Residues of the Fourth Transmembrane Segments of the Na,K-ATPase and the Gastric H,K-ATPase Contribute to Cation Selectivity
We have generated protein chimeras to investigate the role of the fourth transmembrane segments (TM4) of the Na,K- and gastric H,K-ATPases in determining the distinct cation selectivities of these two pumps. Based on a helical wheel analysis, three residues of TM4 of the Na,K-ATPase were changed to...
Gespeichert in:
Veröffentlicht in: | The Journal of biological chemistry 2000-01, Vol.275 (3), p.1749-1756 |
---|---|
Hauptverfasser: | , , , |
Format: | Artikel |
Sprache: | eng |
Schlagworte: | |
Online-Zugang: | Volltext |
Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
Zusammenfassung: | We have generated protein chimeras to investigate the role of the fourth transmembrane segments (TM4) of the Na,K- and gastric H,K-ATPases in determining the distinct cation selectivities of these two pumps. Based on a helical 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 inXenopus laevis oocytes and in the pig kidney epithelial cell line LLC-PK1. Biochemical ATPase assays demonstrated a large sodium-independent ATPase activity at pH 6.0 for the pump carrying the TM4 substitutions, whereas the control constructs exhibited little or no activity in the absence of sodium. Furthermore, at pH 6.0 the K½(Na+) shifted to 1.5 mm for the TM4 construct 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-ATPase. Large increases in K½(K+) were observed for the TM4 construct compared with the control constructs both in two-electrode voltage clamp experiments inXenopus oocytes and in ATPase assays. ATPase assays also revealed a 10-fold shift in vanadate sensitivity for the TM4 construct. Based on these findings, it appears that the three identified TM4 residues play an important role in determining both the specific cation selectivities and the E1/E2 conformational equilibria of the Na,K- and H,K-ATPase. |
---|---|
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.275.3.1749 |