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 (K d ) 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 K d 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.I on coupled symporters, or cotransporters, such as hSGLT1 use electrochemical potential gradients to drive solutes into cells. A common finding for these transporters is that external Na + binds before the substrate. Na + binding induces a conformational change of the protein, resulting in the substrate vestibule becoming open to the external membrane surface. After substrate binding, the two ligands are transported across the membrane and are released into the cytoplasm. The atomic structures of several sodium dependent transporters have been solved [leucine transporter (LeuT), vibrio parahaemolyticus sodium glucose (vSGLT), sodium hydantoin (Mhp1), and sodium betaine (BetP)] (1-6). They share a common structural fold with a five-helix inverted repeat, the "LeuT fold". The substrate binding sites are located in the middle of the protein, isolated from the external and membrane surfaces by hydrophobic gates, and putative Na + sites have been identified. Testing these binding sites is a problem due to the fact that phenomenological kinetic constants for ligand transport (K 0.5 , half-saturation values) are interdependent on each other (7). Here we have developed a method to estimate the intrinsic Na + dissociation constants (K d ) for human sodium glucose cotransport (hSGLT1) that may be broadly applicable to other symporters. We then use this to investigate the importance of hSGLT1 residues predicted to be at or near the two Na + binding sites, Na1 and Na2.The method is based on the fact that voltage-dependent membrane proteins exhibit transient charge movements (capacitive transients) i...

Stochastic steps in secondary active sugar transport

“…S4D). The primary contact is D189, which has a typical Na + to carboxyl oxygen distance less than 3 Å, and the equivalent position in hSGLT1 (D204) was shown to be involved in Na + binding to Na2 (13). Additionally, A62, A63, I65, and S364 all contact the Na + to different degrees.…”

Structural and functional significance of water permeation through cotransporters

“…We have furthermore chemically modified these cysteine side chains with reversible alkylating reagents. Previous studies documented the importance of these side chains in Na + and sugar binding and transport (24)(25)(26).…”

“…WT hSGLT1 and mutant proteins were expressed in Xenopus laevis oocytes as described (24,26). Animal experiments at the University of Copenhagen were performed in accordance with the institutional guidelines mandated by the Danish National Committee for Animal Studies and at University of California, Los Angeles under the approval of the University of California, Los Angeles Animal Research Committee (ARC).…”

“…the SGLT1 capacitive currents (12,24,26,35). Briefly, the membrane potential of the oocyte was held at −50 mV and stepped to various test values (from +50 mV to −150 mV) for 100 ms before returning to −50 mV.…”

Na + coordination at the Na2 site of the Na + /I − symporter

“…The answers to several of these questions, however-such as what the binding order is-remain controversial (46)(47)(48)(49)(50). One topic of particular interest is the allosteric interaction between the two sites.…”