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.     

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.     

Cysteine-scanning mutagenesis of transmembrane segment 11 of the GLUT1 facilitative glucose transporter

The glucose permeation pathway within the GLUT1 facilitative glucose transporter is hypothesized to be formed by the juxtaposition of the hydrophilic faces of several transmembrane alpha-helices. The role of transmembrane segment 11 in forming a portion of this central aqueous channel was investigated using cysteine-scanning mutagenesis in conjunction with sulfhydryl-directed chemical modification. Each of the amino acid residues within transmembrane segment 11 were individually mutated 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 all of these mutants retain measurable transport activity. Four of the cysteine mutants (N411, W412, N415, and F422) had significantly reduced specific activity relative to the C-less protein. Specific activity was increased in five of the mutants (A402, A405, V406, F416, and M420). The solvent accessibility and relative orientation of the residues to the glucose permeation pathway were investigated by determining the sensitivity of the mutant transporters to inhibition by the sulfhydryl-directed reagent p-chloromercuribenzenesulfonate (pCMBS). Cysteine replacement at five positions (I404, G408, F416, G419, and M420) produced transporters that were inhibited by incubation with extracellular pCMBS. All of these residues cluster along a single face of the alpha-helix within the regions showing altered specific activities. These data demonstrate that the exofacial portion of transmembrane segment 11 is accessible to the external solvent and provide evidence for the positioning of this alpha-helix within or near the glucose permeation pathway.     

Analysis of transmembrane segment 10 of the Glut1 glucose transporter by cysteine-scanning mutagenesis and substituted cysteine accessibility.

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, 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 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 alpha-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.     

Crucial effects of amino acid side chain length in transmembrane segment 5 on substrate affinity in yeast glucose transporter Hxt7

We previously identified Asp(340) in transmembrane segment 7 (TM7) as a key determinant of substrate affinity in Hxt7, a high-affinity facilitative glucose transporter of Saccharomyces cerevisiae. To gain further insight into the structural basis of substrate recognition by Hxt7, we performed cysteine-scanning mutagenesis of 21 residues in TM5 of a Cys-less form of Hxt7. Four residues were sensitive to Cys replacement, among which Gln(209) was found to be essential for high-affinity glucose transport activity. The 17 remaining sites were examined further for the accessibility of cysteine to the hydrophilic sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS). Among the Cys mutants, T213C was the only one whose transport activity was completely inhibited by 0.5 mM pCMBS. Moreover, this mutant was protected from pCMBS inhibition by the substrate d-glucose and by 2-deoxy-D-glucose but not by L-glucose, indicating that Thr(213) is situated at or close to a substrate recognition site. The functional role of Thr(213) was further examined with its replacement with each of the other 19 amino acids in wild-type Hxt7. Such replacement generated seven functional transporters with various affinities for glucose. Only three mutants, those with Val, Cys, and Ser at position 213, exhibited high-affinity glucose transport activity. All of these residues possess a side chain length similar to that of Thr, indicating that side chain length at this position is a key determinant of substrate affinity. A working homology model of Hxt7 indicated that Gln(209) and Thr(213) face the central cavity and that Thr(213) is located within van der Waals distance of Asp(340) (TM7).     

Analysis of glucose transporter topology and structural dynamics

Homology modeling and scanning cysteine mutagenesis studies suggest that the human glucose transport protein GLUT1 and its distant bacterial homologs LacY and GlpT share similar structures. We tested this hypothesis by mapping the accessibility of purified, reconstituted human erythrocyte GLUT1 to aqueous probes. GLUT1 contains 35 potential tryptic cleavage sites. Fourteen of 16 lysine residues and 18 of 19 arginine residues were accessible to trypsin. GLUT1 lysine residues were modified by isothiocyanates and N-hydroxysuccinimide (NHS) esters in a substrate-dependent manner. Twelve lysine residues were accessible to sulfo-NHS-LC-biotin. GLUT1 trypsinization released full-length transmembrane helix 1, cytoplasmic loop 6-7, and the long cytoplasmic C terminus from membranes. Trypsin-digested GLUT1 retained cytochalasin B and d-glucose binding capacity and released full-length transmembrane helix 8 upon cytochalasin B (but not D-glucose) binding. Transmembrane helix 8 release did not abrogate cytochalasin B binding. GLUT1 was extensively proteolyzed by alpha-chymotrypsin, which cuts putative pore-forming amphipathic alpha-helices 1, 2, 4, 7, 8, 10, and 11 at multiple sites to release transmembrane peptide fragments into the aqueous solvent. Putative scaffolding membrane helices 3, 6, 9, and 12 are strongly hydrophobic, resistant to alpha-chymotrypsin, and retained by the membrane bilayer. These observations provide experimental support for the proposed GLUT1 architecture; indicate that the proposed topology of membrane helices 5, 6, and 12 requires adjustment; and suggest that the metastable conformations of transmembrane helices 1 and 8 within the GLUT1 scaffold destabilize a sugar translocation intermediate.     

