Transmembrane segment 12 of the Glut1 glucose transporter is an outer helix and is not directly involved in the transport mechanism

A model has been proposed for the exofacial configuration of the Glut1 glucose transporter in which eight transmembrane domains form an inner helical bundle stabilized by four outer helices. The role of transmembrane segment 12, predicted to be an outer helix in this hypothetical model, was examined by cysteine-scanning mutagenesis and the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A previously characterized functional cysteine-less Glut1 molecule was used to produce 21 Glut1 point mutants by changing each residue along helix 12 to a cysteine residue. These mutants were then expressed in Xenopus oocytes, and their protein levels, functional activities, and sensitivities to pCMBS were determined. Strikingly, in contrast to all nine other predicted Glut1 transmembrane helices that have been previously examined by this method, none of the 21 helix 12 single-cysteine mutants exhibited significant inhibition of specific transport activity. Also unlike most other Glut1 transmembrane domains in which solvent-accessible residues lie along a single face of the helix, mutations in five consecutive residues predicted to lie close to the exofacial face of the membrane resulted in sensitivity to pCMBS-induced transport inhibition. These results suggest that helix 12 plays a passive stabilizing role in the structure of Glut1 and is not directly involved in the transport mechanism. Additionally, the pCMBS data indicate that the predicted exoplasmic end of helix 12 is completely exposed to the external solvent when the transporter is in its exofacial configuration.     

The predictive value of transcranial duplex sonography for the clinical diagnosis in undiagnosed parkinsonian syndromes: comparison with SPECT scans

Background  

Transcranial duplex sonography (TCD) of the substantia nigra has emerged as a promising, non-invasive tool to diagnose idiopathic Parkinson's disease (IPD). However, its diagnostic accuracy in patients with undefined parkinsonism remains to be determined.     

Identification of amino acid residues within the C terminus of the Glut4 glucose transporter that are essential for insulin-stimulated redistribution to the plasma membrane

The Glut4 glucose transporter undergoes complex insulin-regulated subcellular trafficking in adipocytes. Much effort has been expended in an attempt to identify targeting motifs within Glut4 that direct its subcellular trafficking, but an amino acid motif responsible for the targeting of the transporter to insulin-responsive intracellular compartments in the basal state or that is directly responsible for its insulin-stimulated redistribution to the plasma membrane has not yet been delineated. In this study we define amino acid residues within the C-terminal cytoplasmic tail of Glut4 that are essential for its insulin-stimulated translocation to the plasma membrane. The residues were identified based on sequence similarity (LXXLXPDEXD) between cytoplasmic domains of Glut4 and the insulin-responsive aminopeptidase (IRAP). Alteration of this putative targeting motif (IRM, insulin-responsive motif) resulted in the targeting of the bulk of the mutant Glut4 molecules to dispersed membrane vesicles that lacked detectable levels of wild-type Glut4 in either the basal or insulin-stimulated states and completely abolished the insulin-stimulated translocation of the mutant Glut4 to the plasma membrane in 3T3L1 adipocytes. The bulk of the dispersed membrane vesicles containing the IRM mutant did not contain detectable levels of any subcellular marker tested. A fraction of the total IRM mutant was also detected in a wild-type Glut4/Syntaxin 6-containing perinuclear compartment. Interestingly, mutation of the IRM sequence did not appreciably alter the subcellular trafficking of IRAP. We conclude that residues within the IRM are critical for the targeting of Glut4, but not of IRAP, to insulin-responsive intracellular membrane compartments in 3T3-L1 adipocytes.     

Analysis of transmembrane segment 8 of the GLUT1 glucose transporter by cysteine-scanning mutagenesis and substituted cysteine accessibility

The GLUT1 glucose transporter has been proposed to form an aqueous substrate translocation pathway via the clustering of several amphipathic transmembrane helices (Mueckler, M., Caruso, C., Baldwin, S. A., Panico, M., Blench, I., Morris, H. R., Allard, W. J., Lienhard, G. E., and Lodish, H. F. (1985) Science 229, 941-945). The possible role of transmembrane helix 8 in the formation of this permeation pathway was investigated using cysteine-scanning mutagenesis and the membrane-impermeant sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). Twenty-one GLUT1 mutants were created from a fully functional cysteine-less parental GLUT1 molecule by successively changing each residue along transmembrane segment 8 to a cysteine. The mutant proteins were then expressed in Xenopus oocytes, and their membrane concentrations, 2-deoxyglucose uptake activities, and sensitivities to pCMBS were determined. Four positions within helix 8, alanine 309, threonine 310, serine 313, and glycine 314, were accessible to pCMBS as judged by the inhibition of transport activity. All four of these residues are clustered along one face of a putative alpha-helix. These results suggest that transmembrane segment 8 of GLUT1 forms part of the sugar permeation pathway. Updated two-dimensional models for the orientation of the 12 transmembrane helices and the conformation of the exofacial glucose binding site of GLUT1 are proposed that are consistent with existing experimental data and homology modeling based on the crystal structures of two bacterial membrane transporters.     

