The long-term objectives of this research are to elucidate some of the mechanisms of ion transport, to determine how changes of the cytosolic mileau modulate ion transport, and to determine how changes of the cellular environment alter transport rate. The focus of this project is the calmodulin activated Ca pump of red cells, which Is a model for plasma membrane Ca pumps in other cells. Recent evidence indicates that the Ca pump mediates Ca/ll exchange and also Ca transport without H movement. Current models of the Ca pump, while including roles for ATP, ADP, phosphate, and Ca, do not explicitly consider protons.
The specific aims of this grant are to examine the effects of extracellular protons and ATP on the red cell Ca pump. Some of the questions to be examined are: l. Are protons important for resetting the pump mechanism after the Ca half cycle? 2. Must Ho bind before ATP can bind to accelerate the return of the transport sites before the next pump cycle can occur? 3. When protons are not transported, what is transported instead? The experimental protocol is to examine the effect of ATP and H on several partial reactions of the Ca pump, including Ca/Ca exchange, pump, including Ca/Ca exchange reversal and phosphointermediate decomposition. One of the goals of a kinetic study is to determine which conformational changes are rate limiting. Regulation of the rate of these conformational changes by alterations in the membrane environment would modulate pump activity. In the red cell it is possible to determine the separate effects of intracellular protons, calcium ions, nucleotide concentrations, and extracellular protons and calcium ions. This is important since, e.g., Cai and Ho are substrates but Cao and Hi are products of the forward pump cycle. A transient rise in Ca is important for signalling in such processes as cardiac, skeletal, and smooth muscle contraction, cell differentiation and cell poliferation. The Ca pump restores Ca to basal levels and therefore assists in terminating the signal. Defects in Ca pump activity would lead to an increase in Ca with subsequent increases in Na and K. These changes would alter key cytoplasmic functions. Such ionic alterations have been implicated in several disease states including renal and cardiac failure, hypertension, and muscular dystrophy. Alterations of the red cell Ca pump have been reported in sickle cell disease.
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