Ru-360 blocked TASK-1 and TASK-3 channel currents. Substitution of external sodium ions with N-methyl-D-glucamine and choline also caused a significant reduction in TASK-1 and TASK-3 currents, demonstrating that maximal conductance through these potassium channels requires the presence of external sodium ions. Whilst Cu2+ blocked TASK-1 and TASK-3 channel currents, TASK-2 currents were not affected by the ion. Mannitol, a scavenger of hydroxyl radicals, did not alter Cu2+ block of TASK-3 currents showing hydroxyl radical production was not the underlying mechanism. Application of thiol oxidant, DTNB (5’,5’-dithio-bis(2 nitrobenzoic acid)), showed a potent block, mimicking that of Cu2+ in size and reversibility. DTNB and Cu2+ block were reversed by disulphide-reducing DTT (dithiothreitol), suggesting thiol rich cysteine residues played a fundamental role in <y, TASK-3 current block by Cu . The standing-outward, voltage-insensitive potassium current in CGNs also showed DTNB and copper sensitivity. Substitutions of TASK-3 cysteine residues to alanine and serine retained copper sensitivity while whole cell current amplitude diminished and a sensitivity to alkaline pH (8.4) was introduced to TASK-3. Point mutation of cysteine 110 was found to be key in facilitating the pH 8.4 potentiation of current. Cu2+ and DTNB were applied to a TASK-2/TASK-3 chimera channel where a robust, albeit reduced, block was observed. The central role of the TASK channels in neuronal excitability is demonstrated by their extensive physiological and cross-species distribution and varied mechanisms of regulation. In this study, the interaction of essential trace element Cu2+ was shown to be a significant mechanism of TASK regulation.