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

Abstract

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 yellow and cyan fluorescent protein. Ca(2+) 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 Ca(2+) regulatory domain of Na(+)/Ca(2+) exchanger in intact hearts. Treatment with the positive inotropic agents ouabain and isoproterenol increased both Ca(2+) transients and Ca(2+)-induced changes in fluorescence resonance energy transfer. 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.