Wolfram syndrome is an autosomal recessive neurodegenerative 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 ␤-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 Mg 2؉ or Ca 2؉ . 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 Ca 2؉ homeostasis. Disruption of this function may place cells at risk to suffer inappropriate death decisions, thus accounting for the progressive ␤-cell loss and neuronal degeneration associated with the disease.

Constitutively active G protein α subunits cause cancer, cholera, Sturge-Weber syndrome, and other disorders. Therapeutic intervention by targeted inhibition of constitutively active Gα subunits in these disorders has yet to be achieved. We found that constitutively active Gα in uveal melanoma (UM) cells was inhibited by the cyclic depsipeptide FR900359 (FR). FR allosterically inhibited guanosine diphosphate-for-guanosine triphosphate (GDP/GTP) exchange to trap constitutively active Gα in inactive, GDP-bound Gαβγ heterotrimers. Allosteric inhibition of other Gα subunits was achieved by the introduction of an FR-binding site. In UM cells driven by constitutively active Gα, FR inhibited second messenger signaling, arrested cell proliferation, reinstated melanocytic differentiation, and stimulated apoptosis. In contrast, FR had no effect on -driven UM cells. FR promoted UM cell differentiation by reactivating polycomb repressive complex 2 (PRC2)-mediated gene silencing, a heretofore unrecognized effector system of constitutively active Gα in UM. Constitutively active Gα and PRC2 therefore provide therapeutic targets for UM. The development of FR analogs specific for other Gα subunit subtypes may provide novel therapeutic approaches for diseases driven by constitutively active Gα subunits or multiple G protein-coupled receptors (GPCRs) where targeting a single receptor is ineffective.

Transmembrane helix 9 of the Glut1 glucose transporter was analyzed by cysteine-scanning mutagenesis and the substituted cysteine accessibility method (SCAM). A cysteine-less (C-less) template transporter containing amino acid substitutions for the six native cysteine residues present in human Glut1 was used to generate a series of 21 mutant transporters by substituting each successive residue in predicted transmembrane segment 9 with a cysteine residue. The mutant proteins were expressed in Xenopus oocytes, and their specific transport activities were directly compared to that of the parental C-less molecule whose function has been shown to be indistinguishable from that of native Glut1. Only a single mutant (G340C) had activity that was reduced (by 75%) relative to that of the C-less parent. These data suggest that none of the amino acid side chains in helix 9 is absolutely required for transport function and that this helix is not likely to be directly involved in substrate binding or translocation. Transport activity of the cysteine mutants was also tested after incubation of oocytes in the presence of the impermeant sulfhydryl-specific reagent, p-chloromercuribenzene sulfonate (pCMBS). Only a single mutant (T352C) exhibited transport inhibition in the presence of pCMBS, and the extent of inhibition was minimal (11%), indicating that only a very small portion of helix 9 is accessible to the external solvent. These results are consistent with the conclusion that helix 9 plays an outer stabilizing role for the inner helical bundle predicted to form the exofacial substrate-binding site. All 12 of the predicted transmembrane segments of Glut1 encompassing 252 amino acid residues and more than 50% of the complete polypeptide sequence have now been analyzed by scanning mutagenesis and SCAM. An updated model is presented for the outward-facing substrate-binding site and relative orientation of the 12 transmembrane helices of Glut1.The members of the GLUT/SLC2A family are membrane proteins that mediate the facilitative transfer of sugars across cellular membranes (1). These proteins belong to the major facilitator superfamily (MFS) 1 that contains nearly 3600 members in all three Kingdoms and represents the largest superfamily of membrane transporters (2-4). GLUT proteins and other MFS proteins are ubiquitously expressed and have been discovered in virtually every organism examined. At least five of the 14 mammalian GLUT proteins are glucose transporters that are expressed and regulated in a cell-specific fashion and that play distinct roles in cellular and organismal glucose homeostasis (5,6). Because glucose is an important or essential nutrient † This research was supported by a grant from the National Institutes of Health (R01 DK43695) for most mammalian cells, defects in its transport are associated with several disease states, including diabetes (7), Glut1 deficiency syndrome (8), and Fanconi-Bickel disease (9). Most of the available kinetic and biophysical data suggest that GLUT proteins operate via ...

