Functional role of conserved transmembrane segment 1 residues in human sodium-dependent vitamin C transporters.
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 (p K a approximately 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.
Varma
Saaket
S
Department of Biochemistry, University at Buffalo, Buffalo, New York 14214, USA.
Campbell
Christine E
CE
Kuo
Shiu-Ming
SM
D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D001692:Biological Transport; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009154:Mutation; D029363:Organic Anion Transporters, Sodium-Dependent / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D060347:Sodium-Coupled Vitamin C Transporters; D013329:Structure-Activity Relationship; D027981:Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D029363:Organic Anion Transporters, Sodium-Dependent; C555162:SLC23A1 protein, human; C555163:SLC23A2 protein, human; D060347:Sodium-Coupled Vitamin C Transporters; D027981:Symporters; D049452:Green Fluorescent Proteins; D006639:Histidine; D001205:Ascorbic Acid