Mapping the urea channel through the rabbit Na(+)-glucose cotransporter SGLT1.

1. The rabbit Na(+)-glucose cotransporter rbSGLT1 and its carboxy-terminal part, C5, which contains transmembrane helices 10-14 of SGLT1 and functions as a low affinity glucose uniporter, were expressed as individual proteins in Xenopus oocytes. Transport of 55 microM urea, ethylene glycol, mannitol and alpha-methyl-D-glucopyranoside (alphaMDG) by control oocytes and by oocytes expressing SGLT1 and C5 was studied by uptake measurements of the 14C-labelled substrates.

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

A truncated human Na(+)/glucose cotransporter (C(5), residues 407-664) was expressed and purified from Escherichia coli using a GST fusion vector and glutathione affinity chromatography. The truncated transporter (C(5)) was cleaved from GST-C(5) by Factor Xa proteolysis and purified by gel filtration chromatography. Up to 1 mg of purified GST-C(5) was obtained from 1 l bacterial culture.

Structure and function of the Na+/glucose cotransporter.

Cotransporters are a major class of membrane transport proteins that are responsible for the accumulation of nutrients, neurotransmitters, osmolytes and ions in cells from bacteria to man. The energy for solute accumulation comes from the proton and/or sodium electrochemical gradients that exist across cell membranes. A major problem in biology is how transport is coupled to these electrochemical potential gradients.

Neutralization of conservative charged transmembrane residues in the Na+/glucose cotransporter SGLT1.

Our goal was to identify pairs of charged residues in the membrane domains of the Na+/glucose cotransporter (SGLT1) that form salt bridges, to obtain information about packing of the transmembrane helices. The strategy was to neutralize Glu225, Asp273, Asp294, and Lys321 in helices 6-8, express the mutants in oocytes, measure [14C]-alphaMDG uptake, and then attempt to find second-site mutations of opposite charge that restored function. alphaMDG uptake by E225A was identical to that by SGLT1, whereas transport was reduced by over 90% for D273A, D294A, and K321A and was not restored in the double mutants D273A/K321A or D294A/K321A.

Five transmembrane helices form the sugar pathway through the Na+/glucose cotransporter.

To test the hypothesis that the C-terminal half of the Na+/glucose cotransporter (SGLT1) contains the sugar permeation pathway, a cDNA construct (C5) coding for rabbit SGLT1 amino acids 407-662, helices 10-14, was expressed in Xenopus oocytes. Expression and function of C5 was followed by Western blotting, electron microscopy, radioactive tracer, and electrophysiological methods. The C5 protein was synthesized in 20-fold higher levels than SGLT1.

Biophysical characteristics of the pig kidney Na+/glucose cotransporter SGLT2 reveal a common mechanism for SGLT1 and SGLT2.

The Na+-dependent, low affinity glucose transporter SGLT2 cloned from pig kidney is 76% identical (at the amino acid level) to its high affinity homologue SGLT1. Using two-microelectrode voltage clamp, we have characterized the presteady-state and steady-state kinetics of SGLT2 expressed in Xenopus oocytes. The kinetic properties of the steady-state sugar-evoked currents as a function of external Na+ and alpha-methyl-D-glucopyranoside (alphaMG) concentrations were consistent with an ordered, simultaneous transport model in which Na+ binds first.

Kinetic and specificity differences between rat, human, and rabbit Na+-glucose cotransporters (SGLT-1).

The Na+ activation and substrate specificity of human, rabbit, and rat Na+-glucose cotransporter (SGLT-1) isoforms were characterized using the Xenopus oocyte expression system and the two-electrode voltageclamp method. We find that there are differences, major and minor, in both the kinetics and substrate specificities between these isoforms; the substrate concentration at half-maximal current (K0.5) for hexoses varies from 0.

Sugar binding to Na+/glucose cotransporters is determined by the carboxyl-terminal half of the protein.

d-Glucose is absorbed across the proximal tubule of the kidney by two Na+/glucose cotransporters (SGLT1 and SGLT2). The low affinity SGLT2 is expressed in the S1 and S2 segments, has a Na+:glucose coupling ratio of 1, a K0.5 for sugar of approximately 2 mM, and a K0.

Arginine-427 in the Na+/glucose cotransporter (SGLT1) is involved in trafficking to the plasma membrane.

To investigate the role of charged intramembrane residues in the function of the rabbit Na+/glucose cotransporter (rbSGLT1) we substituted arginine-427 (R427) by alanine in the putative domain M9 SGLT1. This residue is conserved in all the members of the SGLT1 family. The mutant protein (R427A) was expressed in Xenopus oocytes and, although Western blot analysis revealed that it was produced in amounts comparable to wild-type, no function was measured.

Kinetics of steady-state currents and charge movements associated with the rat Na+/glucose cotransporter.

The rat Na+/glucose cotransporter (SGLT1) was expressed in Xenopus oocytes and steady-state and transient currents were measured using a two-electrode voltage clamp. The maximal glucose induced Na(+)-dependent inward current was approximately 300-500 nA. The apparent affinity constants for sugar (alpha-methyl-D-glucopyranoside; alpha MDG) (K alpha MDG 0.

'Active' sugar transport in eukaryotes.

Sugar transporters in prokaryotes and eukaryotes belong to a large family of membrane proteins containing 12 transmembrane alpha-helices. They are divided into two classes: one facilitative (uniporters) and the other concentrative (cotransporters or symporters). The concentrative transporters are energised by either H+ or Na+ gradients, which are generated and maintained by ion pumps.

SAAT1 is a low affinity Na+/glucose cotransporter and not an amino acid transporter. A reinterpretation.

Recently a member of the Na+/glucose (SGLT1) gene family of cotransporters was isolated from a pig renal cell line and was thought to be the neutral amino acid transporter System A. This cDNA (Kong, C. T.

Sodium/D-glucose cotransporter charge movements involve polar residues.

Na(+)-dependent glucose transporters (SGLT1) exhibit transient carrier currents with a time constant (tau) of 2-20 ms, and the charge transfer (Q) fits the Boltzmann equation. There is a 60-mV negative displacement in the tau/V and Q/V curves between the human and rabbit SGLT1 proteins, and the initial goal was to identify the charges responsible for these differences in kinetics. We have focused on residue 176 in putative transmembrane helix (M4) because this is an aspartic acid in rabbit and asparagine in human.

Sequence comparison of the sodium-D-glucose cotransport systems in rabbit renal and intestinal epithelia.

Using an isolated 1.2 kbp PCR product as specific probe, the rabbit renal cortex sodium-D-glucose cotransport system has been found and cloned from a renal cortex cDNA library. Two overlapping cDNA clones were found, one with a length of 2.

Characterization of an estradiol-stimulated mRNA in the brain of adult male rats.

Differential hybridization of a cDNA library from rat C6 glioma cells with cDNA probes from naive C6 glioma cells and from cells exposed to 17 beta-estradiol identified cDNAs of an mRNA stimulated by 17 beta-estradiol. This mRNA designated ESP1 mRNA, reached maximal levels after 8 h of treatment with 17 beta-estradiol. The stimulation was not suppressed by cycloheximide.