Residues of the fourth transmembrane segments of the Na,K-ATPase and the gastric H,H-ATPase contribute to cation selectivity

We have generated protein chimeras to investigate the role of the fourth tr ansmembrane segments (TM4) of the Na,K- and gastric H,K-ATPases in determin ing the distinct cation selectivities of these two pumps, Based on a helica l wheel analysis, three residues of TM4 of the Na,K-ATPase were changed to their H,K-counterparts. A construct carrying three mutations in TM4 (L319F, N326Y, and T340S) and two control constructs were heterologously expressed in Xenopus laevis oocytes and in the pig kidney epithelial cell line LLC-P K1, Biochemical ATPase assays demonstrated a large sodium-independent ATPas e activity at pH 6.0 for the pump carrying the TM4 substitutions, whereas t he control constructs exhibited little or no activity in the absence of sod ium, Furthermore, at pH 6.0 the K-1/2(Na+) shifted to 1.5 mM for the TM4 co nstruct compared with 9.4 and 5.9 mM for the controls. In contrast, at pH 7 .5 all three constructs had characteristics similar to wild type Na,K-ATPas e, Large increases in K-1/2(K+) were observed for the TM4 construct compare d with the control constructs both in two electrode voltage clamp experimen ts in Xenopus oocytes and in ATPase assays. ATPase assays also revealed a 1 0-fold shift in vanadate sensitivity for the TM4 construct. Based on these findings, it appears that the three identified TM4 residues play an importa nt role in determining both the specific cation selectivities and the E-1/E -2 conformational equilibria of the Na,K- and H,K-ATPase.