MOLECULAR PHYSIOLOGY OF THE SODIUM PUMP

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DESCRIPTION (provided by applicant): The objective of the present work is to obtain a more complete understanding of the molecular physiology of the Na pump or Na,K-ATPase. The Na pump couples the active transport of Na and K ions across the plasma membrane of almost all mammalian cells to the catalysis of ATP hydrolysis. The Na pump is responsible for maintaining normal fluid and electrolyte balance and is essential for renal function and energising reabsorbtion and nutrient uptake. During the past several decades we have learned much about how this oligomeric protein carries out its important function. However, little is known about the control and regulation of the processes which enable the subunits to assemble after synthesis and be delivered as a functioning unit to the plasma membrane. In the present work we will employ the baculovirus-infected insect cell system to introduce Na pump subunits into cells which do not normally express them. We will examine the expression of the sheep alpha1 -subunit (a polytopic membrane protein of 1000 amino acids) and sheep Beta1-subunit (a protein of about 300 amino acids and a single membrane crossing) separately and together and using cell fractionation and metabolic labelling strategies investigate the factors which control their assembly in the endoplasmic reticulum and delivery to the plasma membrane via the golgi apparatus. By utilizing a series of mutations in each sub-unit we will investigate the role of molecular determinants which affect delivery to the plasma membrane, its stability in the membrane and its interactions with cytoskeletal elements. The Na pump is an essential transport system and the site of action of digitalis, the most widely used therapy in cardiac insufficiency. Prospects for improved therapies for cardiac function and for improving impaired renal function will be greatly aided when we have a better understanding of the regulation of the activity of the Na pump in cell membranes.