Cysteine-scanning mutagenesis and substituted cysteine accessibility analysis of transmembrane segment 4 of the Glut1 glucose transporter

A low resolution model has been proposed for the exofacial conformation of the Glut1 glucose transporter in which eight transmembrane segments form an inner helical bundle stabilized by four outer helices. The role of transmembrane segment 4, predicted to be an inner helix in this structural model, was investigated by cysteine-scanning mutagenesis in conjunction with the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A functional, cysteine-less, parental Glut1 molecule was used to produce 21 Glut1 point mutants by individually changing each residue along transmembrane helix 4 to a cysteine. The single cysteine mutants were then expressed in Xenopus oocytes, and their expression levels, transport activities, and sensitivities to pCMBS were determined. In striking contrast to all of the other seven predicted inner helices, none of the 21 helix 4 single-cysteine mutants was demonstrably inhibited by pCMBS. However, cysteine substitution within helix 4 resulted in an unusually high number of severely transport-defective mutants. The low absolute transport activities of two of these mutants (G130C and G134C) were due to their extremely low levels of expression, presumably a result of structural instability and consequent degradation in oocytes, suggesting that these two residues play an important role in maintaining the native structure of Glut1. The other two transport-defective mutants (Y143C and E146C) exhibited low specific transport activities, implying that these two residues play an important role in the transport cycle. Based on these data, we conclude that the exoplasmic end of helix 4 lies outside the inner helical bundle in the exofacial configuration of Glut1.     

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.     

Transmembrane segment 3 of the Glut1 glucose transporter is an outer helix

A model has been proposed for the structure of the Glut1 glucose transporter based on the results of mutagenesis studies and homology modeling in which eight transmembrane segments form an inner helical bundle surrounded by four outer helices. The role of transmembrane segment 3 in this structural model was investigated using cysteine-scanning mutagenesis in conjunction with 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 helix 3 to a cysteine. The single cysteine mutants were then expressed in Xenopus oocytes, and their expression levels, transport activities, and sensitivities to pCMBS were determined. Cysteine substitution at methionine 96 abolished transport activity, whereas substitutions at the other positions resulted in either modest reductions or no significant effect on transport activity. In striking contrast to all other helices that have been examined to date, only one of the 21 helix 3 single-cysteine mutants was inhibited by pCMBS, suggesting that only a small portion of this helix is exposed to the external solvent. This result is consistent with predictions based on our current structural model, in which helix 3 is one of four outer helices that surround the inner helical bundle that comprises the aqueous substrate-binding cavity. An updated two-dimensional model for the orientation of the 12 transmembrane helices and the conformation of the exofacial glucose-binding site of Glut1 is presented that is consistent with existing experimental data.     

Transmembrane helix 7 in the Na+/dicarboxylate cotransporter 1 is an outer helix that contains residues critical for function

Citric acid cycle intermediates, including succinate and citrate, are absorbed across the apical membrane by the NaDC1 Na+/dicarboxylate cotransporter located in the kidney and small intestine. The secondary structure model of NaDC1 contains 11 transmembrane helices (TM). TM7 was shown previously to contain determinants of citrate affinity, and Arg-349 at the extracellular end of the helix is required for transport. The present study involved cysteine scanning mutagenesis of 26 amino acids in TM7 and the associated loops. All of the mutants were well expressed on the plasma membrane, but many had low or no transport activity: 6 were inactive and 7 had activity less than 25% of the parental. Three of the mutants had notable changes in functional properties. F336C had increased transport activity due to an increased Vmax for succinate. The conserved residue F339C had very low transport activity and a change in substrate selectivity. G356C in the putative extracellular loop was the only cysteine mutant that was affected by the membrane-impermeant cysteine reagent, MTSET. However, direct labeling of G356C with MTSEA-biotin gave a weak signal, indicating that this residue is not readily accessible to more bulky reagents. The results suggest that the amino acids of TM7 are functionally important because their replacement by cysteine had large effects on transport activity. However, most of TM7 does not appear to be accessible to the extracellular fluid and is likely to be an outer helix in contact with the lipid bilayer.     

