Jie Wu1, Tomoko Sakaguchi2, Kotoe Takenaka3, Futoshi Toyoda4, Keiko Tsuji2, Hiroshi Matsuura4, Minoru Horie5. 1. Department of Pharmacology, School of Basic Medical Science, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi, China; Department of Cardiovascular and Respiratory Medicine, Shiga University of Medical Science, Otsu, Japan; Department of Physiology, Shiga University of Medical Science, Otsu, Japan. 2. Department of Cardiovascular and Respiratory Medicine, Shiga University of Medical Science, Otsu, Japan. 3. Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan. 4. Department of Physiology, Shiga University of Medical Science, Otsu, Japan. 5. Department of Cardiovascular and Respiratory Medicine, Shiga University of Medical Science, Otsu, Japan. Electronic address: livedoor629@gmail.com.
Abstract
BACKGROUND: KCNQ1-T587M is a C-terminal mutation correlated with severe phenotypes of long QT syndrome (LQTS). However, functional analysis of KCNQ1 channels with the T587M mutation showed a mild genotype in the form of haploinsufficiency in a heterologous expression system. This study sought to explore the molecular mechanism underlying the phenotype-genotype dissociation of LQTS patients carrying the KCNQ1-T587M mutation. METHODS: cDNAs for wild-type (WT) and KCNQ1 mutations (R259C and T587M) were transiently transfected into HEK293 cells stably expressing hERG (hERG-HEK), and whole-cell patch-clamp technique was performed to examine the effect of KCNQ1 mutations on IKr-like currents. In addition, fluorescence resonance energy transfer (FRET) was conducted to demonstrate the molecular interaction between KCNQ1 and hERG when co-expressed in HEK293 cells. RESULTS: KCNQ1-T587M mutation produced a significant (p<0.01) decrease in IKr-like tail current densities without affecting the gating kinetics, while KCNQ1-R259C mutation had no significant effect on the IKr-like tail current densities. Consistent with this result, FRET experiments demonstrated that both KCNQ1-WT and -R259C interacted with hERG in the cytosol and on the plasma membrane; however, the interaction between KCNQ1-T587M and hERG was observed only in the cytosol, and hERG proteins were seldom transported to the cell membrane, suggesting that the KCNQ1-T587M mutation impaired the trafficking of hERG to the cell membrane. CONCLUSIONS: The disruption of hERG trafficking caused by the KCNQ1-T587M mutation is likely the reason why some patients exhibit severe LQTS phenotypes.
BACKGROUND:KCNQ1-T587M is a C-terminal mutation correlated with severe phenotypes of long QT syndrome (LQTS). However, functional analysis of KCNQ1 channels with the T587M mutation showed a mild genotype in the form of haploinsufficiency in a heterologous expression system. This study sought to explore the molecular mechanism underlying the phenotype-genotype dissociation of LQTS patients carrying the KCNQ1-T587M mutation. METHODS: cDNAs for wild-type (WT) and KCNQ1 mutations (R259C and T587M) were transiently transfected into HEK293 cells stably expressing hERG (hERG-HEK), and whole-cell patch-clamp technique was performed to examine the effect of KCNQ1 mutations on IKr-like currents. In addition, fluorescence resonance energy transfer (FRET) was conducted to demonstrate the molecular interaction between KCNQ1 and hERG when co-expressed in HEK293 cells. RESULTS:KCNQ1-T587M mutation produced a significant (p<0.01) decrease in IKr-like tail current densities without affecting the gating kinetics, while KCNQ1-R259C mutation had no significant effect on the IKr-like tail current densities. Consistent with this result, FRET experiments demonstrated that both KCNQ1-WT and -R259C interacted with hERG in the cytosol and on the plasma membrane; however, the interaction between KCNQ1-T587M and hERG was observed only in the cytosol, and hERG proteins were seldom transported to the cell membrane, suggesting that the KCNQ1-T587M mutation impaired the trafficking of hERG to the cell membrane. CONCLUSIONS: The disruption of hERG trafficking caused by the KCNQ1-T587M mutation is likely the reason why some patients exhibit severe LQTS phenotypes.
Authors: Lucía Cócera-Ortega; Ronald Wilders; Selina C Kamps; Benedetta Fabrizi; Irit Huber; Ingeborg van der Made; Anouk van den Bout; Dylan K de Vries; Lior Gepstein; Arie O Verkerk; Yigal M Pinto; Anke J Tijsen Journal: Int J Mol Sci Date: 2022-04-06 Impact factor: 5.923