Rationale: The mutation A341V in the S6 transmembrane segment of KCNQ1 , the α-subunit of the slowly activating delayed-rectifier K + (I Ks ) channel, predisposes to a severe long-QT1 syndrome with sympathetic-triggered ventricular tachyarrhythmias and sudden cardiac death. Objective: Several genetic risk modifiers have been identified in A341V patients, but the molecular mechanisms underlying the pronounced repolarization phenotype, particularly during β-adrenergic receptor stimulation, remain unclear. We aimed to elucidate these mechanisms and provide new insights into control of cAMP-dependent modulation of I Ks . Methods and Results: We characterized the effects of A341V on the I Ks macromolecular channel complex in transfected Chinese hamster ovary cells and found a dominant-negative suppression of cAMP-dependent Yotiao-mediated I Ks upregulation on top of a dominant-negative reduction in basal current. Phosphomimetic substitution of the N-terminal position S27 with aspartic acid rescued this loss of upregulation. Western blot analysis showed reduced phosphorylation of KCNQ1 at S27, even for heterozygous A341V, suggesting that phosphorylation defects in some (mutant) KCNQ1 subunits can completely suppress I Ks upregulation. Functional analyses of heterozygous KCNQ1 WT:G589D and heterozygous KCNQ1 WT:S27A, a phosphorylation-inert substitution, also showed such suppression. Immunoprecipitation of Yotiao with KCNQ1 -A341V (in the presence of KCNE1 ) was not different from wild-type. Conclusions: Our results indicate the involvement of the KCNQ1 -S6 region at/or around A341 in cAMP-dependent stimulation of I Ks , a process that is under strong dominant-negative control, suggesting that tetrameric KCNQ1 phosphorylation is required. Specific long-QT1 mutations, including heterozygous A341V, disable this regulation.