Conformational Changes of a Ca2+-Binding Domain of the Na+ -Ca2+ Exchanger Monitored by FRET in Transgenic Zebrafish Heart

The Na(+) -Ca(2+) exchanger is the major Ca(2+) extrusion mechanism in cardiac myocytes. The activity of the cardiac Na(+)-Ca(2+) exchanger is dynamically regulated by intracellular Ca(2+). Previous studies indicate that Ca(2+) binding to a high-affinity Ca(2+)-binding domain (CBD1) in the large intracellular loop is involved in regulation. We generated transgenic zebrafish with cardiac-specific expression of CBD1 linked to YFP and CFP. Ca(2+)-binding to CBD1 induces conformational changes as detected by FRET. With this transgenic fish model, we were able to monitor conformational changes of the Ca(2+) regulatory domain of NCX in intact hearts. Treatment with the positive inotropic agents ouabain and isoproterenol increased both Ca(2+) transients and Ca(2+)-induced changes in FRET. The results indicate that Ca(2+) regulation of the Na(+)-Ca(2+) exchanger domain CBD1 changes with inotropic state. The transgenic fish models will be useful to further characterize the regulatory properties of the Na(+)-Ca(2+) exchanger in vivo. Key words: Ca2+-binding domain, sodium-calcium exchange, zebrafish, FRET.