Cysteine residues located within the core domain of human vitamin C transporter, hSVCT2, are essential for its structural and functional properties.

SVCT2 is the most ubiquitous Vitamin C (Ascorbic Acid, AA) transporter, essential in cell function and disease, but its structural and transport mechanistic details remain unclear. Using the 3D structures of related transporters, including UapA and mouse SVCT1 (mSVCT1), a model for human SVCT2 (hSVCT2) was proposed, consisting of 14 transmembrane segments (TMS). Four of these TMS are positioned between the core and gate domains, with 14 cysteine residues located within these domains. To explore their role, HEK-293 cells expressing hSVCT2-GFP were treated with reducing (DTT) and alkylating agents (PCMB and NEM). Mutants of SVCT2 were created by replacing cysteine residues with serine, and their effects on protein expression, membrane localization, and transport were analyzed. Results revealed that cysteine oxidation did not impact SVCT2 activity, but treatments with PCMB or NEM completely inhibited AA transport by decreasing Vmax, without affecting Km or sodium cooperativity. Mutant transporters with cysteine substitutions at specific residues (113, 129, 160, 187, and 401) showed significantly reduced AA transport, but protein sorting remained unaffected. These findings provide new insights into the role of cysteines in hSVCT2 function and offer a foundation for further structural and functional analysis of this physiologically critical transporter.