Chih-Chieh Yu1, Jum-Suk Ko2, Tomohiko Ai3, Wen-Chin Tsai4, Zhenhui Chen5, Michael Rubart6, Matteo Vatta7, Thomas H Everett5, Alfred L George8, Peng-Sheng Chen9. 1. Krannert Institute of Cardiology and Division of Cardiology, Department of Medicine, Indiana University, Indianapolis, Indiana; Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan. 2. Krannert Institute of Cardiology and Division of Cardiology, Department of Medicine, Indiana University, Indianapolis, Indiana; Division of Cardiology, Department of Internal Medicine, Wonkwang University School of Medicine and Hospital, Iksan, Korea. 3. Krannert Institute of Cardiology and Division of Cardiology, Department of Medicine, Indiana University, Indianapolis, Indiana; Department of Molecular Pathogenesis, Medical Research Institute; Department of Emergency Medicine, Tokyo Medical and Dental University, Tokyo, Japan. 4. Krannert Institute of Cardiology and Division of Cardiology, Department of Medicine, Indiana University, Indianapolis, Indiana; Hualien Tzu-Chi General Hospital, Hualien City, Taiwan. 5. Krannert Institute of Cardiology and Division of Cardiology, Department of Medicine, Indiana University, Indianapolis, Indiana. 6. Krannert Institute of Cardiology and Division of Cardiology, Department of Medicine, Indiana University, Indianapolis, Indiana; Department of Pediatrics, Riley Heart Research Center. 7. Krannert Institute of Cardiology and Division of Cardiology, Department of Medicine, Indiana University, Indianapolis, Indiana; Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana. 8. Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, Illinois. 9. Krannert Institute of Cardiology and Division of Cardiology, Department of Medicine, Indiana University, Indianapolis, Indiana. Electronic address: chenpp@iu.edu.
Abstract
BACKGROUND: Apamin-sensitive small-conductance calcium-activated potassium (SK) channels are gated by intracellular Ca(2+) through a constitutive interaction with calmodulin. OBJECTIVE: We hypothesize that arrhythmogenic human calmodulin mutations impede activation of SK channels. METHODS: We studied 5 previously published calmodulin mutations (N54I, N98S, D96V, D130G, and F90L). Plasmids encoding either wild-type or mutant calmodulin were transiently transfected into human embryonic kidney 293 cells that stably express subtype 2 of SK protein channels (SK2 cells). Whole-cell voltage-clamp recording was used to determine apamin-sensitive current densities. We also performed optical mapping studies in normal murine hearts to determine the effects of apamin in hearts with (n=7) or without (n=3) pretreatment with sea anemone toxin. RESULTS: SK2 cells transfected with wild-type calmodulin exhibited an apamin-sensitive current density of 33.6 pA/pF (31.4-36.5 pA/pF) (median and confidence interval 25th-75th percentile), which was significantly higher than that observed for cells transfected with N54I (17.0 pA/pF [14.0-27.7 pA/pF]; P = .016), F90L (22.6 pA/pF [20.3-24.3 pA/pF]; P = .011), D96V (13.0 pA/pF [10.9-15.8 pA/pF]; P = .003), N98S (13.7 pA/pF [8.8-20.4 pA/pF]; P = .005), and D130G (17.6 pA/pF [13.8-24.6 pA/pF]; P = .003). The decrease in SK2 current densities was not associated with a decrease in membrane protein expression or intracellular distribution of the channel protein. Apamin increased the ventricular action potential duration at 80% repolarization (from 79.6 ms [63.4-93.3 ms] to 121.8 ms [97.9-127.2 ms]; P = .010) in hearts pretreated with anemone toxin but not in control hearts. CONCLUSION: Human arrhythmogenic calmodulin mutations impede the activation of SK2 channels in human embryonic kidney 293 cells.
BACKGROUND: Apamin-sensitive small-conductance calcium-activated potassium (SK) channels are gated by intracellular Ca(2+) through a constitutive interaction with calmodulin. OBJECTIVE: We hypothesize that arrhythmogenichumancalmodulin mutations impede activation of SK channels. METHODS: We studied 5 previously published calmodulin mutations (N54I, N98S, D96V, D130G, and F90L). Plasmids encoding either wild-type or mutant calmodulin were transiently transfected into humanembryonic kidney 293 cells that stably express subtype 2 of SK protein channels (SK2 cells). Whole-cell voltage-clamp recording was used to determine apamin-sensitive current densities. We also performed optical mapping studies in normal murine hearts to determine the effects of apamin in hearts with (n=7) or without (n=3) pretreatment with sea anemone toxin. RESULTS:SK2 cells transfected with wild-type calmodulin exhibited an apamin-sensitive current density of 33.6 pA/pF (31.4-36.5 pA/pF) (median and confidence interval 25th-75th percentile), which was significantly higher than that observed for cells transfected with N54I (17.0 pA/pF [14.0-27.7 pA/pF]; P = .016), F90L (22.6 pA/pF [20.3-24.3 pA/pF]; P = .011), D96V (13.0 pA/pF [10.9-15.8 pA/pF]; P = .003), N98S (13.7 pA/pF [8.8-20.4 pA/pF]; P = .005), and D130G (17.6 pA/pF [13.8-24.6 pA/pF]; P = .003). The decrease in SK2 current densities was not associated with a decrease in membrane protein expression or intracellular distribution of the channel protein. Apamin increased the ventricular action potential duration at 80% repolarization (from 79.6 ms [63.4-93.3 ms] to 121.8 ms [97.9-127.2 ms]; P = .010) in hearts pretreated with anemone toxin but not in control hearts. CONCLUSION:Humanarrhythmogeniccalmodulin mutations impede the activation of SK2 channels in humanembryonic kidney 293 cells.
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