Ademuyiwa S Aromolaran1, Prakash Subramanyam1, Donald D Chang1, William R Kobertz2, Henry M Colecraft3. 1. Department of Physiology and Cellular Biophysics, Columbia University, College of Physicians and Surgeons, 1150 St. Nicholas Avenue, 504 Russ Berrie, New York, NY 10032, USA. 2. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School Worcester, MA 01605, USA. 3. Department of Physiology and Cellular Biophysics, Columbia University, College of Physicians and Surgeons, 1150 St. Nicholas Avenue, 504 Russ Berrie, New York, NY 10032, USA hc2405@columbia.edu.
Abstract
AIMS: Long QT syndrome 1 (LQT1) mutations in KCNQ1 that decrease cardiac IKs (slowly activating delayed rectifier K(+) current) underlie ventricular arrhythmias and sudden death. LQT1 mutations may suppress IKs by preventing KCNQ1 assembly, disrupting surface trafficking, or inhibiting gating. We investigated mechanisms underlying how three LQT1 mutations in KCNQ1 C-terminus assembly domain (R555H/G589D/L619M) decrease IKs in heterologous cells and cardiomyocytes. METHODS AND RESULTS: In Chinese hamster ovary (CHO) cells, mutant KCNQ1 + KCNE1 channels either produced no currents (G589D/L619M) or displayed markedly reduced IKs with a right-shifted voltage-dependence of activation (R555H). When co-expressed with wild-type (wt) KCNQ1, the mutant KCNQ1s displayed varying intrinsic dominant-negative capacities that were affected by auxiliary KCNE1. All three mutant KCNQ1s assembled with wt KCNQ1 as determined by fluorescence resonance energy transfer (FRET). We developed an optical quantum dot labelling assay to measure channel surface density. G589D/R555H displayed substantial reductions in surface density, which were either partially (G589D) or fully (R555H) rescued by wt KCNQ1. Unexpectedly, L619M showed no trafficking defect. In adult rat cardiomyocytes, adenovirus-expressed homotetrameric G589D/L619M + KCNE1 channels yielded no currents, whereas R555H + KCNE1 produced diminished IKs with a right-shifted voltage-dependence of activation, mimicking observations in CHO cells. In contrast to heterologous cells, homotetrameric R555H channels showed no trafficking defect in cardiomyocytes. CONCLUSION: Distinct LQT1 mutations in KCNQ1 assembly domain decrease IKs using unique combinations of biophysical and trafficking mechanisms. Functional deficits in IKs observed in heterologous cells are mostly, but not completely, recapitulated in adult rat cardiomyocytes. A 'methodological chain' combining approaches in heterologous cells and cardiomyocytes provides mechanistic insights that may help advance personalized therapy for LQT1 mutations. Published on behalf of the European Society of Cardiology. All rights reserved.
AIMS: Long QT syndrome 1 (LQT1) mutations in KCNQ1 that decrease cardiac IKs (slowly activating delayed rectifier K(+) current) underlie ventricular arrhythmias and sudden death. LQT1 mutations may suppress IKs by preventing KCNQ1 assembly, disrupting surface trafficking, or inhibiting gating. We investigated mechanisms underlying how three LQT1 mutations in KCNQ1 C-terminus assembly domain (R555H/G589D/L619M) decrease IKs in heterologous cells and cardiomyocytes. METHODS AND RESULTS: In Chinese hamster ovary (CHO) cells, mutant KCNQ1 + KCNE1 channels either produced no currents (G589D/L619M) or displayed markedly reduced IKs with a right-shifted voltage-dependence of activation (R555H). When co-expressed with wild-type (wt) KCNQ1, the mutant KCNQ1s displayed varying intrinsic dominant-negative capacities that were affected by auxiliary KCNE1. All three mutant KCNQ1s assembled with wt KCNQ1 as determined by fluorescence resonance energy transfer (FRET). We developed an optical quantum dot labelling assay to measure channel surface density. G589D/R555H displayed substantial reductions in surface density, which were either partially (G589D) or fully (R555H) rescued by wt KCNQ1. Unexpectedly, L619M showed no trafficking defect. In adult rat cardiomyocytes, adenovirus-expressed homotetrameric G589D/L619M + KCNE1 channels yielded no currents, whereas R555H + KCNE1 produced diminished IKs with a right-shifted voltage-dependence of activation, mimicking observations in CHO cells. In contrast to heterologous cells, homotetrameric R555H channels showed no trafficking defect in cardiomyocytes. CONCLUSION: Distinct LQT1 mutations in KCNQ1 assembly domain decrease IKs using unique combinations of biophysical and trafficking mechanisms. Functional deficits in IKs observed in heterologous cells are mostly, but not completely, recapitulated in adult rat cardiomyocytes. A 'methodological chain' combining approaches in heterologous cells and cardiomyocytes provides mechanistic insights that may help advance personalized therapy for LQT1 mutations. Published on behalf of the European Society of Cardiology. All rights reserved.
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