Reconstitution of phosphoinositide 3-kinase-dependent insulin signaling in a cell-free system

Early insulin signaling events were examined in a novel cell-free assay utilizing subcellular fractions derived from 3T3-L1 adipocytes. The following cellular processes were observed in vitro in a manner dependent on insulin, time of incubation, and exogenous ATP: 1) autophosphorylation and activation of the insulin receptor; 2) tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1); 3) association of tyrosine-phosphorylated IRS-1 with phosphoinositide 3-kinase; 4) activation of the kinase Akt via its phosphorylation on Thr-308 and Ser-473; and 5) phosphorylation of glycogen synthase kinase-3 by activated Akt. The activation of Akt in vitro was abolished in the presence of the phosphoinositide 3-kinase inhibitor, wortmannin, thus recapitulating the most notable regulatory feature of Akt observed in vivo. Evidence is presented indicating that the critical spatial compartmentalization of signaling molecules necessary for efficient signal transduction is likely to be preserved in the cell-free system. Additionally, data are provided demonstrating that full Akt activation in this system is dependent on plasma membrane-associated IRS-1, cannot be mediated by robust cytosol-specific tyrosine phosphorylation of IRS-1, and occurs in the complete absence of detectable IRS-2 phosphorylation in the cytosol and plasma membrane.     

Wolframin expression induces novel ion channel activity in endoplasmic reticulum membranes and increases intracellular calcium

Wolfram syndrome is an autosomal recessive neuro-degenerative disorder associated with juvenile onset non-autoimmune diabetes mellitus and progressive optic atrophy. The disease has been attributed to mutations in the WFS1 gene, which codes for a protein predicted to possess 9-10 transmembrane segments. Little is known concerning the function of the WFS1 protein (wolframin). Endoglycosidase H digestion, immunocytochemistry, and subcellular fractionation studies all indicated that wolframin is localized to the endoplasmic reticulum in rat brain hippocampus and rat pancreatic islet beta-cells, and after ectopic expression in Xenopus oocytes. Reconstitution of wolframin from oocyte membranes into planar lipid bilayers demonstrated that the protein induced a large cation-selective ion channel that was blocked by Mg2+ or Ca2+. Inositol triphosphate was capable of activating channels in the fused bilayers that were similar to channel components induced by wolframin expression. Expression of wolframin also increased cytosolic calcium levels in oocytes. Wolframin thus appears to be important in the regulation of intracellular Ca2+ homeostasis. Disruption of this function may place cells at risk to suffer inappropriate death decisions, thus accounting for the progressive beta-cell loss and neuronal degeneration associated with the disease.     

Cysteine-scanning mutagenesis of transmembrane segment 7 of the GLUT1 glucose transporter

The human erythrocyte facilitative glucose transporter (Glut1) is predicted to contain 12 transmembrane spanning alpha-helices based upon hydropathy plot analysis of the primary sequence. Five of these helices (3, 5, 7, 8, and 11) are capable of forming amphipathic structures. A model of GLUT1 tertiary structure has therefore been proposed in which the hydrophilic faces of several amphipathic helices are arranged to form a central aqueous channel through which glucose traverses the hydrophobic lipid bilayer. In order to test this model, we individually mutated each of the amino acid residues in transmembrane segment 7 to cysteine in an engineered GLUT1 molecule devoid of all native cysteines (C-less). Measurement of 2-deoxyglucose uptake in a Xenopus oocyte expression system revealed that nearly all of these mutants retain measurable transport activity. Over one-half of the cysteine mutants had significantly reduced specific activity relative to the C-less protein. The solvent accessibility and relative orientation of the residues within the helix was investigated by determining the sensitivity of the mutant transporters to inhibition by the sulfhydryl directed reagent p-chloromercuribenzene sulfonate (pCMBS). Cysteine replacement at six positions (Gln(282), Gln(283), Ile(287), Ala(289), Val(290), and Phe(291)), all near the exofacial side of the cell membrane, produced transporters that were inhibited by incubation with extracellular pCMBS. Residues predicted to be near the cytoplasmic side of the cell membrane were minimally affected by pCMBS. These data demonstrate that the exofacial portion of transmembrane segment 7 is accessible to the external solvent and provide evidence for the positioning of this alpha-helix within the glucose permeation pathway.