Effects of E119, D123, D130, and D185 mutations on Zn²⁺ potency. (A) Normalized Zn²⁺ concentration responses are shown for E119H-H140A, E119H-H193A, E119H-H140A-H193A, and E119A-H193A. Colored lines represent fits of the mean data to a Hill function: E119H-H140A (solid magenta line, IC₅₀ = 0.67 µM, n_H = 1.2), E119H-H193A (solid green line, IC₅₀ = 4.2 µM, n_H = 0.7), E119H-H140A-H193A (solid navy blue line, IC₅₀ = 7.6 µM, n_H = 0.7), and E119A-H193A (solid black line, IC₅₀ = 273.7 µM, n_H = 0.8). Dashed lines represent fits to Hv1 E119H, E119A, and H140A-H193A, as shown in Fig. 3. (B) Normalized Zn²⁺ concentration responses are shown for D123H, D123H-H140A, and D123H-H193A. Colored lines represent fits of the mean data to a Hill function: D123H (solid gray line, IC₅₀ = 2.5 µM, n_H = 0.8), D123H-H140A (solid purple line, IC₅₀ = 20.5 µM, n_H = 0.5), and D123H-H193A (solid pink line, IC₅₀ = 28.6 µM, n_H = 0.9). In B–D, dashed lines represent fits to WT Hv1, H140A, and H193A data, as shown in Fig. 2. (C) Normalized Zn²⁺ concentration responses are shown for D130H, D130H-H140A, and D130H-H193A. Colored lines represent fits of the mean data to a Hill function: D130H (solid olive line, IC₅₀ = 2.4 µM, n_H = 0.7), D130H-H140A (solid pink line, IC₅₀ = 39.7 µM, n_H = 0.5), and D130H-H193A (solid aqua line, IC₅₀ = 5.6 µM, n_H = 0.6). (D) Normalized Zn²⁺ concentration responses are shown for D185H, H140A-D185H, D185H-H193A, and D185A-H193A. Colored lines represent fits of the mean data to a Hill function: D185H (solid cyan line, IC₅₀ = 6.5 µM, n_H = 0.6), H140A-D185H (solid pink line, IC₅₀ = 18.0 µM, n_H = 0.6), D185H-H193A (solid green line, IC₅₀ = 26.7 µM, n_H = 0.6), and D185A-H193A (solid royal blue line, IC₅₀ = 99.2 µM, n_H = 0.7). Data points represent means ± SEM.