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Lys‐84 is located in the translocation pathway of the Na + /dicarboxylate cotransporter
Author(s) -
Weerachayaphorn Jittima,
Pajor Ana M
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a530-c
Subject(s) - cotransporter , chemistry , cysteine , substrate (aquarium) , mutagenesis , mutant , steric effects , transmembrane domain , biochemistry , helix (gastropod) , symporter , stereochemistry , membrane , biophysics , enzyme , transporter , biology , organic chemistry , ecology , snail , gene , sodium
The Na + /dicarboxylate cotransporter, NaDC1, transports Na + with citric acid cycle intermediates, such as succinate and citrate. Transmembrane helix (TM) 3 is highly conserved and may be important for succinate and glutarate transport. The goal of this study was to determine whether TM3 forms part of the substrate permeation pathway. The extracellular half of TM3 from Ile‐98 to Arg‐112, and Lys‐84, were studied by cysteine‐scanning mutagenesis. The cysteine mutants were expressed in HRPE cells and assayed by Transport Specificity Ratio (TSR) analysis, a measure of relative catalytic efficiency with succinate and citrate. Mutants V100C, L105C, R108C and A110C were inactive. TSR analysis suggests TM3 contains determinants for catalytic efficiency at positions 84, 101, 103, 106, 111. Mutants were tested for sensitivity to the membrane‐impermeant cysteine reagent, MTSES, but only K84C was inactivated. K84C was only accessible to the outside medium in Na + and had substrate protection from MTSES labeling, probably due to steric hindrance, rather than large‐scale conformational change. K84C may be found near the substrate binding site and its accessibility to the outside depends on conformational state. Our data suggest that TM3 contains residues involved in substrate recognition. Lys‐84, originally predicted to be inside the cell at the base of TM3, is probably in the transport pathway of NaDC1. (NIH grant DK46269.)