Cysteinyl residues participate in regulation of SVCT1‐mediated L‐ascorbic acid transport

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.