The first transmembrane segment of the dopamine D2 receptor: accessibility in the binding-site crevice and position in the transmembrane bundle

The binding site of the dopamine D2 receptor, like that of homologous G-protein-coupled receptors (GPCRs), is contained within a water-accessible crevice formed among its seven transmembrane segments (TMs). Using the substituted-cysteine-accessibility method (SCAM), we are mapping the residues that contribute to the surface of this binding-site crevice. We have now mutated to cysteine, one at a time, 21 consecutive residues in TM1. Six of these mutants reacted with charged sulfhydryl reagents, whereas bound antagonist only protected N52(1.50)C from reaction. Except for A38(1.36)C, none of the substituted cysteine mutants in the extracellular half of TM1 appeared to be accessible. Pro(1.48) is highly conserved in opsins, but absent in catecholamine receptors, and the high-resolution rhodopsin structure showed that Pro(1.48) bends the extracellular portion of TM1 inward toward TM2 and TM7. Analysis of the conversation of residues in the extracellular portion of TM1 of opsins showed a pattern consistent with alpha-helical structure with a conserved face. In contrast, this region in catecholamine receptors is poorly conserved, suggesting a lack of critical contacts. Thus, in catecholamine receptors in the absence of Pro(1.48), TM1 may be straighter and therefore further from the helix bundle, consistent with the apparent lack of conserved contact residues. When examined in the context of a model of the D2 receptor, the accessible residues in the cytoplasmic half of TM1 are at the interface with TM7 and with helix 8 (H8). We propose the existence of critical contacts of TM1, TM7, and H8 that may stabilize the inactive state of the receptor.     

Transmembrane segment 6 of the Glut1 glucose transporter is an outer helix and contains amino acid side chains essential for transport activity

Experimental data and homology modeling suggest a structure for the exofacial configuration of the Glut1 glucose transporter in which 8 transmembrane helices form an aqueous cavity in the bilayer that is stabilized by four outer helices. The role of transmembrane segment 6, predicted to be an outer helix in this model, was examined by cysteine-scanning mutagenesis and the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzene-sulfonate (pCMBS). A fully functional Glut1 molecule lacking all 6 native cysteine residues was used as a template to produce a series of 21 Glut1 point mutants in which each residue along helix 6 was individually changed to cysteine. These mutants were expressed in Xenopus oocytes, and their expression levels, functional activities, and sensitivities to inhibition by pCMBS were determined. Cysteine substitutions at Leu(204) and Pro(205) abolished transport activity, whereas substitutions at Ile(192), Pro(196), Gln(200), and Gly(201) resulted in inhibition of activity that ranged from approximately 35 to approximately 80%. Cysteine substitutions at Leu(188), Ser(191), and Leu(199) moderately augmented specific transport activity relative to the control. These results were dramatically different from those previously reported for helix 12, the structural cognate of helix 6 in the pseudo-symmetrical structural model, for which none of the 21 single-cysteine mutants exhibited reduced activity. Only the substitution at Leu(188) conferred inhibition by pCMBS, suggesting that most of helix 6 is not exposed to the external solvent, consistent with its proposed role as an outer helix. These data suggest that helix 6 contains amino acid side chains that are critical for transport activity and that structurally analogous outer helices may play distinct roles in the function of membrane transporters.     

Identification of a Key Residue Determining Substrate Affinity in the Yeast Glucose Transporter Hxt7: A TWO-DIMENSIONAL COMPREHENSIVE STUDY*

We previously identified Asn³³¹ in transmembrane segment 7 (TM7) as a key residue determining substrate affinity in Hxt2, a moderately high-affinity facilitative glucose transporter of Saccharomyces cerevisiae. To gain further insight into the structural basis of substrate recognition by yeast glucose transporters, we have now studied Hxt7, whose affinity for glucose is the highest among the major hexose transporters. The functional role of Asp³⁴⁰ in Hxt7, the residue corresponding to Asn³³¹ of Hxt2, was examined by replacing it with each of the other 19 amino acids. Such replacement of Asp³⁴⁰ generated transporters with various affinities for glucose, with the affinity of the Cys³⁴⁰ mutant surpassing that of the wild-type Hxt7. To examine the structural role of Asp³⁴⁰ in the substrate translocation pathway, we performed cysteine-scanning mutagenesis of the 21 residues in TM7 of a functional Cys-less Hxt7 mutant in conjunction with exposure to the hydrophilic sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS). The transport activity of the D340C mutant of Cys-less Hxt7, in which Asp³⁴⁰ is replaced with Cys, was completely inhibited by pCMBS, indicating that Asp³⁴⁰ is located in a water-accessible position. This D340C mutant showed a sensitivity to pCMBS that was ∼70 times that of the wild-type Hxt7, and it was protected from pCMBS inhibition by the substrates d-glucose and 2-deoxy-d-glucose but not by l-glucose. These results indicate that Asp³⁴⁰ is situated at or close to a substrate recognition site and is a key residue determining high-affinity glucose transport by Hxt7, supporting the notion that yeast glucose transporters share a common mechanism for substrate recognition.     