A valine-to-isoleucine mutation at amino acid residue 197 of Glut2 or the equivalent residue 165 of Glut1 has been shown to impair glucose transport activity. This mutation was originally discovered in the Glut2 gene of a patient with type 2 diabetes. We investigated the mechanism of the effect of this mutation on transport activity via the analysis of Glut1 mutants expressed in Xenopus oocytes combined with cysteine substitution mutagenesis and the use of cysteine-reactive chemical probes. Aliphatic side chain substitutions at position 165 that were bulkier than the native valine residue inhibited glucose transport activity, whereas substitutions of less bulky side chains had little effect on transport, suggesting a role for steric hindrance. A cysteine residue was introduced at position 165 of a functional, cysteine-less Glut1 construct, and this mutant was then tested for inhibition of transport activity by a membrane-impermeant sulfhydryl-specific reagent (p-chloromercuribenzenesulfonate). p-Chloromercuribenzenesulfonate inhibited activity of the Cys 165 mutant when it was added to the external buffer but not when it was injected directly into oocytes, indicating that this residue is accessible from the external solvent but not from the cytoplasm. Competition experiments indicated that Cys 165 lies near the exofacial substrate-binding site or directly in the sugar permeation pathway. These data provide evidence that the side chain of Val 165 , which resides in the middle of transmembrane helix 5, juts into the aqueous permeation pathway of Glut1, probably between the exofacial substrate-binding site and the outer vestibule of the pathway.

Transmembrane segment 5 of the Glut1 glucose transporter has been proposed to form an amphipathic transmembrane helix that lines the substrate translocation pathway (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). This hypothesis was tested using cysteine-scanning mutagenesis in conjunction with the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A series of 21 mutants was created from a fully functional, cysteine-less, parental Glut1 molecule by changing each residue within putative transmembrane segment 5 to cysteine. Each mutant was then expressed in Xenopus oocytes and its steadystate protein level, 2-deoxyglucose uptake activity, and sensitivity to pCMBS were measured. All 21 mutants exhibited measurable transport activity, although several of the mutants exhibited reduced activity due to a corresponding reduction in steady-state protein. Six of the amino acid side chains within transmembrane segment 5 were clearly accessible to pCMBS in the external medium, as determined by inhibition of transport activity, and a 7th residue showed inhibition that lacked statistical significance because of the extremely low transport activity of the corresponding mutant. All 7 of these residues were clustered along one face of a putative ␣-helix, proximal to the exoplasmic surface of the plasma membrane. These results comprise the first experimental evidence for the existence of an amphipathic transmembrane ␣-helix in a glucose transporter molecule and strongly suggest that transmembrane segment 5 of Glut1 forms part of the sugar permeation pathway.

The Glut1 glucose transporter has been proposed to form an aqueous sugar translocation pathway through the lipid bilayer via the clustering of several 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 participation of transmembrane helix 10 in the formation of this putative aqueous tunnel was tested using cysteine-scanning mutagenesis in conjunction with the membrane-impermeant, sulfhydryl-specific reagent, pchloromercuribenzenesulfonate (pCMBS). A series of 21 mutants was created from a fully functional, cysteineless, parental Glut1 molecule by changing each residue within putative transmembrane segment 10 to cysteine. Each mutant was then expressed in Xenopus oocytes, and its plasma membrane content, 2-deoxyglucose uptake activity, and sensitivity to pCMBS were measured. Helix 10 exhibited a highly distinctive reaction profile to scanning mutagenesis whereby cysteine substitution at residues within the cytoplasmic N-terminal half of the helix tended to increase specific transport activity, whereas substitution at residues within the exoplasmic C-terminal half of the helix tended to decrease specific transport activity. Four residues within helix 10 were clearly accessible to pCMBS as judged by inhibition or stimulation of transport activity. All four of these residues were clustered along one face of a putative ␣-helix. These results combined with previously published data suggest that transmembrane segment 10 of Glut1 forms part of the sugar permeation pathway. Two-dimensional models for the conformation of the 12 transmembrane helices and the exofacial glucose-binding site of Glut1 are proposed that are consistent with existing experimental data.

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 ␣-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.Passive transport of glucose across the plasma membrane of mammalian cells is mediated by members of the GLUT (SLC2a) family of membrane glycoproteins (reviewed in Refs.

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, sulfhydrylspecific 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 transport of glucose across mammalian cell membranes is mediated by proteins produced by the Glut gene family (reviewed in Refs. 1-3), part of the major facilitator superfamily (MFS) 2 that contains several thousand members, present in virtually every organism examined thus far (4). MFS proteins are involved in the transport of a diverse collection of low molecular weight molecules across membranes (5).Glut1 is expressed at its highest known levels in the human erythrocyte and is the only member of the Glut protein family to have been purified (6, 7). It remains one of the most extensively studied and thoroughly characterized members of the MFS (8). Based on analysis of the protein sequence deduced from a human cDNA clone, Glut1 was the first MFS protein predicted to possess 12 transmembrane helices (9), a major feature of most if not all members of this superfamily. A number of experimental observations, most notably a comprehensive glycosylation-scanning mutagenesis study (10), are consistent with this structural prediction. Several of the 12 predicted transmembrane helices are amphipathic, an observation that led to the suggestion that these helices form an exposed cavity in the membrane-buried portion of the protein that participates in the binding and translocation of glucose across the lipid bilayer (9). Hydrogen bond-forming amino acid side chains within the...