Dominant-Negative Control of cAMP-Dependent I _Ks Upregulation in Human Long-QT Syndrome Type 1

Abstract

Rationale:The mutation A341V in the S6 transmembrane segment of KCNQ1, the ␣-subunit of the slowly activating delayed-rectifier K ؉ (I Ks ) channel, predisposes to a severe long-QT1 syndrome with sympathetictriggered 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 ␤-adrenergic receptor stimulation, remain unclear. We aimed to elucidate these mechanisms and provide new insights into control of cAMPdependent 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. Key Words: ion channels Ⅲ long-QT syndrome Ⅲ potassium Ⅲ torsade de pointes T he slowly activating delayed-rectifier K ϩ current (I Ks ) contributes importantly to cardiac repolarization. In large mammals, including humans, it has a small amplitude under basal isolated-myocyte conditions but forms a sizable repolarization reserve that is recruited when the action potential duration (APD) prolongs and during ␤-adrenergic receptor (␤AR) stimulation. 1,2 I Ks is carried by a macromolecular channel complex consisting of a homotetramer of pore-forming ␣-subunits encoded by KCNQ1 (Kv7.1), KCNE1 ␤-subunits, 3,4 and the regulatory A-kinase anchoring protein Yotiao, which binds to the KCNQ1 C-terminus. 5 There are also multiple interactions with other proteins. During ␤AR stimulation, when cAMP levels rise, phosphorylation of KCNQ1 at N-terminal position S27 is controlled by protein kinase A (PKA) and protein phosphatase 1 that are localized to the complex by Yotiao, thereby providing local control of I Ks enhancement. 5 Anchored PKA also phosphorylates Yotiao itself, thereby further enhancing I Ks . 6 An intact C-terminus of KCNE1 is critical for the PKA-dependent upregulation of I Ks . 7 Congenital defects (long-QT syndrome types 1 and 5; LQT1, LQT5; Jervell and Lange-Nielsen syndrome), pharmacological inhibition, 8 and acquired loss of I Ks , 9 can all result in QT-interval prolongation and enhanced susceptibility to ventricular tachyarrhythmias, notably torsade de pointes. These arrhythmias occur predominantly during conditions of exerc...

Long QT mutations disrupt IKS regulation by PKA and PIP2 at the same KCNQ1 helix C-KCNE1 interface

“…The LQT1 mutation A341V in KCNQ1 segment 6 has recently been shown to preserve the KCNQ1-yotiao interaction but to reduce cAMP-induced KCNQ1 phosphorylation at S27 and subsequent I KS current upregulation (Heijman et al, 2012). The LQT5 mutation D76N in KCNE1 C-terminus spared the cAMPmediated KCNQ1 phosphorylation at S27 but failed to upregulate its resulting I KS current.…”

“…8C). To date, only one LQT1 mutation A341V in KCNQ1 (Heijman et al, 2012) has been found to disrupt KCNQ1 phosphorylation at S27, while preserving the KCNQ1-yotiao interaction. Interestingly, the LQT5 mutant P127T has been described both as a single mutation (Splawski et al, 2000) and as being additionally associated with the LQT1 mutation A341V in another proband who presented with syncope and longer QTc interval than his parents (Westenskow et al, 2004).…”

Ionic Mechanisms Underlying the Effects of Vasoactive Intestinal Polypeptide on Canine Atrial Myocardium

“…35 The observed VIP effects on repolarizing currents are consistent with the known consequences of cAMP/PKA signaling pathway activation. 36,37 However, it was unexpected that VIP suppresses I Na because activation of cAMP/PKA pathway would increase I Na .…”

Synergistic Restoring Effects of Isoproterenol and Magnesium on KCNQ1-Inhibited Bradycardia Cell Models Cultured in Microelectrode Array

“…This suggests an association with vagal mechanisms [5]. Moreover, the Iks channel is regulated by signals generated in the β-adrenergic receptor (β-AR) [6]; notably, KCNQ1 mutations impair the cellular response to β-adrenergic stimulation [7]. Also, it has been reported that β-adrenergic agonists and magnesium modulate Iks in frog cardiomyocytes [8].…”