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 α1-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 μM ouabain were isolated, and the Na+, K+, ATP and pH dependencies of the Na+,K+-ATPase activity measured in the presence of 10 μM 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+ (K 0.5 (Na+) 13.04 mM), relative to the wild type (K 0.5 (Na+) 7.13 mM). By contrast, the apparent affinity for Na+ displayed by the Leu 332→Ala mutant was increased (K 0.5 (Na+) 3.92 mM). Either of the mutants exhibited lower apparent affinity for K+ relative to the wild type (K 0.5 (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 (K 0.5 (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.
