Probing the regulation of TASK potassium channels by PI(4,5)P2 with switchable phosphoinositide phosphatases

TASK channels are background K+ channels that contribute to the resting conductance in many neurons. A key feature of TASK channels is the reversible inhibition by Gq-coupled receptors, thereby mediating the dynamic regulation of neuronal activity by modulatory transmitters. The mechanism that mediates channel inhibition is not fully understood. While it is clear that activation of G alpha(q) is required, the immediate signal for channel closure remains controversial. Experimental evidence pointed to either phospholipase C (PLC)-mediated depletion of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P-2) as the cause for channel closure or to a direct inhibitory interaction of active G alpha(q) with the channel. Here, we address the role of PI(4,5)P-2 for G-protein-coupled receptor (GPCR)-mediated TASK inhibition by using recently developed genetically encoded tools to alter phosphoinositide (PI) concentrations in the living cell. When expressed in CHO cells, TASK-1-and TASK-3-mediated currents were not affected by depletion of plasma membrane PI(4,5)P-2 either via the voltage-activated phosphatase Ci-VSP or via chemically triggered recruitment of a PI(4,5)P-2-5'-phosphatase. Depletion of both PI(4,5)P-2 and PI(4) P via membrane recruitment of a novel engineered dual-specificity phosphatase also did not inhibit TASK currents. In contrast, each of these methods produced robust inhibition of the bona fide PI(4,5)P-2-dependent channel KCNQ4. Efficient depletion of PI(4,5)P-2 and PI(4) P was further confirmed with a fluorescent phosphoinositide sensor. Moreover, TASK channels recovered normally from inhibition by co-expressed muscarinic M1 receptors when resynthesis of PI(4,5)P-2 was prevented by depletion of cellular ATP. These results demonstrate that TASK channel activity is independent of phosphoinositide concentrations within the physiological range. Consequently, Gq-mediated inhibition of TASK channels is not mediated by depletion of PI(4,5)P-2.