The slow delayed rectifier K(+) current (I(Ks)) is a major determinant of action potential repolarization in the heart. I(Ks) channels are formed by coassembly of pore-forming KCNQ1 alpha-subunits and ancillary KCNE1 beta-subunits. Two gain of function mutations in KCNQ1 subunits (S140G and V141M) have been associated with atrial fibrillation (AF). Previous heterologous expression studies found that both mutations caused I(Ks) to be instantaneously activated, presumably by preventing channel closure. The purpose of this study was to refine our understanding of the channel gating defects caused by these two mutations located in the S1 domain of KCNQ1. Site-directed mutagenesis was used to replace S140 or V141 with several other natural amino acids. Wild-type and mutant channels were heterologously expressed in Xenopus oocytes and channel function was assessed with the two-microelectrode voltage clamp technique. Long intervals between voltage clamp pulses revealed that S140G and V141M KCNQ1-KCNE1 channels are not constitutively active as previously reported, but instead exhibit extremely slow deactivation. The slow component of I(Ks) deactivation was decreased 62-fold by S140G and 140-fold by the V141M mutation. In addition, the half-point for activation of these mutant I(Ks) channels was approximately 50 mV more negative than wild-type channels. Other substitutions of S140 or V141 in KCNQ1 caused variable shifts in the voltage dependence of activation, but slowed I(Ks) deactivation to a much lesser extent than the AF-associated mutations. Based on a published structural model of KCNQ1, S140 and V141 are located near E160 in S2 and R237 in S4, two charged residues that could form a salt bridge when the channel is in the open state. In support of this model, mutational exchange of E160 and R237 residues produced a constitutively open channel. Together our findings suggest that altered charge-pair interactions within the voltage sensor module of KCNQ1 subunits may account for slowed I(Ks) deactivation induced by S140 or V141.