Structure-function activity of the human sodium-dependent multivitamin transporter: role of His&#185;&#185;&#8309; and His²⁵⁴.
Intestinal absorption of biotin occurs via a Na(+)-dependent carrier-mediated process that involves the sodium-dependent multivitamin transporter (SMVT; product of the Slc5a6 gene). The SMVT system is exclusively expressed at the apical membrane domain of the polarized intestinal epithelial cells. Whereas previous studies from our laboratory and others have characterized different physiological and biological aspects of SMVT, little is currently known about its structure-function activity relationship. Using site-directed mutagenesis approach, we examined the role of the positively charged histidine (His) residues of the human SMVT (hSMVT) in transporting the negatively charged biotin. Of the seven conserved (across species) His residues in the hSMVT polypeptide, only His¹¹⁵ and His²⁵⁴ were found to be important for the function of hSMVT as their mutation led to a significant reduction in carrier-mediated biotin uptake. This inhibition was mediated via a significant reduction in the maximal velocity (V(max)), but not the apparent Michaelis constant (K(m)), of the biotin uptake process and was not related to the charge of the His residue. The inhibition was also not due to changes in transcriptional or translational efficiency of the mutated hSMVT compared with wild-type carrier. However, surface biotinylation assay showed a significant reduction in the level of expression of the mutated hSMVT at the cell surface, a finding that was further confirmed by confocal imaging. Our results show important role for His¹¹⁵ and His²⁵⁴ residues in hSMVT function, which is most probably mediated via an effect on level of hSMVT expression at the cell membrane.
Ghosal
Abhisek
A
Department of Medicine, University of California, Irvine, USA.
Said
Hamid M
HM
D000595:Amino Acid Sequence; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins; D009154:Mutation; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D027981:Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; D012333:RNA, Messenger; D027981:Symporters; C119981:biotin transporter