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

Abstract

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-[14C]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-[14C]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-[14C]Asc uptake by 57–65%, and increased > 6-fold the K0.5 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.