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Mads Schak Toustrup-Jensen

Na+,K+-ATPase amino acids involved in transport of the 3rd sodium ion

Publikation: KonferencebidragPosterForskning

Available evidence indicates that two of the three Na+ ions bound in the E1 form occupy approximately the same positions as the K+ ions in E2, but the location of the third Na+ ion is unsolved. We have previously found a marked decrease in Na+ affinity for activation of phosphorylation in the human α3 mutant D923N, which is associated with RDP [1]. D923 is located in the cytoplasmic half of transmembrane helix M8 in a putative transport channel between M5, M7, M8 and M10. The external K+ sites behaved wild type (wt)-like in the mutant, suggesting that D923 is associated with the third Na+ ion. I have mutated several residues of the rat α1 isoform related to the channel structure and have characterized the effects on Na+ and K+ affinities and the E2-E1 and E1P-E2P conformational transitions of the pump cycle. Mutation D928N of rat α1, equivalent to D923N of human α3, shows a conspicuous reduction of apparent Na+ affinity without effect on external K+ affinity. D928L shows a large reduction of apparent Na+ affinity, even though the E2-E1 conformational equilibrium is shifted strongly in favor of the Na+ binding E1 form. Located deeper in the channel than D923/928 is Q856. Mutants Q856L, Q856A and Q856E exhibit ~24-,~9- and ~4-fold reduction of Na+ affinity, respectively, relative to wt, and the Q856 mutants display wt-like interaction with K+ at the E2P sites, thus supporting the hypothesis that the channel containing D923/928 and Q856 is a transport pathway for the third Na+ ion. I mutated C932 close to the proposed channel inlet. C932F reduced Na+ affinity ~73-fold. This fits into a model, where the bulky phenylalanine prevents Na+ from entering the channel.
Udgivelsesår23 sep. 2012
StatusUdgivet - 23 sep. 2012
Begivenhed3rd International Workshop on Expression, Structure and Function of Membrane Proteins - Plesso Didattico of the University of Florence, Florence, Italien
Varighed: 23 sep. 201227 sep. 2012


Workshop3rd International Workshop on Expression, Structure and Function of Membrane Proteins
LokationPlesso Didattico of the University of Florence

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