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).
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