The long-term objectives of this research are to elucidate some of the mechanisms of ion transport, to determine how changes of the cellular environment alter transport rate. The focus of this project is the calmodulin activated Ca pump of human red cells. The Ca pump offers an unusual opportunity for structure-function studies; a portion of the C terminal of the pump, binds to another portion of the pump to inhibit transport. The red cell Ca pump is also a model for plasma membrane Ca pumps from other cells. Experiments on similar modes of operation of the Na pump are planned in order to provide a basis for a comparison between these two pumps and to determine if the mechanism is conserved.
The specific aims are to: I. examine other modes of operation. In particular to compare the modulation of the rate of Na pump mediated ATP/ADP exchange and Na/Na exchange with the analogous modes of the Ca pump. These modes provide information about the order of ligand binding and release during the steady state operation of the Na (or Ca) portion of the pump cycle. II. characterize chemical modifiers and inhibitors. In particular to determine the relation between the eosin inhibition site, the FITC site and the ATP site. III. characterize auto-inhibitory peptide interactions. In particular to determine some electrostatic and steric constraints on the interaction between the C-terminal auto-inhibitory peptide, C20W, and the proteolysis-activated Ca pump. These 3 complimentary approaches will provide a foundation for evaluation of future chemical and genetic manipulations. For example, the conformations involved in Ca/Ca exchange may reduce the reactivity or accessibility of the lysine with which FITC reacts. The experimental protocol is to examine the effect of ligands, inhibitors and peptides on Ca uptake into inside out vesicles made from human red blood cells and to examine other modes of operation of the Ca pump. A transient rise in Ca is important for signalling in such processes as muscle contraction, cell differentiation and cell proliferation. 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 alterations of 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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