Long QT syndrome is an inherited disorder of cardiac repolarization caused by mutations in cardiac ion channel genes, including KVLQT1. In this study, the functional consequences of three long QT-associated missense mutations in KvLQT1 (R243C, W248R, E261K) were characterized using the Xenopus oocyte heterologous expression system and two-microelectrode voltage clamp techniques. These mutations are located in or near the intracellular linker between the S4 and S5 transmembrane domains, a region implicated in activation gating of potassium channels. The E261K mutation caused loss of function and did not interact with wildtype KvLQT1 subunits. R243C or W248R KvLQT1 subunits formed functional channels, but compared with wild-type KvLQT1 current, the rate of activation was slower, and the voltage dependence of activation and inactivation was shifted to more positive potentials. Co expression of minK and KvLQT1 channel subunits induces a slow delayed rectifier K ؉ current, I Ks , characterized by slow activation and a markedly increased magnitude compared with current induced by KvLQT1 subunits alone. Coexpression of minK with R243C or W248R KvLQT1 subunits suppressed current, suggesting that coassembly of mutant subunits with minK prevented normal channel gating. The decrease in I Ks caused by loss of function or altered gating properties explains the prolonged QT interval and increased risk of arrhythmia and sudden death associated with these mutations in KVLQT1.The slow delayed rectifier K ϩ current, I Ks , 1 is one of several outward currents that modulate repolarization of cardiac action potentials (1, 2). The channel mediating I Ks is formed by coassembly of two subunits, minK and KvLQT1 (3, 4). Mutations in KVLQT1 (also called KCNQ1) cause long QT syndrome (LQT) (5), an inherited disorder of ventricular repolarization that predisposes affected individuals to cardiac arrhythmias and sudden death (6). The physiologic consequences of some mutations have been studied by heterologous expression of KvLQT1 in Xenopus oocytes or mammalian cells (7-10). Most missense mutations in KVLQT1 cause loss of channel function when mutant subunits are expressed alone and a dominantnegative effect when coexpressed with wild-type (WT) KvLQT1 subunits. However, some mutations alter the function of Kv-LQT1 channels, and a study of their biophysical properties has led to novel insights into the structural basis of channel function. For example, the LQT-associated mutation V254M caused loss of function but accelerated activation of KvLQT1 channel current when mutant subunits were coexpressed with WT Kv-LQT1 subunits (8). V254M is located in the cytosolic loop connecting the S4 and S5 transmembrane domains of KvLQT1. Missense mutations in the S4-S5 linker have also been reported to accelerate activation and deactivation of human ether-a-go-go-related gene (HERG) channels (11). Furthermore, the S4-S5 linker determines the rate of deactivation of chimeric channels constructed from Kv2.1 and Kv3.1 (12). Together, these studies indicate th...