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J. Biol. Chem., Vol. 276, Issue 32, 29961-29968, August 10, 2001
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From the Department of Physiology and Biophysics, University of
Texas Medical Branch, Galveston, Texas 77555
The Na+/dicarboxylate
co-transporter, NaDC-1, couples the transport of sodium and Krebs cycle
intermediates, such as succinate and citrate. Previous studies
identified two functionally important amino acids, Glu-475 and Cys-476,
located in transmembrane domain (TMD) 9 of NaDC-1. In the present
study, each amino acid in TMD-9 was mutated to cysteine, one at a time,
and the accessibility of the membrane-impermeant reagent
[2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) to the
replacement cysteines was determined. Cysteine substitution was
tolerated at all but five of the sites: the A461C mutant was not
present at the plasma membrane, whereas the F473C, T474C, E475C, and
N479C mutants were inactive proteins located on the plasma membrane.
Cysteine substitution of four residues found near the extracellular
surface of TMD-9 (Ser-478, Ala-480, Ala-481, and Thr-482) resulted in
proteins that were sensitive to inhibition by MTSET. The accessibility
of MTSET to the four substituted cysteines was highest in the presence
of the transported cations, sodium or lithium, and low in choline. The
four mutants also exhibited substrate protection of MTSET
accessibility. The MTSET accessibility to S478C, A481C, and A480C was
independent of voltage. In contrast, T482C was more accessible to MTSET
in choline buffer at negative holding potentials, but there was no effect of voltage in sodium buffer. In conclusion, TMD-9 may be involved in transducing conformational changes between the
cation-binding sites and the substrate-binding site in NaDC-1, and it
may also form part of the translocation pathway through the transporter.
Conformationally Sensitive Residues in Transmembrane Domain 9 of
the Na+/dicarboxylate Co-transporter*
*
This work was supported by National Institutes of Health
Grants DK46269 and DK02429.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Physiology
and Biophysics, University of Texas Medical Branch, Galveston, TX
77555-0641. Tel.: 409-772-3434; Fax: 409-772-3381; E-mail: ampajor@utmb.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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