TY  - JOUR
A1  - Mager, Thomas
A1  - Rimon, Abraham
A1  - Padan, Etana
A1  - Fendler, Klaus
T1  - Transport mechanism and pH regulation of the Na+/H+ antiporter NhaA from Escherichia coli : an electrophysiological study
T2  - The journal of biological chemistry
N2  - Using an electrophysiological assay the activity of NhaA was tested in a wide pH range from pH 5.0 to 9.5. Forward and reverse transport directions were investigated at zero membrane potential using preparations with inside-out and right side-out-oriented transporters with Na+ or H+ gradients as the driving force. Under symmetrical pH conditions with a Na+ gradient for activation, both the wt and the pH-shifted G338S variant exhibit highly symmetrical transport activity with bell-shaped pH dependences, but the optimal pH was shifted 1.8 pH units to the acidic range in the variant. In both strains the pH dependence was associated with a systematic increase of the Km for Na+ at acidic pH. Under symmetrical Na+ concentration with a pH gradient for NhaA activation, an unexpected novel characteristic of the antiporter was revealed; rather than being down-regulated, it remained active even at pH as low as 5. These data allowed a transport mechanism to advance based on competing Na+ and H+ binding to a common transport site and a kinetic model to develop quantitatively explaining the experimental results. In support of these results, both alkaline pH and Na+ induced the conformational change of NhaA associated with NhaA cation translocation as demonstrated here by trypsin digestion. Furthermore, Na+ translocation was found to be associated with the displacement of a negative charge. In conclusion, the electrophysiological assay allows the revelation of the mechanism of NhaA antiport and sheds new light on the concept of NhaA pH regulation.
Y1  - 2011
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/25544
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-255444
UR  - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3123120/
SN  - 0021-9258
SN  - 1083-351X
N1  - © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
VL  - 286
IS  - 26
SP  - 23570
EP  - 23581
PB  - American Society for Biochemistry and Molecular Biology
CY  - Bethesda, Md.
ER  -