Slo2.1 channels with hydrophobic substitutions of Glu275 are nonfunctional. The peak magnitude of currents recorded at the end of 0.3-s pulses to 0 mV in response to 1 and 6 mM NFA are plotted as a function of the GES hydrophobicity scale value for free energy transfer from water to membrane (Engelman et al., 1986) for the indicated Glu275 substitution (in single letter code). WT channels are indicated as “E.” I_Slo2.1 for E275D channels (“D”) represents constitutive activity (no NFA) in oocytes injected with 1 ng cRNA and recorded after 3 d (n = 5). Currents in WT channels and for all other mutant channels were measured in oocytes 2 d after injection with 10 ng of Slo2.1 cRNA (for WT channels, n = 10; for mutant channels, n = 5). Error bars indicate mean ± SEM.