Substitutions of the pore helix residue Phe240 induce a spectrum of constitutive channel activity. (A) NFA further activates F240L and F240V channels, but not F240C channel currents. Currents were elicited with 300-ms pulses to 0 mV from a V_h of −80 mV. Arrows indicate 0 current level. (B) Relationship between constitutive channel activity of Phe240 mutant channels and hydrophilicity of the introduced residue (indicated by single letter code). I_c-rel for WT (“F”) and mutant channels (n = 4–12) are plotted as a function of solvent parameter (hydrophilicity) values (Levitt, 1976) for the indicated Phe240 substitution. The line represents a linear regression fit of data (adjusted R² = 0.41). (C) I-V_t relationships for F240C Slo2.1 channel currents measured from oocytes bathed in KCM211 or K104 extracellular solution in the absence and presence of 1 mM NFA (n = 6). (D) I-V_t relationships for WT and F240C Slo2.1 channel currents recorded before and after maximal increase in outward currents induced by NaCl loading from recording pipettes (n = 9). Error bars indicate mean ± SEM.