Cysteine-scanning mutagenesis of transmembrane segment 1 of glucose transporter GLUT1: extracellular accessibility of helix positions

Transmembrane segment 1 of the cysteine-less GLUT1 glucose transporter was subjected to cysteine-scanning mutagenesis. The majority of single-cysteine mutants were functional transporters, as assessed by 2-deoxy-d-glucose uptake or 3-O-methyl-d-glucose transport. Substitution of cysteine for Leu-21, Gly-22, Ser-23, Gln-25, and Gly-27, however, led to uptake rates that were less than 10% of that of the nonmutated cysteine-less GLUT1. NEM, a membrane-permeable agent, was used to identify positions that are sensitive to transport alteration by sulfhydryl reagents, whereas uptake modification by the membrane-impermeant pCMBS indicated accessibility to water-soluble solutes from the external cell environment. Twelve of the 21 single-cysteine mutants were significantly (p < 0.01) affected by NEM, and on the basis of this sensitivity, four positions were identified by pCMBS to form a water-accessible surface within helix 1. The pCMBS-sensitive positions are localized at the exofacial C-terminal end along a circumference of the helix.     

An acidic amino acid transmembrane helix 10 residue conserved in the neurotransmitter:sodium:symporters is essential for the formation of the extracellular gate of the γ-aminobutyric acid (GABA) transporter GAT-1

GAT-1 mediates transport of GABA together with sodium and chloride in an electrogenic process enabling efficient GABAergic transmission. Biochemical and modeling studies based on the structure of the bacterial homologue LeuT are consistent with a mechanism whereby the binding pocket is alternately accessible to either side of the membrane and which predicts that the extracellular part of transmembrane domain 10 (TM10) exhibits aqueous accessibility in the outward-facing conformation only. In this study we have engineered cysteine residues in the extracellular half of TM10 of GAT-1 and probed their state-dependent accessibility to sulfhydryl reagents. In three out of four of the accessible cysteine mutants, the inhibition of transport by a membrane impermeant sulfhydryl reagent was diminished under conditions expected to increase the proportion of inward-facing transporters, such as the presence of GABA together with the cotransported ions. A conserved TM10 aspartate residue, whose LeuT counterpart participates in a "thin" extracellular gate, was found to be essential for transport and only the D451E mutant exhibited residual transport activity. D451E exhibited robust sodium-dependent transient currents with a voltage-dependence indicative of an increased apparent affinity for sodium. Moreover the accessibility of an endogenous cysteine to a membrane impermeant sulfhydryl reagent was enhanced by the D451E mutation, suggesting that sodium binding promotes an outward-facing conformation of the transporter. Our results support the idea that TM10 of GAT-1 lines an accessibility pathway from the extracellular space into the binding pocket and plays a role in the opening and closing of the extracellular transporter gate.     

Glycine-rich transmembrane helix 10 in the staphylococcal tetracycline transporter TetA(K) lines a solvent-accessible channel

The staphylococcal TetA(K) tetracycline exporter is classified within the major facilitator superfamily of transport proteins and contains 14 alpha-helical transmembrane segments (TMS). Using cysteine-scanning mutagenesis, 27 amino acid residues across and flanking putative TMS 10 of the TetA(K) transporter were individually replaced with cysteine. The level of solvent accessibility to each of the targeted amino acid positions was determined as a measure of fluorescein maleimide reactivity and demonstrated that TMS 10 of TetA(K) has a cytoplasmic boundary at G313 and is likely to extend from at least V298 on the periplasmic side. TMS 10 was found to be amphiphilic containing at least partially solvent accessible amino acid residues along the length of one helical face, suggesting that this helix may line a solvent-exposed channel. Functional analyses of these cysteine mutants demonstrated a significant role for a number of amino acid residues, including a predominance of glycine residues which were further analyzed by alanine substitution. These residues are postulated to allow interhelical interactions between TMS 10 and distal parts of TetA(K) that are likely to be required for the tetracycline transport mechanism in TetA(K) and may be a general feature required by bacterial tetracycline transporters for activity.     

