Functional consequences of alterations to Pro328 and Leu332 located in the 4th transmembrane segment of the alpha-subunit of the rat kidney Na+,K(+)-ATPase.
Site-specific mutagenesis was used to analyse the functional roles of the residues Pro328 and Leu332 located in the conserved PEGLL motif of the predicted transmembrane helix M4 in the alpha 1-subunit of the ouabain resistant rat kidney Na+,K(+)-ATPase. cDNAs encoding either of the Na+,K(+)-ATPase mutants Pro328-->Ala and Leu332-->Ala, and wild type, were cloned into the expression vector pMT2 and transfected into COS-1 cells. Ouabain-resistant clones growing in the presence of 10 microM ouabain were isolated, and the Na+,K+, ATP and pH dependencies of the Na+,K(+)-ATPase activity measured in the presence of 10 microM ouabain were analysed. Under these conditions the exogenous expressed Na+,K(+)-ATPase contributed more than 95% of the Na+,K(+)-ATPase activity. The Pro328-->Ala mutant displayed a reduced apparent affinity for Na+ (K0.5 (Na+) 13.04 mM), relative to the wild type (K0.5 (Na+) 7.13 mM). By contrast, the apparent affinity for Na+ displayed by the Leu332-->Ala mutant was increased (K0.5 (Na+) 3.92 mM). Either of the mutants exhibited lower apparent affinity for K+ relative to the wild type (K0.5 (K+) 2.46 mM for Pro328-->Ala and 1.97 mM for Leu332-->Ala, compared with 0.78 mM for wild type). Both mutants exhibited higher apparent affinity for ATP than the wild type (K0.5 (ATP) 0.086 mM for Pro328-->Ala and 0.042 mM for Leu332-->Ala, compared with 0.287 mM for wild type). The influence of pH was in accordance with an acceleration of the E2 (K)-->E1 transition in the mutants relative to the wild type. These data are consistent with a role of Pro328 and Leu332 in the stabilization of the E2 form and of Pro328 in Na+ binding. The possible role of the mutated residues in K+ binding is discussed.
Vilsen
B
B
Danish Biomembrane Research Centre, Institute of Physiology, University of Aarhus.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D004247:DNA; D005260:Female; D007668:Kidney / Q000201:enzymology* / Q000648:ultrastructure; D007930:Leucine / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010042:Ouabain / Q000494:pharmacology; D011392:Proline / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D017208:Rats, Wistar; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D010042:Ouabain; D004247:DNA; D011392:Proline; D000254:Sodium-Potassium-Exchanging ATPase; D007930:Leucine