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 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.
Department of Physiology, Cardiovascular Research Laboratories, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1760, USA.
Yi
Y
Xie
Michela
M
Ottolia
Scott A
SA
John
Jau-Nian
JN
Chen
Kenneth D
KD
Philipson
D000818:Animals; D030801:Animals, Genetically Modified; D002110:Caffeine / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D016373:DNA, Antisense / Q000235:genetics; D004285:Dogs; D004625:Embryo, Nonmammalian / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer; D006321:Heart / Q000187:drug effects / Q000502:physiology*; D007545:Isoproterenol / Q000494:pharmacology; D009200:Myocardial Contraction / Q000187:drug effects / Q000502:physiology; D009543:Nifedipine / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D010446:Peptide Fragments / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D019831:Sodium-Calcium Exchanger / Q000737:chemistry / Q000502:physiology*; D020107:Troponin T / Q000235:genetics; D015027:Zebrafish / Q000235:genetics / Q000502:physiology*; D029961:Zebrafish Proteins / Q000235:genetics / Q000502:physiology*
D016373:DNA, Antisense; D010446:Peptide Fragments; D019831:Sodium-Calcium Exchanger; D020107:Troponin T; D029961:Zebrafish Proteins; C480681:tnnt2a protein, zebrafish; D002110:Caffeine; D010042:Ouabain; D009543:Nifedipine; D007545:Isoproterenol