Advances in knowledge of magnesium physiology and biochemistry indicate that Mg2+ may be an intracellular regulatory ligand. Mg2+ activates or inhibits numerous important enzyme systems at specific Mg2+ binding sites. Mg2+ transport systems are regulated by a variety of hormones in a manner that will cause changes in intracellular Mg2+ content and probably free concentration within specific compartments. In addition, Mg2+ deficiency results alters the regulation of at least one Mg2+- regulated system, adenylate cyclase. To further study the role of intracellular Mg2+, this proposal will focus three aspects of Mg2+ biochemistry in eukaryotic cells. First, the cellular regulation of Mg2+ influx will be studied. Protein kinase C, cyclic AMP-dependent protein kinase and a third pathway via the beta-adrenergic receptor but independent of cyclic AMP are known to modulate the rate of Mg2+ influx and possibly its intracellular compartmentation. The interaction of these three pathways and the mechanism by which they alter Mg2+ influx will be studied in our model system, the murine S49 lymphoma cell These studies will allow insight into the potential role(s) of hormonal regulation of Mg2+ transport and intracellular Mg2+. Second, to gain further insight into Mg2+'s potential intracellular role(s), the effect(s) of Mg2+ deficiency on cell response to hormone will be studied. Clonal lines of the S49 lymphoma cell able to grow in 30 muM Mg2+ versus a normal Mg2+ concentration of 800 muM have been isolated; they exhibit decreased cell Mg2+ content and compromised cyclic AMP synthesis in response several agonists. We will investigate which component(s) of the receptor-cyclase-kinase pathway may be defective in Mg2+ deficiency and whether other abnormal cell responses are present. Finally, the only currently available tool for study of Mg2+ transport is the very short-lived isotope, 28Mg, thus placing severe limitations on the experimental approaches available for study of the actual mechanism of Mg2+ transport. Our development of a strain of the bacterium Salmonella typhimurium in which all genes for Mg2+ transport have been deleted provides an opportunity to develop an important additional tool to study the mechanism of Mg2+ transport, namely the gene(s) for a Mg2+ transport protein. We will screen a yeast genomic library in the S. typhimurium strain to identify a eukaryotic gene(s) for Mg2+ transport by complementation and thus allowing an eventual approach to study of the mechanism of Mg2+ transport.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026340-10
Application #
3273831
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1980-01-01
Project End
1990-12-31
Budget Start
1990-01-01
Budget End
1990-12-31
Support Year
10
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Gibson, M M; Bagga, D A; Miller, C G et al. (1991) Magnesium transport in Salmonella typhimurium: the influence of new mutations conferring Co2+ resistance on the CorA Mg2+ transport system. Mol Microbiol 5:2753-62
Snavely, M D; Miller, C G; Maguire, M E (1991) The mgtB Mg2+ transport locus of Salmonella typhimurium encodes a P-type ATPase. J Biol Chem 266:815-23
Snavely, M D; Gravina, S A; Cheung, T T et al. (1991) Magnesium transport in Salmonella typhimurium. Regulation of mgtA and mgtB expression. J Biol Chem 266:824-9
Hmiel, S P; Snavely, M D; Florer, J B et al. (1989) Magnesium transport in Salmonella typhimurium: genetic characterization and cloning of three magnesium transport loci. J Bacteriol 171:4742-51
Snavely, M D; Florer, J B; Miller, C G et al. (1989) Magnesium transport in Salmonella typhimurium: 28Mg2+ transport by the CorA, MgtA, and MgtB systems. J Bacteriol 171:4761-6
Snavely, M D; Florer, J B; Miller, C G et al. (1989) Magnesium transport in Salmonella typhimurium: expression of cloned genes for three distinct Mg2+ transport systems. J Bacteriol 171:4752-60
Maguire, M E (1988) Magnesium and cell proliferation. Ann N Y Acad Sci 551:201-15;discussion 215-7
Maguire, M E (1987) Hormonal regulation of magnesium uptake: differential coupling of membrane receptors to magnesium uptake. Magnesium 6:180-91
Hmiel, S P; Snavely, M D; Miller, C G et al. (1986) Magnesium transport in Salmonella typhimurium: characterization of magnesium influx and cloning of a transport gene. J Bacteriol 168:1444-50
Grubbs, R D; Maguire, M E (1986) Regulation of magnesium but not calcium transport by phorbol ester. J Biol Chem 261:12550-4

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