title: Sarcolemmal Ca(2+)-entry through L-type Ca(2+) channels controls the profile of Ca(2+)-activated Cl(-) current in canine ventricular myocytes.
abstract: Ca(2+)-activated Cl(-) current (ICl(Ca)) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. We aimed to study the profile of ICl(Ca) systematically under physiological conditions (normal Ca(2+) cycling and AP voltage-clamp) as well as in conditions designed to change [Ca(2+)]i. The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Cav1.2 was also studied. The profile of ICl(Ca), identified as a 9-anthracene carboxylic acid-sensitive current under AP voltage-clamp conditions, contained an early fast outward and a late inward component, overlapping early and terminal repolarizations, respectively. Both components were moderately reduced by ryanodine, while fully abolished by BAPTA, but not EGTA. [Ca(2+)]i was monitored using Fura-2-AM. Setting [Ca(2+)]i to the systolic level measured in the bulk cytoplasm (1.1μM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Ca(2+)-entry through L-type Ca(2+) channels was essential for activation of ICl(Ca). TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels, when assessed using immunolabeling and confocal microscopy in both canine myocytes and human ventricular myocardium. Activation of ICl(Ca) in canine ventricular cells requires Ca(2+)-entry through neighboring L-type Ca(2+) channels and is only augmented by SR Ca(2+)-release. Substantial activation of ICl(Ca) requires high Ca(2+) concentration in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA.
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Debrecen, H-4012 Debrecen, Nagyerdei krt 98, P.O. Box 22, Hungary; Faculty of Pharmacy, University of Debrecen, H-4012 Debrecen, Nagyerdei krt 98, P.O. Box 22, Hungary.
authors.forename: Balázs
authors.initials: B
authors.lastname: Horváth
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Debrecen, H-4012 Debrecen, Nagyerdei krt 98, P.O. Box 22, Hungary.
authors.forename: Krisztina
authors.initials: K
authors.lastname: Váczi
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Debrecen, H-4012 Debrecen, Nagyerdei krt 98, P.O. Box 22, Hungary.
authors.forename: Bence
authors.initials: B
authors.lastname: Hegyi
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Debrecen, H-4012 Debrecen, Nagyerdei krt 98, P.O. Box 22, Hungary; MTA-DE Momentum, Laboratory of Protein Dynamics, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, H-4012 Debrecen, Nagyerdei krt 98, P.O. Box 22, Hungary.
authors.forename: Mónika
authors.initials: M
authors.lastname: Gönczi
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Debrecen, H-4012 Debrecen, Nagyerdei krt 98, P.O. Box 22, Hungary.
authors.forename: Beatrix
authors.initials: B
authors.lastname: Dienes
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Debrecen, H-4012 Debrecen, Nagyerdei krt 98, P.O. Box 22, Hungary.
authors.forename: Kornél
authors.initials: K
authors.lastname: Kistamás
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Debrecen, H-4012 Debrecen, Nagyerdei krt 98, P.O. Box 22, Hungary.
authors.forename: Tamás
authors.initials: T
authors.lastname: Bányász
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Debrecen, H-4012 Debrecen, Nagyerdei krt 98, P.O. Box 22, Hungary; Division of Sport Physiology, Department of Physiology, Faculty of Medicine, University of Debrecen, H-4012 Debrecen, Nagyerdei krt 98, P.O. Box 22, Hungary.
authors.forename: János
authors.initials: J
authors.lastname: Magyar
authors.affiliation: Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Szeged, H-6720 Szeged, Dóm tér 12, P.O. Box 427, Hungary.
authors.forename: István
authors.initials: I
authors.lastname: Baczkó
authors.affiliation: Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Szeged, H-6720 Szeged, Dóm tér 12, P.O. Box 427, Hungary; MTA-SZTE Research Group of Cardiovascular Pharmacology, Hungarian Academy of Sciences, H-6720 Szeged, Dóm tér 12, P.O. Box 427, Hungary.
authors.forename: András
authors.initials: A
authors.lastname: Varró
authors.affiliation: Department of Medical Biology, Faculty of Medicine, University of Szeged, H-6720 Szeged, Somogyi Béla utca 4, P.O. Box 427, Hungary.
authors.forename: György
authors.initials: G
authors.lastname: Seprényi
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Debrecen, H-4012 Debrecen, Nagyerdei krt 98, P.O. Box 22, Hungary.
authors.forename: László
authors.initials: L
authors.lastname: Csernoch
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Debrecen, H-4012 Debrecen, Nagyerdei krt 98, P.O. Box 22, Hungary; Department of Dental Physiology and Pharmacology, Faculty of Dentistry, University of Debrecen, H-4012 Debrecen, Nagyerdei krt 98, P.O. Box 22, Hungary.
authors.forename: Péter P
authors.initials: PP
authors.lastname: Nánási
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Debrecen, H-4012 Debrecen, Nagyerdei krt 98, P.O. Box 22, Hungary; Department of Dental Physiology and Pharmacology, Faculty of Dentistry, University of Debrecen, H-4012 Debrecen, Nagyerdei krt 98, P.O. Box 22, Hungary. Electronic address: szentandrassy.norbert@med.unideb.hu.
authors.forename: Norbert
authors.initials: N
authors.lastname: Szentandrássy
mesh_terms: D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D015415:Biomarkers; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D018118:Chloride Channels / Q000378:metabolism*; D004285:Dogs; D055724:Electrophysiological Phenomena; D006352:Heart Ventricles / Q000378:metabolism*; D006801:Humans; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D018118:Chloride Channels
keywords: Action potential voltage-clamp; Bestrophin-3; Ca(2+)-activated Cl(−) current; Canine cardiomyocytes; Human cardiomyocytes; TMEM16A