Abstract
Ca2+-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 Ca2+ cycling and AP voltage-clamp) as well as in conditions designed to change [Ca2+]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. [Ca2+]i was monitored using Fura-2-AM. Setting [Ca2+]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). Ca2+-entry through L-type Ca2+ 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 Ca2+-entry through neighboring L-type Ca2+ channels and is only augmented by SR Ca2+-release. Substantial activation of ICl(Ca) requires high Ca2+ concentration in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA.
Highlights
The role of anions in cardiac electrophysiology received far less attention as physiological and pharmacological research has focused mainly on cation channels [1] until the discovery of the cAMPactivated Cl− current [2, 3]
Our results indicate that blockade of sarcoplasmic reticulum (SR) Ca2+-release may decrease, but fails to eliminate the activation of ICl(Ca) in canine ventricular cells confirming the critical importance of transsarcolemmal Ca2+-entry through L-type Ca2+ channels (LTCCs) in controlling ICl(Ca)
ICl(Ca) was defined as a 9-anthracene carboxylic acid (9-AC)-sensitive current (I9-AC), i.e. as a difference current obtained by measuring the current before and after application of 0.5 mmol/L 9-AC during step depolarizations to a range of voltages from −60 to +60 mV
Summary
The role of anions in cardiac electrophysiology received far less attention as physiological and pharmacological research has focused mainly on cation channels [1] until the discovery of the cAMPactivated Cl− current [2, 3]. The aims of our research were (1) to characterize ICl(Ca) profiles under actual cardiac APs in canine ventricular cardiomyocytes, in a species with cardiac APs and ion currents most resembling to those of human [28, 29], (2) to study the Ca2+- and rate-dependence of the current, (3) to confirm Cl− as the charge carrier, (4) to examine the role of transsarcolemmal Ca2+-entry and CICR in activation of ICl(Ca) (5) to reveal the expression pattern of Bestrophin-3 and TMEM16A and (6) to visualize their cellular appearance regarding co-localization with each other as well as with pore forming subunit of L-type Ca2+ channel (Cav1.2). TMEM16A and Bestrophin-3 showed strong co-localization with one another and with Cav1.2 channels in both canine and human left ventricular myocardium
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