Kinetics of charge translocation in the passive downhill uptake mode of the Na+/H+ antiporter NhaA of Escherichia coli.
The Na+/H+ antiporter NhaA is the main Na+ extrusion system in E. coli. Using direct current measurements combined with a solid supported membrane (SSM), we obtained electrical data of the function of NhaA purified and reconstituted in liposomes. These measurements demonstrate NhaA's electrogenicity, its specificity for Li+ and Na+ and its pronounced pH dependence in the range pH 6.5-8.5. The mutant G338S, in contrast, presents a pH independent profile, as reported previously. A complete right-side-out orientation of the NhaA antiporter within the proteoliposomal membrane was determined using a NhaA-specific antibody based ELISA assay. This allowed for the first time the investigation of NhaA in the passive downhill uptake mode corresponding to the transport of Na+ from the periplasmic to the cytoplasmic side of the membrane. In this mode, the transporter has kinetic properties differing significantly from those of the previously investigated efflux mode. The apparent Km values were 11 mM for Na+ and 7.3 mM for Li+ at basic pH and 180 mM for Na+ and 50 mM for Li+ at neutral pH. The data demonstrate that in the passive downhill uptake mode pH regulation of the carrier affects both apparent Km as well as turnover (Vmax).
Zuber
D
D
Max Planck Institut für Biophysik, Max von Laue Strasse 3, D-60438 Frankfurt/Main, Germany.
Krause
R
R
Venturi
M
M
Padan
E
E
Bamberg
E
E
Fendler
K
K
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001693:Biological Transport, Active / Q000502:physiology; D004594:Electrophysiology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport / Q000502:physiology; D007700:Kinetics; D008081:Liposomes; D009154:Mutation / Q000235:genetics; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D000939:Epitopes; D029968:Escherichia coli Proteins; D008081:Liposomes; C102839:NhaA protein, E coli; D017923:Sodium-Hydrogen Exchangers; D012964:Sodium