Abstract

Increased permeability to Ca2+ in response to a depolarizing membrane potential occurs in a wide variety of excitable cell types. In some cell this permeability increase is either blocked or enhanced by dihydropyridines, leading to the hypothesis that the dihydropyridine binding site is part of or associated with the voltage-dependent calcium channel. We propose to purify the DHP binding protein from bovine ventricular tissue and to characterize its subunit composition. This will be accomplished using the binding radioactive DHPS to follow the purification. Purification procedures will utilize conventional column chromatography, HPLC and FPLC techniques, affinity chromatography, and ultracentrifugation. In a collaborative effort, we will use reconstitution techniques to establish whether the DHP binding protein is also the voltage- dependent calcium channel. Important to this purification and characterization of the cardiac DHP binding protein will be the production of antibody and toxin probes specific for this protein. One approach to the preparation of specific antibodies will be to use the subunits of the skeletal muscle DHP binding protein as antigens. We will also use the partially-purified cardiac preparation. Probes of the DHP binding protein will be used to purify it further, to determine the subunit composition, and to analyze the arrangement of the subunits in the membrane.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL037028-04
Application #
3352529
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1985-12-01
Project End
1992-07-31
Budget Start
1988-08-01
Budget End
1989-07-31
Support Year
4
Fiscal Year
1988
Total Cost
Indirect Cost

  Institution

Name
Baylor College of Medicine
Department
Type
Schools of Medicine
DUNS #
074615394
City
Houston
State
TX
Country
United States
Zip Code
77030

  Publications

Callaway, C; Seryshev, A; Wang, J P et al. (1994) Localization of the high and low affinity [3H]ryanodine binding sites on the skeletal muscle Ca2+ release channel. J Biol Chem 269:15876-84
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
Hamilton, S L; Codina, J; Hawkes, M J et al. (1991) Evidence for direct interaction of Gs alpha with the Ca2+ channel of skeletal muscle. J Biol Chem 266:19528-35
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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