Increased permeability to Ca2+ in response to a depolarizing membrane potential occurs in a wide variety of cell types. In some cells this permeability increase is blocked by dihydropyridines. We propose to characterize the dihydropyridine binding site of rat cardiac tissue and to determine its subcellular location (sarcoplasmic reticulum, sarcolemma or T-tubular membranes). The first phase of this study will involve an analysis of the binding of [3H]-nitrendipine to these membranes. The calculated binding site densities in the sarcolemma will be compared to the calcium channel densities determined electrophysiologically. The binding protein will be solubilized and characterized in detergent solution. Photoaffinity derivatives of some of the calcium channel antagonists will be synthesized, assayed for activity and used, in radioactive forms, to identify and compare the specific binding proteins in the membranes. Using combined electrophoretic, chromatographic and immunological techniques, we propose to purify and characterize biochemically the antagonist binding protein. In a collaborative effort, we will use reconstitution to establish its relationship (or identify) to the voltage dependent calcium channel.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL037028-02
Application #
3352528
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1985-12-01
Project End
1987-07-31
Budget Start
1986-08-01
Budget End
1987-07-31
Support Year
2
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Type
Schools of Medicine
DUNS #
074615394
City
Houston
State
TX
Country
United States
Zip Code
77030
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Wang, J P; Needleman, D H; Hamilton, S L (1993) Relationship of low affinity [3]ryanodine binding sites to high affinity sites on the skeletal muscle Ca2+ release channel. J Biol Chem 268:20974-82
Hawkes, M J; Nelson, T E; Hamilton, S L (1992) [3H]ryanodine as a probe of changes in the functional state of the Ca(2+)-release channel in malignant hyperthermia. J Biol Chem 267:6702-9
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Hazarika, P; Sheldon, A; Kaetzel, M A et al. (1991) Regulation of the sarcoplasmic reticulum Ca(2+)-release channel requires intact annexin VI. J Cell Biochem 46:86-93
Diaz-Munoz, M; Hamilton, S L; Kaetzel, M A et al. (1990) Modulation of Ca2+ release channel activity from sarcoplasmic reticulum by annexin VI (67-kDa calcimedin). J Biol Chem 265:15894-9
Kaetzel, M A; Hazarika, P; Diaz-Munoz, M et al. (1990) Annexins: a subcellular localization and reconstitution approach to elucidate cellular function. Biochem Soc Trans 18:1108-10
Chu, A; Diaz-Munoz, M; Hawkes, M J et al. (1990) Ryanodine as a probe for the functional state of the skeletal muscle sarcoplasmic reticulum calcium release channel. Mol Pharmacol 37:735-41
Hamilton, S L; Alvarez, R M; Fill, M et al. (1989) [3H]PN200-110 and [3H]ryanodine binding and reconstitution of ion channel activity with skeletal muscle membranes. Anal Biochem 183:31-41
Hamilton, S L; Hawkes, M J; Brush, K et al. (1989) Subunit composition of the purified dihydropyridine binding protein from skeletal muscle. Biochemistry 28:7820-8

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