Functional Role of Conserved Transmembrane Segment 1 Residues in Human Sodium-Dependent Vitamin C Transporters

• Published in: Biochemistry (Easton) 2008-03, Vol.47 (9), p.2952-2960
• Main Authors: Varma, Saaket, Campbell, Christine E, Kuo, Shiu-Ming
• Format: Article
• Language: English
• Subjects: Animals; Ascorbic Acid - metabolism; Biological Transport; Cell Line; CHO Cells; Cricetinae; Cricetulus; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Histidine - chemistry; Histidine - genetics; Histidine - metabolism; Humans; Hydrogen-Ion Concentration; Microscopy, Confocal; Microscopy, Fluorescence; Models, Biological; Mutation; Organic Anion Transporters, Sodium-Dependent - chemistry; Organic Anion Transporters, Sodium-Dependent - genetics; Organic Anion Transporters, Sodium-Dependent - metabolism; Protein Structure, Tertiary; Sodium-Coupled Vitamin C Transporters; Structure-Activity Relationship; Symporters - chemistry; Symporters - genetics; Symporters - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi701666q

Sodium-dependent vitamin C transporters, SVCT1 and SVCT2, are the only two known proteins for the uptake of ascorbate, the active form of vitamin C. Little structural information is available for SVCTs, although a transport activity increase from pH 5.5 to 7.5 suggests a functional role of one or more conserved histidines (pK a ≈ 6.5). Confocal fluorescence microscopy and uptake kinetic analyses were used here to characterize cells transfected with mutants of EGFP-tagged hSVCTs. Mutating any of the four conserved histidine residues (His51, 147, 210, or 354) in hSVCT1 to alanine did not affect the apical membrane localization in polarized MDCK cells. His51Ala (in putative transmembrane segment 1, TM1) was the only mutation that resulted in a significant loss of ascorbate transport and an increase in apparent Km with no significant effect on Vmax. The corresponding mutation in hSVCT2, His109Ala, also led to a loss of transport activity. Among eight other mutations of His51 in hSVCT1, significant sodium-dependent ascorbate transport activity was only observed with asparagine or tyrosine replacement. Thus, our results suggest that uncharged His51, directly or indirectly, contributes to substrate binding through the hydrogen bond. His51 cannot account for the observed pH dependence as neutral amino acid substitutions failed to abolish the pH-dependent activity increase. The importance of TM1 is further strengthened by the comparable loss of sodium-dependent ascorbate transport activity upon the mutation of adjacent conserved Gln50 and the apparent change in substrate specificity in the hSVCT1-His51Gln mutation, which showed a specific increase in sodium-independent dehydroascorbate transport.