Abstract-Nearly a hundred different KCNQ1 mutations have been reported as leading to the cardiac long QT syndrome, characterized by prolonged QT interval, syncopes, and sudden death. We have previously shown that phosphatidylinositol-4,5-bisphosphate (PIP 2 ) regulates the KCNQ1-KCNE1 complex. In the present study, we show that PIP 2 affinity is reduced in three KCNQ1 mutant channels (R243H, R539W, and R555C) associated with the long QT syndrome. In giant excised patches, direct application of PIP 2 on the cytoplasmic face of the three mutant channels counterbalances the loss of function. Reintroduction of a positive charge by application of methanethiosulfonate ethylammonium on the cytoplasmic face of R555C mutant channels also restores channel activity. The channel affinity for a soluble analog of PIP 2 is decreased in the three mutant channels. By using a model that describes the KCNQ1-KCNE1 channel behavior and by fitting the relationship between the kinetics of deactivation and the current amplitude obtained in whole-cell experiments, we estimated the PIP 2 binding and dissociation rates on wild-type and mutant channels. The dissociation rate of the three mutants was higher than for the wild-type channel, suggesting a decreased affinity for PIP 2 . PIP 2 binding was magnesium-dependent, and the PIP 2 -dependent equilibrium constant in the absence of magnesium was higher with the wild-type than with the mutant channels. Altogether, our data suggest that a reduced PIP 2 affinity of KCNQ1 mutants can lead to the long QT syndrome. Key Words: KCNQ1 Ⅲ KCNE1 Ⅲ phosphatidylinositol-4,5-bisphosphate Ⅲ long QT syndrome K CNQ1 is the pore-forming subunit of the channel complex (composed of KCNQ1 and its regulator KCNE1) underlying the delayed rectifier potassium current I Ks , a key modulator of the action potential duration in the human heart. 1,2 Nearly 100 KCNQ1 mutations have been reported to cause the long QT syndrome, a genetic disease characterized by prolonged cardiac repolarization, cardiac arrhythmias, and a high risk of sudden death. 3,4 Phosphaditylinositol-4,5-bisphosphate (PIP 2 ) is an important intracellular regulator of many ion channels and transporters. [5][6][7][8] We showed recently that intracellular PIP 2 regulates KCNQ1-KCNE1 channel activity in such a way that PIP 2 stabilizes the open state of the channels, leading to an increased current amplitude, slowed deactivation kinetics, and a shift in the activation curve toward negative potentials. 9 In general, it is known that positively charged amino acids are implicated in channel-PIP 2 interactions. 10 -13 Because various KCNQ1 mutations producing neutralization of a residue in an intracellular segment of the channel protein lead to reduced current amplitude, accelerated deactivation, and shift in the activation curve toward positive potentials, 14,15 we hypothesized that mutations neutralizing a positive charge (eg, R243H, R539W, and R555C) may act through an impaired channel-PIP 2 interaction. We thus compared the effects of PIP 2 on wild-typ...