Fig. 3.

Point mutations of Leu-209 alter constitutive activity of Slo2.1 channels. (A) Slo2.2-based homology model of the S5-S6 region of a Slo2.1 subunit, highlighting the close proximity of Leu-209 in S5 and Phe-240 at the base of the pore helix (PH). (B) [NFA]-response relationships for wt (same data as in Fig. 1D) and three Leu-209 mutant channels. For L209A channels, EC₅₀ = 1.4 ± 0.3 mM, n_H = 1.6 ± 0.5 (n = 5). For L209N channels, EC₅₀ = 0.28 ± 0.04 mM, n_H = 0.60 ± 0.09 (n = 6). L209E channels do not respond to 1 or 6 mM NFA (n = 5), indicating that these mutant channels are maximally activated. (C–E) Representative currents (upper panels) recorded from individual oocytes expressing indicated mutant Slo2.1 channels under control conditions and I–V_t relationships (lower panels) for multiple oocytes before and after application of 1 and 6 mM NFA (L209A, n = 8; L209N, n = 5; L209E, n = 6). (F) Peak outward current measured at 0 mV in the absence (control) and presence of 1 and 6 mM NFA for channels containing Leu-209 substitutions as indicated on x-axis (n = 5–10). Amino acid substitutions are listed from left to right in order of decreasing hydrophobicity. (G) The variation in normalized constitutive current (I_c-rel) is contour plotted (z-axis) as a function of volume and membrane propensity of the residue substituted for Leu-209. The aa volume (x-axis) represents the average volume of a residue buried in a protein [1]. The amino acid membrane propensity (y-axis) is a knowledge-based hydropathy scale derived from an extensive transmembrane helix database and optimization algorithm described in detail by Punta and Maritan [4], where a negative value indicates a high membrane propensity. The native residue, Leu-209 (L) is indicated in red text. Error bars indicate ± S.E. (panels C–E) or + S.E. (panel F).