Abstract 583: Identification of Target Proteins for Neuronal Calcium Sensor-1 (NCS-1) in the Heart
Introduction: Changes in intracellular Ca2+ concentration regulate numerous cellular functions that are mediated by Ca2+ binding to a large family of EF-hand Ca2+ sensor proteins. We previously reported that one of them, neuronal calcium sensor-1 (NCS-1) was present in both cytosol and membrane fractions of rat heart. Adenovirus-mediated gene delivery of NCS-1 to isolated ventricular myocytes results in an increase in myocardial contractile function, with a slight alteration of sarcoplasmic reticular function.
Aims and Methods: To further investigate the role of NCS-1 in the regulation of cardiac contractility, we used the yeast two-hybrid system to identify novel protein interactions among cardiac E-C coupling-associated proteins and NCS-1. Human NCS-1 was used as a bait to screen a human heart cDNA library by yeast mating.
Results: 120 positive colonies were initially obtained under high stringency mating selection. DNA inserts of 47 true positive clones were sequenced; 27 clones contained unique DNA sequences, including 4 unknown genes. The remaining 23 identified proteins are primarily expressed in the cytoplasm, with fewer proteins localized at the plasma membrane. Cellular functions of these proteins include membrane transport, metabolism energy pathways, cell growth/maintenance, signal transduction/cell communication and structural/cytoskeletal-associated proteins. Interestingly, the yeast two hybrid studies show that NCS-1 interacts with Troponin I (cTnI), a key inhibitory protein involved in the regulation of E-C coupling in cardiac muscle. cTnI inhibits sarcomere contraction by interfering with actin-myosin interaction. Co-immunoprecipitation studies in cardiomyocytes also suggest an interaction between NCS-1 and Troponin I.
Conclusion: Our results deonstrate that NCS-1 is involved in multiple aspects of Ca2+ signaling in the heart. Furthermore, identification of a possible protein-protein interaction between Troponin I and NCS-1 provides novel insights into a cellular mechanism by which NCS-1 can augment cardiac contractile function.