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Cysteinyl residues participate in regulation of SVCT1‐mediated L‐ascorbic acid transport
Author(s) -
Illing Anthony C,
Shawki Ali,
Eyster Thomas W,
Mackenzie Bryan
Publication year - 2006
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.20.5.a840
Subject(s) - ascorbic acid , cotransporter , chemistry , xenopus , membrane transport , mutagenesis , transporter , dehydroascorbic acid , biochemistry , cysteine , ion transporter , vitamin c , mutation , membrane , gene , enzyme , food science , organic chemistry , sodium
Intestinal and renal absorption of vitamin C (L‐ascorbic acid, L‐Asc) is mediated by the Na + /L‐Asc cotransporter SVCT1 (SLC23A1). We are exploring the molecular mechanisms and structure‐function of SVCT1 using radiotracer and voltage‐clamp techniques in cRNA‐injected Xenopus oocytes. Uptake of 100 μM L‐[ 14 C]Asc was inhibited by 1‐h preincubation with sulfhydryl‐reactive agents; for example, 1 mM NEM inhibited uptake by 99 ± 1% versus untreated, 19 ± 4 (SD) pmol.min 1 . Since pCMB (membrane‐permeant) inhibited L‐[ 14 C]Asc transport to the same extent as pCMBS (impermeant), we targeted all three exofacial cysteinyl residues for site‐directed mutagenesis. Individual C71A, C129A, and C342A mutations decreased L‐[ 14 C]Asc uptake by 57–65%, and increased > 6‐fold the K 0.5 for L‐Asc (each ≈0.4 mM, compared with 60 ± 10 μM in wildtype). Double and triple mutations abolished transport activity. Critical exofacial cysteinyl residues in SVCT1 may therefore participate in regulation of vitamin C transport activity by sensing the cell's redox environment.