Several neurotransmitters, including acetylcholine, regulate neuronal tone by suppressing a non‐inactivating low‐threshold voltage‐gated potassium current generated by the M‐channel. Agonist dependent control of the M‐channel is mediated by calmodulin, activation of anchored protein kinase C (PKC), and depletion of the phospholipid messenger phosphatidylinositol 4,5‐bisphosphate (PIP2). In this report, we show how this trio of second messenger responsive events acts synergistically and in a stepwise manner to suppress activity of the M‐current. PKC phosphorylation of the KCNQ2 channel subunit induces dissociation of calmodulin from the M‐channel complex. The calmodulin‐deficient channel has a reduced affinity towards PIP2. This pathway enhances the effect of concomitant reduction of PIP2, which leads to disruption of the M‐channel function. These findings clarify how a common lipid cofactor, such as PIP2, can selectively regulate ion channels.

M-channel inhibitors, especially XE991, are being used increasingly in animal experiments; however, insufficient characterization of XE991 at times confounds the interpretation of results when using this compound. Here, we demonstrate that XE991 and linopirdine are state-dependent inhibitors that favor the activated-subunit of neuronal Kv7/KCNQ channels. We performed patch-clamp experiments on homomeric Kv7.2 or heteromeric Kv7.2/3 channels expressed in Chinese hamster ovary cells to characterize XE991 and linopirdine. Neither inhibitor was efficacious around the resting membrane potential of cells in physiologic conditions. Inhibition of Kv7.2 and Kv7.2/3 channels by XE991 was closely related with channel activation. When the voltage dependence of activation was left-shifted by retigabine or right-shifted by the mutation, Kv7.2(R214D), the shift in half-activation voltage proportionally coincided with the shift in the half-effective potential for XE991 inhibition. Inhibition kinetics during XE991 wash-in was facilitated at depolarized potentials. Ten-minute washout of XE991 resulted in ∼30% current recovery, most of which was attributed to surface transport of Kv7.2 channels. Linopirdine also exhibited similar inhibition characteristics, with the exception of near- complete current recovery after washout at depolarized potentials. Inhibition kinetics of both XE991 and linopirdine was not as sensitive to changes in voltage as would be predicted by open- channel inhibition. Instead, they were well explained by binding to a single activated subunit. The characteristics of XE991 and linopirdine should be taken into account when these M-channel inhibitors are used in experiments.