Fig. 1.

Mutation of a single residue in TM2 systematically increases K2P channel activity. a Sequence conservation along transmembrane helix 2 (TM2 helix) computed from 66 vertebrate, insect, and nematode K2P channels, represented using WebLogo 3 ⁵³. b Current–voltage relationships obtained at pH 7.4 in X. laevis oocytes by injection of cRNA encoding wild-type (black squares) and TM2.6 mutant channels (red squares). TM2.6 mutations are indicated in red next to corresponding current traces. Insets for hTASK1, mTRAAK, mTRESK, and hTWIK1 AA represent wild-type channel currents at a reduced scale. rTWIK2 LY and hTHIK2 5RA harbor additional mutations in intracellular trafficking signals that allow increased surface expression^10,52. Each point represents the mean ± standard error of the mean, numbers in parentheses represent the number of oocytes tested for each condition. Injected cRNA amounts and incubation times are reported in Supplementary Table 1. c Relative surface expression for wild-type and TM2.6 mutant channels using flow cytometry. HA/GFP-tagged wild-type (black) and mutant K2P channels (red) were expressed in HEK cells. Relative surface expression was determined by measuring the median fluorescence intensity of HA-positive cells (labeled in non-permeabilizing conditions) relative to the median fluorescence intensity of the GFP signal (total channel content). Center lines, medians; open circles, means; box limits, 25th and 75th percentiles; whiskers, standard deviation. Kruskal–Wallis, Dunn’s multiple comparison test, p > 0.05