The loop between transmembrane helices 6 and 7 (L6/7) of P-type ATPases has been suggested to be important for the functional linkage of ion binding and enzyme phosphorylation or to be a site of initial cation binding. To investigate the role of L6/7 in Na,K-ATPase, alanine substitutions were made for charged and conserved residues in L6/7 of the human alpha1 subunit and the proteins were expressed in yeast for analysis. All mutants except the triple mutant E825A/E828A/D830A bound ouabain. Although the equilibrium dissociation constant for ouabain binding by most mutants was similar to the wild-type value, the K(d) of R837A for ouabain binding was approximately 15-fold higher than the wild-type K(d). (18)O exchange measurements indicated that the apparent affinity of this mutant for Pi was reduced about 3-fold. The concentration dependence of KCl inhibition of ouabain binding or of NaCl inhibition of ouabain binding revealed 2-4-fold changes in the apparent affinity for cations in the E825A, E828A, and R837A mutants. The E825A and E828A mutants lost the ability to bind ouabain after extraction with 0.1% SDS or after brief heating, indicating that these mutations affected the stability of the enzyme. The ATPase activity of the other mutants was measured after extraction of crude yeast membranes with 0.1% SDS. For all mutants except R834A, R837A, and R848A, the activity was at least 50% of wild-type activity.

Alanine substitutions were made for 15 amino acids in the cytoplasmic loop between transmembrane helices 6 and 7 (L6/7) of the human ␣ 1 -subunit of Na,K-ATPase. Most mutations reduced Na,K-ATPase activity by less than 50%; however, the mutations R834A, R837A, and R848A reduced Na,K-ATPase activity by 75, 89, and 66%, respectively. Steady-state phosphoenzyme formation from ATP was reduced in mutants R834A, R837A, and R848A, and R837A also had a faster E 2 P 3 E 2 dephosphorylation rate compared with the wild-type enzyme. Effects of L6/7 mutations on the phosphorylation domain of the protein were also demonstrated by 18 O exchange, which showed that intrinsic rate constants for P i binding and/or reaction with the protein were altered. Although most L6/7 mutations had no effect on the interaction of Na ؉ or K ؉ with Na,K-ATPase, the E825A, E828A, R834A, and R837A mutations reduced the apparent affinity of the enzyme for both Na 3؉ , indicating that Br-TITU 3؉ does not bind to charged residues in L6/7. This observation makes it unlikely that L6/7 functions as a cytoplasmic cation binding site in Na,K-ATPase, and together with the effects of L6/7 mutations on phosphate interactions with the enzyme suggests that L6/7 is important in stabilizing the phosphorylation domain and its relationship to the ion binding sites of the protein.Na,K-ATPase, also called the sodium pump, is a heterodimeric protein consisting of ␣-and ␤-subunits. It pumps 3 Na ϩ out of the cell and 2 K ϩ into the cell during each transport cycle at the expense of ATP hydrolysis, and this activity maintains transmembrane gradients of the ions. Na,K-ATPase belongs to the P-type ATPase family in which the mechanism of action involves formation of a transient, covalently phosphorylated intermediate during the reaction cycle. Other P-type ATPases include sarcoplasmic reticulum and plasma membrane Ca-ATPase, H,K-ATPase found in stomach and colon, and several prokaryotic transport enzymes (1-3). These P-type ATPases have similar structural organization, and amino acid sequences near the sites of ATP binding, phosphorylation, and Mg 2ϩ binding, and residues attributed to cation binding in the transmembrane domain are highly conserved (3, 4). The catalytic ␣-subunit of Na,K-ATPase is composed of 10 transmembrane helices (TM).1 Proteolysis experiments (5, 6) and sitedirected mutagenesis studies indicate that the cation occlusion sites are located within the transmembrane domain, and carboxyl and other oxygen-containing side chain of residues within TM 4, 5, 6, and 8 are crucial for occlusion of cations (7-12).According to the crystal structure of Ca-ATPase (13, 14), the parts of the polypeptide that are exposed in the cytoplasm are organized into three interacting domains designated A, N, and P. The P (phosphorylation) domain is composed of the intracellular loop between TM4 and TM5, with the N-terminal part (roughly Asn 330 -Asn 359 ) connecting to TM4, and the C-terminal part (roughly Lys 605 -Asp 737 ) connecting to TM5. The aspartate residue (Asp 351 ) ...