Most voltage-gated potassium (Kv) channels undergo C-type inactivation during sustained depolarization. The voltage dependence and other mechanistic aspects of this process are debated, and difficult to elucidate because of concomitant voltage-dependent activation. Here, we demonstrate that MinK-KCNQ1 (I(Ks)) channels with an S6-domain mutation, F340W in KCNQ1, exhibit constitutive activation but voltage-dependent C-type inactivation. F340W-I(Ks) inactivation was sensitive to extracellular cation concentration and species, and it altered ion selectivity, suggestive of pore constriction. The rate and extent of F340W-I(Ks) inactivation and recovery from inactivation were voltage-dependent with physiologic intracellular ion concentrations, and in the absence or presence of external K(+), with an estimated gating charge, z(i), of approximately 1. Finally, double-mutant channels with a single S4 charge neutralization (R231A,F340W-I(Ks)) exhibited constitutive C-type inactivation. The results suggest that F340W-I(Ks) channels exhibit voltage-dependent C-type inactivation involving S4, without the necessity for voltage-dependent opening, allosteric coupling to voltage-dependent S6 transitions occurring during channel opening, or voltage-dependent changes in ion occupancy. The data also identify F340 as a critical hub for KCNQ1 gating processes and their modulation by MinK, and present a unique system for further mechanistic studies of the role of coupling of C-type inactivation to S4 movement, without contamination from voltage-dependent activation.