Inhibition of KCNQ2/3 channels by HN38 and XE991 depends on the conformation of the outer vestibule

Recent studies identified HN38 as a novel KCNQ2 channel inhibitor. However, to date no study has carefully examined HN38 in regards to its mechanism of action or determined whether it inhibits KCNQ2/3 channels. To address these questions, we used heterologous expression of human KCNQ2/3 channels in HEK293T cells. Consistent with previous reports, we found that HN38 almost completely blocked KCNQ2 channel activity. This inhibition was independent of the presence of the KCNQ1-5 auxiliary neuronal subunit beta-secretase 1 (BACE-1). Similar to its parent compound, retigabine, HN38 required the presence of KCNQ2 tryptophan W236 for inhibition. Surprisingly, we found that HN38 maximally inhibited KCNQ2/3 channels, as well as the KCNQ2/3-mediated M-current in CA1 pyramidal neurons, by approximately 40%. This incomplete block of KCNQ2/3 channels by HN38 appears to be partially due to the conformation of the KCNQ2/3 outer vestibule and in particular the outer turret lysine 259 of KCNQ3 channels. We conclude that the KCNQ3 outer vestibule conformation regulates the ability of blockers, like HN38 as well as XE991, to inhibit KCNQ2/3 channels, which should be considered for the design of new KCNQ2/3 channels compounds.