The mechanism by which mammalian cells regulate sodium, potassium, calcium and pH in response to hormones and other environmental factors has been extensively studied at the physiological level but is poorly understood at the biochemical level. The long term goal of this grant has been to understand the biochemical mechanism by which cation transport across the plasma membrane of mammalian cells is regulated by purifying the transport systems and regulatory systems to homogeneity. The two systems which have been the focus of this grant in the past few years and which will continue to be the focus are the (Na,K) ATPase, the enzyme primarily responsible for establishing cation gradients in most cells and an extracellullar ATP receptor which rapidly abolishes plasma membrane cation gradients. The study of the (Na,K)ATPase will focus on the mechanism by which expression of a novel """"""""ouabain resistance gene"""""""" results in a totally ouabain insensitive (Na,K)ATPase in the plasma membrane of green monkey CV-1 fibroblasts. This gene was isolated by the ability to confer ouabain resistance on green monkey fibroblasts upon transfection of genomic DNA from murine fibroblasts. A 6.5 kbase fragment of murine genomic DNA has cloned into a plasmid and shown to confer ouabain resistance in recipients cells. The gene has been partially sequenced and the predicted coding region has no homology to the alpha or beta subunits of the (Na,K)ATPase. The goal of this grant is to complete the gene sequence, isolate a cDNA clone, verify that expression of the cDNA clone also confers ouabain resistance, obtain antibodies against the expressed protein and determine the function of the gene product. The physiological relevance of this gene is not understood but possible functions include 1) the ability to confer tissue specific resistance to circulating inhibitors of the pump, 2) the ability to alter cation transport stoichiometries in specific tissues. Since the (Na,K)ATPase is almost always defined as the ouabain sensitive component of cation transport or ATPase activity the completely ouabain resistant form of the pump found in cells expressing the ouabain resistance gene could have easily been overlooked in past studies. A second goal is to isolate the extracellular receptor for ATP which regulates plasma membrane sodium, calcium and potassium fluxes. Extracellular ATP is much more effective than any other hormone or growth regulator in stimulating plasma membrane cation fluxes in Friend erythroleukemia cells, murine T cells and rat parotid cells. Although there are reports that extracellular ATP is a membrane permeabilizing agent in some cells, this is clearly not so in the cells listed above: small organic anions do not become permeant. A 50% maximal effect is observed at 10 uM ATP4- and it appears likely that ATP4- which is sorted at very high concentration (>100 mM) in many secretory vesicles is acting locally as a hormone in synergy with the other hormones and/or transmitter with which it is stored.
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