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 (1.1 μM) 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.
ASJC Scopus subject areas
- Molecular Biology
- Cardiology and Cardiovascular Medicine