The goal of this research is to understand, at the molecular level, how Ca is accumulated and released by subspecialized regions of sarcoplasmic reticulum (SR) in cardiac muscle. Five key cardiac SR proteins regulating intracellular Ca flux will be studied: phospholamban, a free SR protein regulating the Ca pump and recently shown to have Ca channel activity; the ryanodine receptor, the primary Ca release channel of junctional SR; calsequestrin, the intraluminal Ca binding protein localized to junctional SR; the 26-kDa calsequestrin-binding protein, a protein which may anchor calsequestrin to the junctional SR membrane; and 100-kDa and 90-kDa proteins, putative Ca storage proteins localized to free SR. Studies on phospholamban will address membrane protein topology and basic mechanisms of action. We will determine whether phospholamban regulates the Ca pump directly (by a protein:protein interaction) or indirectly (by altering membrane surface charge), and whether the Ca channel activity of phospholamban is necessary for Ca pump modulation. These studies will be facilitated by expressing native and mutated phospholamban in cultured atrial tumor cells. Three important regulatory sites of the cardiac ryanodine receptor will be sequenced and characterized functionally: a unique phosphorylation site for multifunctional Ca2+/calmodulin-dependent protein kinase; the calmodulin-binding site(s); and calpain II cleavage sites. All three classes of sites modulate channel activity, and localization and characterization of these sites will give new insight into topological domains required for channel function. In a collaborative study, cardiac calsequestrin will be crystallized and the three dimensional structure determined to localize the Ca binding sites, the Ca-regulated hydrophobic domain, and the unique C-terminal tail. The 26-kDa calsequestrin-binding protein will be purified and sequenced, in order to determine whether this protein anchors calsequestrin to the junctional face membrane. Finally, the 100-kDa and 90-kDa proteins, which may be intraluminal proteins adapted for Ca handling inside free SR, will be purified and sequenced. Comparison of these latter proteins to calsequestrin should yield new information on the significance and function of intraluminal Ca storage proteins which are localized to discrete SR regions. Completion of the studies described will increase our understanding of the role of the SR in basic mechanisms of excitation- contraction coupling in cardiac muscle.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL028556-13
Application #
2216310
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1983-01-01
Project End
1996-12-31
Budget Start
1995-01-01
Budget End
1995-12-31
Support Year
13
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Pharmacology
Type
Schools of Medicine
DUNS #
005436803
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Chopra, Nagesh; Kannankeril, Prince J; Yang, Tao et al. (2007) Modest reductions of cardiac calsequestrin increase sarcoplasmic reticulum Ca2+ leak independent of luminal Ca2+ and trigger ventricular arrhythmias in mice. Circ Res 101:617-26
Knollmann, Bjorn C; Chopra, Nagesh; Hlaing, Thinn et al. (2006) Casq2 deletion causes sarcoplasmic reticulum volume increase, premature Ca2+ release, and catecholaminergic polymorphic ventricular tachycardia. J Clin Invest 116:2510-20
Kirchhefer, Uwe; Hanske, Gabriela; Jones, Larry R et al. (2006) Overexpression of junctin causes adaptive changes in cardiac myocyte Ca(2+) signaling. Cell Calcium 39:131-42
Kirchhefer, Uwe; Baba, Hideo A; Hanske, Gabriela et al. (2004) Age-dependent biochemical and contractile properties in atrium of transgenic mice overexpressing junctin. Am J Physiol Heart Circ Physiol 287:H2216-25
Gyorke, Inna; Hester, Nichole; Jones, Larry R et al. (2004) The role of calsequestrin, triadin, and junctin in conferring cardiac ryanodine receptor responsiveness to luminal calcium. Biophys J 86:2121-8
Kirchhefer, Uwe; Jones, Larry R; Begrow, Frank et al. (2004) Transgenic triadin 1 overexpression alters SR Ca2+ handling and leads to a blunted contractile response to beta-adrenergic agonists. Cardiovasc Res 62:122-34
Yang, Alexander; Sonin, Dimitry; Jones, Larry et al. (2004) A beneficial role of cardiac P2X4 receptors in heart failure: rescue of the calsequestrin overexpression model of cardiomyopathy. Am J Physiol Heart Circ Physiol 287:H1096-103
Tijskens, Pierre; Jones, Larry R; Franzini-Armstrong, Clara (2003) Junctin and calsequestrin overexpression in cardiac muscle: the role of junctin and the synthetic and delivery pathways for the two proteins. J Mol Cell Cardiol 35:961-74
Kirchhefer, Uwe; Neumann, Joachim; Bers, Donald M et al. (2003) Impaired relaxation in transgenic mice overexpressing junctin. Cardiovasc Res 59:369-79
Kirchhefer, Uwe; Baba, Hideo A; Kobayashi, Yvonne M et al. (2002) Altered function in atrium of transgenic mice overexpressing triadin 1. Am J Physiol Heart Circ Physiol 283:H1334-43

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