Abstract

TWIK-related spinal cord K⁺ channel (TRESK) is the most recently cloned two-pore-domain potassium (2PK⁺) channel, regulated by the calcium/calmodulin-dependent protein phosphatase calcineurin. Functional identification of endogenous TRESK and its distinction from the other 2PK⁺ channels, producing similar background K⁺ current, are impeded by the lack of specific inhibitors. Therefore, we searched for antagonists selective against TRESK among the mouse 2PK⁺ channels by screening more than 200 substances. Mibefradil, zinc, and mercuric ions inhibited TRESK expressed in Xenopus laevis oocytes with IC₅₀ values lower than 10 μM. The specificity of the identified agents was determined by measuring their effects on mouse TALK-1, TASK-1, TASK-2, TASK-3, THIK-1, TRAAK, TREK-1, and TREK-2. Mibefradil failed to discriminate well among the functional 2PK⁺ channels; however, Zn²⁺ and Hg²⁺ exerted a significantly stronger inhibitory effect on TRESK than on the other channels. Sensitivity to zinc but insensitivity to ruthenium red were distinctive features of TRESK. Whereas both Zn²⁺ and Hg²⁺ were selective blockers of TRESK among the mouse 2PK⁺ channels, human TRESK was resistant to Zn²⁺; it was blocked only by Hg²⁺. His132 of mouse TRESK was partly responsible for this difference. Mouse TRESK expressed in COS-7 cells was also inhibited by Zn²⁺ and Hg²⁺, and TRESK single-channel current was diminished in outside-out patches, indicating that the action of the ions was membrane-delimited, most probably targeting the channel itself. Thus, both Zn²⁺ and Hg²⁺ are expected to inhibit endogenous TRESK in isolated mouse cells, and these ions can be applied to identify the calcineurin-activated 2PK⁺ channel in its natural environment.