Cysteine Cross-linking Defines the Extracellular Gate for the Leishmania donovani Nucleoside Transporter 1.1 (LdNT1.1)

Equilibrative nucleoside transporters are a unique family of proteins that enable uptake of nucleosides/nucleobases into a wide range of eukaryotes and internalize a myriad of drugs used in the treatment of cancer, heart disease, AIDs, and parasitic infections. In previous work we generated a structural model for such a transporter, the LdNT1.1 nucleoside permease from the parasitic protozoan Leishmania donovani, using ab initio computation. The model suggested that aromatic residues present in transmembrane helices 1, 2, and 7 interact to form an extracellular gate that closes the permeation pathway in the inward-open conformation. Mutation of residues Phe-48_TM1 and Trp-75_TM2 abrogated transport activity, consistent with such prediction. In this study cysteine mutagenesis and oxidative cross-linking were combined to analyze proximity relationships of helices 1, 2, and 7 in LdNT1.1. Disulfide bond formation between introduced paired cysteines at the interface of such helices (A61C_TM1/F74C_TM2, A61C_TM1/G350C_TM7, and F74C_TM2/G350C_TM7) was analyzed by transport measurement and gel mobility shifts upon oxidation with Cu (II)-(1,10-phenanthroline)₃. In all cases cross-linking inhibited transport. However, if LdNT1.1 ligands were included during cross-linking, inhibition of transport was reduced, suggesting that ligands moved the three gating helices apart. Moreover, all paired cysteine mutants exhibited a mobility shift upon oxidation, corroborating the formation of a disulfide bond. These data support the notion that helices 1, 2, and 7 constitute the extracellular gate of LdNT1.1, thus further validating the computational model and the previously demonstrated importance of F48_TM1 and Trp-75_TM2 in tethering together helices that are part of the gate.     

Transmembrane segment 5 of the Glut1 glucose transporter is an amphipathic helix that forms part of the sugar permeation 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 steady-state 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 alpha-helix, proximal to the exoplasmic surface of the plasma membrane. These results comprise the first experimental evidence for the existence of an amphipathic transmembrane alpha-helix in a glucose transporter molecule and strongly suggest that transmembrane segment 5 of Glut1 forms part of the sugar permeation pathway.     

Cysteine-scanning mutagenesis study of the sixth transmembrane segment of the Na,K-ATPase alpha subunit

The accessibility of the residues of the sixth transmembrane segment (TM) of the Bufo marinus Na,K-ATPase alpha subunit was explored by cysteine scanning mutagenesis. Methanethiosulfonate reagents reached only the two most extracellular positions (T803, D804) in the native conformation of the Na,K-pump. Palytoxin induced a conductance in all mutants, including D811C, T814C and D815C which showed no active electrogenic transport. After palytoxin treatment, four additional positions (V805, L808, D811 and M816) became accessible to the sulfhydryl reagent. We conclude that one side of the sixth TM helix forms a wall of the palytoxin-induced channel pore and, probably, of the cation pathway from the extracellular side to one of their binding sites.     

A novel topology model of the human Na(+)/H(+) exchanger isoform 1

The membrane topology of the human Na(+)/H(+) exchanger isoform 1 (NHE1) was assessed by substituted cysteine accessibility analysis. Eighty-three cysteine residues were individually introduced into a functional cysteineless NHE1, and these mutants were expressed in the exchanger-deficient PS120 cells. The topological disposition of introduced cysteines was determined by labeling with a biotinylated maleimide in the presence or absence of preincubation with the membrane-impermeable sulfhydryl reagent, 2-trimethylammoniumethyl-methanethiosulfonate in streptolysin O-permeabilized or nonpermeabilized cells. We proposed a new model for the topology of NHE1 that is significantly different from the model derived from hydropathy analysis. In this model, NHE1 is composed of 12 transmembrane segments (TMs) with the N and C termini located in the cytosol. The large, last extracellular loop in the membrane domain of the original model was suggested to comprise an intracellular loop, a new transmembrane segment (TM11), and an extracellular loop in the new model. Interestingly, cysteines at 183 and 184 and at 324 and 325 mapped to intracellular loops connecting TMs 4 and 5 (IL2) and TMs 8 and 9 (IL4), respectively, were accessible to sulfhydryl reagents from the outside. Furthermore, exchange activities of two mutants, R180C and Q181C, within IL2 were markedly inhibited by external MTSET. These data suggest that part of IL2 or IL4 may be located in a pore-lining region that is accessible from either side of the membrane and involved in ion transport.