V659 couples voltage sensing and gating. (A) Conductance–voltage relation for V659C mutant fit with a Boltzmann equation reveals constitutive conductance over negative potentials and shallower slope (z). (B) Scaled V659C tail currents evoked at −100 mV after a pulse to +60 mV show dramatically slowed deactivation. Dotted line indicates zero current level. (C) Plot of time constants obtained from fits to deactivating currents on a log scale plotted as a function of voltage for V659C. (D) Steady-state current versus voltage relations for a variety of substitutions made at V659. Mutations to A, F, or T were fit with a standard Boltzmann function and show constitutive conductance and shallower slope. V659I shows an intermediate phenotype that was fit with a double Boltzmann equation (see Materials and methods). n = 3–5 for each data point. (E) Scaled V659 mutant tail currents evoked at −100 mV after pulses to +60 mV. (F) Average deactivation time constants on a log scale versus voltage for V659 mutants. n = 3–5 for each data point. (G) Deactivation time constants versus side chain volume for V659 mutants. Amino acids with side chains significantly larger or smaller than the native valine show slowed deactivation. Nonfunctional mutants (V659D, V659K, V659N, and V659P) are included as open triangles above the line at the top of the y axis at appropriate side chain volume. Side chain volume obtained from Zamyatnin (1972). (H) Deactivation time constant versus hydrophobicity of side chains substituted at V659. Nonfunctional mutants (V659D, V659K, V659N, and V659P) are included as open triangles at the top of the y axis at the x axis location of appropriate hydrophobicity. Hydrophobicity scale adapted from Black and Mould (1991).