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. Ca²⁺-binding domain; sodium/calcium exchange; zebrafish; fluorescence resonance energy transfer