Five Transmembrane Helices Form the Sugar Pathway through the Na+/Glucose Cotransporter

Abstract

To test the hypothesis that the C-terminal half of the Na ؉ /glucose cotransporter (SGLT1) contains the sugar permeation pathway, a cDNA construct (C 5 ) coding for rabbit SGLT1 amino acids 407-662, helices 10 -14, was expressed in Xenopus oocytes. Expression and function of C 5 was followed by Western blotting, electron microscopy, radioactive tracer, and electrophysiological methods. The C 5 protein was synthesized in 20-fold higher levels than SGLT1. The particle density in the protoplasmic face of the oocyte plasma membrane increased 2-fold after C 5 -cRNA injection compared with noninjected oocytes. The diameters of the C 5 particles were heterogeneous (4.8 ؎ 0.3, 7.1 ؎ 1.2, and 10.3 ؎ 0.8 nm) in contrast to the endogenous particles (7.6 ؎ 1.2 nm). C 5 increased the ␣-methyl-D-glucopyranoside (␣MDG) uptake up to 20-fold above that of noninjected oocytes and showed an apparent K 0.5 ␣MDG of 50 mM and a turnover of ϳ660 s ؊1. Influx was independent of Na ؉ with transport characteristics similar to those of SGLT1 in the absence of Na2) inhibited by phloretin, K i PT ‫؍‬ ϳ500 M, and 3) insensitive to phlorizin. These results indicate that C 5 behaves as a specific low affinity glucose uniporter. Preliminary studies with three additional constructs, hC 5 (the human equivalent of C 5 ), hC 4 (human SGLT1 amino acids 407-648, helices 10 -13), and hN 13 (amino acids 1-648, helices 1-13), further suggest that helices 10 -13 form the sugar permeation pathway for SGLT1.Cotransporters are a major class of membrane proteins that use cation (Na ϩ , H ϩ , and Li ϩ ) gradients to drive the uphill transport of solutes and water into cells. Although many of these proteins have been cloned and subjected to structure/ function analyses, it has been problematic to draw definitive conclusions about their structure or to identify ligand binding sites and permeation pathways. This is largely due to the difficulty in obtaining structural information at the atomic level. Today, most of our understanding comes from indirect methods such as the comparison of the primary and secondary structure and function of homologous proteins, and functional analysis of chimeric proteins. Here we have studied the functional properties of protein truncations to probe the pathway for sugar transport through the Na ϩ -dependent glucose transporter, SGLT1 (1, 2).SGLT1 is a protein of 662 residues that contains 14 transmembrane helices (3). The structure and function of cloned SGLT1 isoforms and homologs, such as SMIT1 and SNST1, have been compared (4, 5), and the major differences were found to be concentrated toward the C termini. Functional analysis of a SGLT2-SGLT1 chimera (6) suggested that a Cterminal fraction of SGLT1 (residues 410 -662, helices 10 -14) determines both the sugar affinity and selectivity. To test if the terminal five helices of SGLT1 actually form the pathway for sugar transport, we expressed and functionally analyzed the truncated protein (residues 407-662) in Xenopus oocytes. It behaves as a specific low affinity glucose uniport...

Molecular Analysis of the SGLT2 Gene in Patients with Renal Glucosuria

“…If transferred to the brush border membrane at all, then the protein predicted by this mutation would have only the seven N-terminally located transmembranous domains, whereas domains 8 to 14 would be missing. Such a mutation has been shown to result in a nonfunctional glucose transporter because it lacks the transmembranous domains 10 to 13 that have been demonstrated to be essential for sugar binding and sugar translocation (15,19,20). It is interesting, however, that one patient (patient 06-1) homozygous for a truncating mutation showed a considerably lower glucose excretion, suggesting some glucose reabsorption by an unknown alternative mechanism.…”

Purification and functional reconstitution of a truncated human Na+/glucose cotransporter (SGLT1) expressed in E. coli

“…In this study, human C 5 was expressed as a fusion protein in Escherichia coli and puri¢ed by a¤nity and gel ¢ltration chromatography. Functional reconstitution of C 5 as well as the fusion protein (GST-C 5 ) in proteoliposomes revealed similar transport characteristics as described for C 5 when expressed in Xenopus oocytes [4].…”

“…Replacement of the C-terminus of the low a¤nity glucose transporter SGLT2 with the corresponding sequence of the high a¤nity SGLT1 in an SGLT2/SGLT1 chimera increased 10-fold the a¤nity for KMDG and like SGLT1 the chimera transported D-galactose and 3-O-methyl-glucose [3]. In addition, expression of the ¢ve C-terminal helices of SGLT1 (C 5 , residues 407^664) in Xenopus laevis oocytes exhibited a sugar uniporter phenotype [4]. Furthermore, mutation of glutamine 457 residing in C 5 to arginine (Q457R) causes glucose-galactose-malabsorption by blocking sugar translocation [5].…”

Alternative splicing of the rat sodium/bile acid transporter changes its cellular localization and transport properties

“…For example, selective truncation of the Na ϩ -glucose cotransporter SGLT1, a protein that normally comprises 13 transmembrane domains, generates a Na ϩ -independent carrier with properties similar to a facilitative glucose carrier. Indeed, truncated constructs encoding only five or four carboxyl-terminal transmembrane domains of the rabbit or human SGLT1, respectively, were still capable of mediating glucose influx (20).…”