Search for the ouabain-binding site of Na,K-ATPase.
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RU Radboud Universiteit Nijmegen, 10 januari 2007
Promotor : Pont, J.J.H.H.M. de Co-promotor : Koenderink, J.B.
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SubjectUMCN 5.4: Renal disorders
Na,K-ATPase is an integral membrane protein found in almost all plasma membranes of higher eukaryotic cells. It maintains the electrochemical gradients present across the plasma membrane of the cells by catalyzing ATP-dependent transport of sodium and potassium ions. This enzyme is composed of two subunits, a catalytic a-subunit that crosses the membrane ten times and an accessory ß-subunit that crosses the membrane once. Cardiac glycosides such as ouabain specifically inhibit Na,K-ATPase activity. Cardiac glycosides are mainly used in the treatment of congestive heart failure and arrhythmias. The localization of the ouabain-binding site on Na,K-ATPase has been studied for many years, but the amino acids involved in direct binding are still unknown. Gastric and non-gastric H,K-ATPase, like Na,K-ATPase, belong to the P2-type ATPases and have a similar subunit composition and structure. Although the catalytic subunits of these three enzymes are about 63% identical, the former enzyme is not inhibited by ouabain. Thus those amino acids that differ might be important for the specificity of ouabain binding. Two polar but uncharged amino acids (Gln111 and Asn122) at the border of the first extracellular loop have been found are responsible for the high ouabain-sensitivity of non-rodent Na,K-ATPase. Using chimera-based experimental strategy, we demonstrated that a gastric H,K-ATPase that contained Glu312, Val314, Ile315, Gly319, Phe783, Thr797, and Asp804 of Na,K-ATPase bound ouabain with the same affinity as the native enzyme. In addition, we identified that Glu312, Glu312, Pro778, Leu795 and Cys802 of Na,K-ATPase are crucial for obtaining a high affinity ouabain binding site into non-gastric H,K-ATPase. Based on crystal structure of Ca2+-ATPase we constructed a homology model for the ouabain-binding site of Na,K-ATPase in which most of the found amino acids as well as several earlier postulated amino acids play a crucial role.
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