Premium
Essential role of a conserved transmembrane histidine residue in the activity of two homologous sodium‐dependent vitamin C transporters, SVCT1 and SVCT2
Author(s) -
Varma Saaket,
Kuo ShiuMing
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a244-c
Subject(s) - histidine , symporter , chemistry , biochemistry , mutant , transporter , transmembrane domain , wild type , sodium , residue (chemistry) , amino acid , gene , organic chemistry
The functional motifs of SVCTs are not known. The increase in the activity of SVCTs from pH 6.5 to 8.00 suggested a role of histidine residues. This work tested the hypothesis through point mutations of SVCT1‐EGFP and SVCT2‐EGFP. The membrane localization of mutants and their activities were determined through confocal microscopy and uptake assay, respectively, in MDCK cells. Amongst the four conserved histidine residues of SVCT1, H51, H147, H210 and H354, only H51A mutation significantly decreased sodium‐dependent uptake of AsA and increased Km without affecting membrane localization. H51N, but not C, Q, K, R, or D replacement, partially restored AsA uptake. In addition, compared to the wildtype and H51C, H51K showed a steeper increase in the activity at alkaline pH where lysine is more deprotonated. These observations suggested an important contribution of an uncharged amine group at position 51. H51Q mutant had significantly increased sodium‐independent dehydroascorbate (DHA) uptake but not the uptake of unrelated phenylalanine. Corresponding mutation in SVCT2‐EGFP, H109A, also showed a significant loss in the activity. This histidine is part of two highly conserved residues QH in nucleobase: cation symporter‐2 (NCS2) family which include SVCTs. QH to HQ mutation decreased AsA uptake in SVCT1‐EGFP. Overall, our results suggest that QH motif might be important for the function of all NCS2 family members. Supported by Mark Diamond Research Fund.