The long-term objective of this project is to elucidate mechanisms of ion transport across the inner membrane of mitochondria.
Specific aims are: (1) To study the mechanism and regulation of the uncoupling protein (UcP) of brown adipose tissue mitochondria both in intact mitochondria and in proteoliposomes reconstituted with the purified 32 kDa uncoupling protein. Brown adipose tissue provides heat to small mammals and human infants, who are very susceptible to cold stress. The mechanism of heat production is broadly understood and relies on the regulated transport of H+ ions through UcP, an energy-dissipating (therefore heat-producing) protein found uniquely in this tissue. This project introduced fluorescent probe spectroscopy to study transport by UcP, a powerful technique that has produced major new findings and should lead to rapid advances in our understanding of this unique protein. (2) To study the function of mutagenized UcP. UcP belongs to an important gene family that includes the phosphate transporter and adenine nucleotide exchanger of mitochondria. Although its amino acid sequence is known, it has not been possible to exploit this information because so little is known about its transport properties. Armed with new insights into its function and transport kinetics, this project will begin a study of the chemistry of ligand interaction on a molecular level. This study will use mutagenized UcP supplied by a collaborator who has successfully overexpressed the functionally active protein in yeast cells using UcP cDNA in a yeast expression vector. (3) To study the pleiotropic effects of local anesthetics on mitochondrial ion transport and to resolve their mechanism of uncoupling. Bupivicaine is a new local anesthetic that can produce serious toxicity in patients. It has been found to uncouple oxidative phosphorylation in mitochondria, and this has been a suggested basis of its toxicity. There is controversy in the literature over the mechanism of uncoupling, and this project will attempt to resolve this by measurements of ion transport and respiration of mitochondria.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM031086-09A2
Application #
3279004
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1982-07-01
Project End
1995-11-30
Budget Start
1991-12-15
Budget End
1992-11-30
Support Year
9
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Toledo
Department
Type
Schools of Medicine
DUNS #
807418939
City
Toledo
State
OH
Country
United States
Zip Code
43614
Grover, G J; Garlid, K D (2000) ATP-Sensitive potassium channels: a review of their cardioprotective pharmacology. J Mol Cell Cardiol 32:677-95
Garlid, K D (2000) The state of water in biological systems. Int Rev Cytol 192:281-302
Garlid, K D; Jabyyrek, M; Jezek, P et al. (2000) How do uncoupling proteins uncouple? Biochim Biophys Acta 1459:383-9
Jabyyrek, M; Varecha, M; Gimeno, R E et al. (1999) Transport function and regulation of mitochondrial uncoupling proteins 2 and 3. J Biol Chem 274:26003-7
Jezek, P; Zackova, M; Rehakova, Z et al. (1999) Existence of uncoupling protein-2 antigen in isolated mitochondria from various tissues. FEBS Lett 455:79-82
Garlid, K D; Jabyyrek, M; Jezek, P (1998) The mechanism of proton transport mediated by mitochondrial uncoupling proteins. FEBS Lett 438:10-4
Jezek, P; Modriansky, M; Garlid, K D (1997) Inactive fatty acids are unable to flip-flop across the lipid bilayer. FEBS Lett 408:161-5
Jezek, P; Modriansky, M; Garlid, K D (1997) A structure-activity study of fatty acid interaction with mitochondrial uncoupling protein. FEBS Lett 408:166-70
Yarov-Yarovoy, V; Paucek, P; Jabyyrek, M et al. (1997) The nucleotide regulatory sites on the mitochondrial KATP channel face the cytosol. Biochim Biophys Acta 1321:128-36
Garlid, K D; Paucek, P; Yarov-Yarovoy, V et al. (1997) Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels. Possible mechanism of cardioprotection. Circ Res 81:1072-82

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