Abstract

and specific aims): The focus of project is the plasma membrane Ca pump (PMCa). The PM Ca pump is one of the three primary mechanisms for the removal of cytoplasmic calcium and thus often plays a pivotal role in terminating signal transduction in many cells. Defects in Ca pump activity would lead to an increase in Ca, with subsequent changes in Na and K. These changes would alter key cytoplasmic functions. Hypertension, sickle cell disease, ischemia/reperfusion injury, excitotoxicity, stroke, and Alzheimer's disease show alterations in cellular Ca homeostasis. The PMCa offers several advantages for structure function studies of EP type pumps: Unlike the Na pump, PMCa is a single subunit. In contrast to the SR Ca pump, parts of the protein are accessible from the extracellular media and this offers advantages for somatic cell selection procedures. This application is focused on the extracellular release steps. Changes in the IC50 for extracellular Ca inhibition could result from changes in the extracellular release steps, alterations of an access channel or changes in translocation steps or a combination of these.
Aim 1 is to determine whether extracellular Ca release occurs through a high field access channel.
Aim 2 is to compare extracellular CA and Mg inhibition of the ferret cardiac calcium pump with rat calcium pump when expressed in xenopus oocytes and to determine which steps are altered to account for the different sensitivity to extracellular Ca.
Aim 3 is to select Ca-out resistant pumps generated by random mutagenesis of the PMCa cDNA (without other alterations of cellular genetics).

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK037512-16
Application #
6380549
Study Section
Special Emphasis Panel (ZRG2-LBPA (01))
Program Officer
Badman, David G
Project Start
1986-08-01
Project End
2003-07-31
Budget Start
2001-08-01
Budget End
2003-07-31
Support Year
16
Fiscal Year
2001
Total Cost
$212,957
Indirect Cost

  Institution

Name
University of Missouri-Columbia
Department
Pharmacology
Type
Schools of Medicine
DUNS #
112205955
City
Columbia
State
MO
Country
United States
Zip Code
65211

  Publications

Gatto, Craig; Milanick, Mark (2009) Red blood cell Na pump: Insights from species differences. Blood Cells Mol Dis 42:192-200
Reifenberger, Matthew S; Arnett, Krista L; Gatto, Craig et al. (2008) The reactive nitrogen species peroxynitrite is a potent inhibitor of renal Na-K-ATPase activity. Am J Physiol Renal Physiol 295:F1191-8
Ogan, Jeffrey T; Reifenberger, Matthew S; Milanick, Mark A et al. (2007) Kinetic characterization of Na,K-ATPase inhibition by Eosin. Blood Cells Mol Dis 38:229-37
Gatto, Craig; Arnett, Krista L; Milanick, Mark A (2007) Divalent cation interactions with Na,K-ATPase cytoplasmic cation sites: implications for the para-nitrophenyl phosphatase reaction mechanism. J Membr Biol 216:49-59
Reifenberger, Matthew S; Arnett, Krista L; Gatto, Craig et al. (2007) Extracellular terbium and divalent cation effects on the red blood cell Na pump and chrysoidine effects on the renal Na pump. Blood Cells Mol Dis 39:7-13
Gatto, Craig; Helms, Jeff B; Prasse, Megan C et al. (2006) Similarities and differences between organic cation inhibition of the Na,K-ATPase and PMCA. Biochemistry 45:13331-45
Dunham, Philip B; Kelley, Scott J; Logue, Paul J et al. (2005) Na+-inhibitory sites of the Na+/H+ exchanger are Li+ substrate sites. Am J Physiol Cell Physiol 289:C277-82
Gatto, Craig; Helms, Jeff B; Prasse, Megan C et al. (2005) Kinetic characterization of tetrapropylammonium inhibition reveals how ATP and Pi alter access to the Na+-K+-ATPase transport site. Am J Physiol Cell Physiol 289:C302-11
Helms, Jeff B; Arnett, Krista L; Gatto, Craig et al. (2004) Bretylium, an organic quaternary amine, inhibits the Na,K-ATPase by binding to the extracellular K-site. Blood Cells Mol Dis 32:394-400
MacDiarmid, Colin W; Milanick, Mark A; Eide, David J (2003) Induction of the ZRC1 metal tolerance gene in zinc-limited yeast confers resistance to zinc shock. J Biol Chem 278:15065-72

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