title: Functional identification and characterization of sodium binding sites in Na symporters.
abstract: Sodium cotransporters from several different gene families belong to the leucine transporter (LeuT) structural family. Although the identification of Na(+) in binding sites is beyond the resolution of the structures, two Na(+) binding sites (Na1 and Na2) have been proposed in LeuT. Na2 is conserved in the LeuT family but Na1 is not. A biophysical method has been used to measure sodium dissociation constants (Kd) of wild-type and mutant human sodium glucose cotransport (hSGLT1) proteins to identify the Na(+) binding sites in hSGLT1. The Na1 site is formed by residues in the sugar binding pocket, and their mutation influences sodium binding to Na1 but not to Na2. For the canonical Na2 site formed by two -OH side chains, S392 and S393, and three backbone carbonyls, mutation of S392 to cysteine increased the sodium Kd by sixfold. This was accompanied by a dramatic reduction in the apparent sugar and phlorizin affinities. We suggest that mutation of S392 in the Na2 site produces a structural rearrangement of the sugar binding pocket to disrupt both the binding of the second Na(+) and the binding of sugar. In contrast, the S393 mutations produce no significant changes in sodium, sugar, and phlorizin affinities. We conclude that the Na2 site is conserved in hSGLT1, the side chain of S392 and the backbone carbonyl of S393 are important in the first Na(+) binding, and that Na(+) binding to Na2 promotes binding to Na1 and also sugar binding.
authors.affiliation: Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1751.
authors.forename: Donald D F
authors.initials: DD
authors.lastname: Loo
authors.forename: Xuan
authors.initials: X
authors.lastname: Jiang
authors.forename: Edurne
authors.initials: E
authors.lastname: Gorraitz
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Hirayama
authors.forename: Ernest M
authors.initials: EM
authors.lastname: Wright
mesh_terms: D001665:Binding Sites / Q000235:genetics*; D001703:Biophysics / Q000379:methods; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism*; D051269:Sodium-Glucose Transporter 1 / Q000235:genetics / Q000378:metabolism*
chemical_list: C495674:SLC5A1 protein, human; D051269:Sodium-Glucose Transporter 1; D012964:Sodium