Conformational changes of a Ca2+-binding domain of the Na+/Ca2+ exchanger monitored by FRET in transgenic zebrafish heart

The Na+/Ca2+ exchanger is the major Ca2+ extrusion mechanism in cardiac myocytes. The activity of the cardiac Na+/Ca2+ exchanger is dynamically regulated by intracellular Ca2+. Previous studies indicate that Ca2+ binding to a high-affinity Ca2+-binding domain (CBD1) in the large intracellular loop is involved in regulation. We generated transgenic zebrafish with cardiac-specific expression of CBD1 linked to yellow and cyan fluorescent protein. Ca2+ binding to CBD1 induces conformational changes, as detected by fluorescence resonance energy transfer. With this transgenic fish model, we were able to monitor conformational changes of the Ca2+ regulatory domain of Na+/Ca2+ exchanger in intact hearts. Treatment with the positive inotropic agents ouabain and isoproterenol increased both Ca2+ transients and Ca2+-induced changes in fluorescence resonance energy transfer. The results indicate that Ca2+ regulation of the Na+/Ca2+ exchanger domain CBD1 changes with inotropic state. The transgenic fish models will be useful to further characterize the regulatory properties of the Na+/Ca2+ exchanger in vivo.