Contribution to Tl⁺, K⁺, and Na⁺ Binding of Asn⁷⁷⁶, Ser⁷⁷⁵, Thr⁷⁷⁴, Thr⁷⁷², and Tyr⁷⁷¹ in Cytoplasmic Part of Fifth Transmembrane Segment in α-Subunit of Renal Na,K-ATPase

Abstract

The sequence Y771TLTSNIPEIT781P in the fifth transmembrane segment of the alpha-subunit of Na,K-ATPase is unique among cation pump proteins. Here, in search of the molecular basis for Na,K specificity, alanine and conservative substitutions were directed to six oxygen-carrying residues in this segment. The contribution of the residues to cation binding was estimated from direct binding of Tl+ [Nielsen, et al. (1998) Biochemistry 37, 1961-1968], K+ displacement of ATP binding at equilibrium, and Na+-dependent phosphorylation from ATP in the presence of oligomycin. As an intrinsic control, substitution of Thr781 had no effect on Tl+(K+) or Na+ binding. There are several novel observations from this work. First, the carboxamide group of Asn776 is equally important for binding Tl+(K+) or Na+, whereas a shift of the position of the carboxamide of Asn776 (Asn776Gln) causes a large depression of Na+ binding without affecting the binding of Tl+(K+). Second, Thr774 is important for Na+ selectivity because removal of the hydroxyl group reduces the binding of Na+ with no effect on binding of Tl+(K+). Removal of the methyl groups of Thr774 or Thr772 reduces binding of both Tl+(K+) and Na+, whereas the hydroxyl group of Thr772 does not contribute to cation binding. Furthermore, the hydroxyl groups of Ser775 and Tyr771 are important for binding both Tl+(K+) and Na+. The data suggest that rotating or tilting of the cytoplasmic part of the fifth transmembrane segment may adapt distances between coordinating groups and contribute to the distinctive Na+/K+ selectivity of the pump.

Importance of Conserved α-Subunit Segment709GDGVND for Mg2+ Binding, Phosphorylation, and Energy Transduction in Na,K-ATPase

“…This agrees with the prevention of selective tryptic or chymotryptic cleavage of exposed bonds near the TGES segment after transition to the E 2 P form (2). Transmission of structural changes from the P domain to M4 and M5 may alter distances between cation coordinating residues to adapt from sites that are selective for Na ϩ with a diameter of 1.9 Å to sites that prefer K ϩ with a diameter of 2.7 Å (12,22 action, the Asp 710 3 Ala mutation does increase the K1 ⁄2 for phosphate, but after correction for the change in affinity for ouabain, there is an increase of the estimated apparent affinity for phosphate (Table V). For both wild type and mutations, the apparent affinities for phosphate are low, in the millimolar range, in the E 2 P⅐ouabain complex (Table V).…”

Electrogenicity of Na,K- and H,K-ATPase Activity and Presence of a Positively Charged Amino Acid in the Fifth Transmembrane Segment

“…DISCUSSION We have taken advantage of the functional differences and the high degree of sequence homology between the H,K-and Na,K-ATPases to attempt to identify the determinant of the electrogenicity of cation transport by the group IIc P-ATPases. In the fifth transmembrane segment, several amino acid residues have been shown to play a role in cation binding in both the H,K-and Na,K-ATPases (22,23,(25)(26)(27)(28)(29)(30)(31) and also in SERCA (32). The middle of the fifth transmembrane segment region is highly similar between the H,K-and Na,K-ATPases (Fig.…”

“…Only the S782A mutant had a very small but significant 86 Rb uptake of 2.6 Ϯ 1.1 pmol/min, whereas the wild-type Na,KATPase expressed a transport activity of 45.1 Ϯ 8.6 pmol/min, similar to that of the wild-type H,K-ATPase. A low affinity for extracellular K ϩ has been observed with mutants of the corresponding position (Ser 775 ) in ␣ 1 Na,K-ATPase from other species (21)(22)(23). Assuming a similar effect of homologous mutations in the Bufo ␣ 1 Na,K-pump, we studied the transport function of the Ser 782 mutants at a higher (40 mM) concentration of K ϩ .…”

Functional Properties of Na,K-ATPase, and Their Structural Implications, as Detected with Biophysical Techniques

“…Based on these precedents intensive mutagenesis studies of Ca-ATPase and Na,K-ATPase of such residues within transmembrane segments have been carried out [1,14]. For Na,K-ATPase, which is the main topic of this essay, there is evidence that E327 in M4, S775, T774, N776 and E779 in M5, D804 and D808 in M6 are involved in monovalent cation binding and occlusion [43,68,70,73].…”