Characterization of Recombinant Human Cardiac KCNQ1/KCNE1 Channels (I Ks) Stably Expressed in HEK 293 Cells |
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Authors: | Ming-Qing Dong Chu-Pak Lau Zhan Gao Gea-Ny Tseng Gui-Rong Li |
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Affiliation: | (1) Department of Medicine and Research Centre of Heart, Brain, Hormone, and Healthy Aging, Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong, People’s Republic of China;(2) Department of Physiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia |
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Abstract: | The present study was designed to characterize pharmacological, biophysical and electrophysiological properties of the recombinant human cardiac I Ks (KCNQ1/KCNE1) channels at physiological temperature. Human cardiac KCNQ1 and KCNE1 genes were cotransfected into HEK 293 cells, and a cell clone stably expressing both genes was selected. Membrane currents were recorded using a perforated patch-clamp technique. The typical I Ks was slowly activated upon depolarization voltages in HEK 293 cells stably expressing human cardiac KCNQ1 and KCNE1 genes, and the current was inhibited by I Ks blockers HMR 1556 and chromanol 293B, with 50% inhibitory concentrations (IC50s) of 83.8 nM and 9.2 μM, respectively. I Ks showed a significant temperature-dependent increase in its magnitude upon elevating bath temperature to 36°C from room temperature (21°C). The current was upregulated by the β-adrenoceptor agonist isoproterenol, and the effect was reversed by H89. In addition, I Ks was inhibited by Ba2+ in a concentration-dependent manner (IC50 = 1.4 mM). Action potential clamp revealed a “bell-shaped” time course of I Ks during the action potential, and maximal peak current was seen at the plateau of the action potential. A significant use- and frequency-dependent increase of I Ks was observed during a train of action potential clamp. These results indicate that the recombinant human cardiac I Ks stably expressed in HEK 293 cells is similar to native I Ks in drug sensitivity and regulated by Ba2+ and β-adrenoceptor via the cyclic adenosine monophosphate/protein kinase A pathway. Importantly, the current exhibits significant temperature dependence, a bell-shaped time course during action potential and prominent use- or frequency-dependent accumulation during a train of action potentials. |
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Keywords: | Electrophysiology Ion channel Slowly activating delayed rectifier potassium current Recombinant human cardiac KCNQ1/KCNE1 HEK 293 cell Perforated recording |
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