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Cys-loop receptor neurotransmitter-gated ion channels are pentameric assemblies of subunits that contain three domains: extracellular, transmembrane, and intracellular. The extracellular domain forms the agonist binding site. The transmembrane domain forms the ion channel. The cytoplasmic domain is involved in trafficking, localization, and modulation by cytoplasmic second messenger systems but its role in channel assembly and function is poorly understood and little is known about its structure. The intracellular domain is formed by the large (>100 residues) loop between the α-helical M3 and M4 transmembrane segments. Putative prokaryotic Cys-loop homologues lack a large M3M4 loop. We replaced the complete M3M4 loop (115 amino acids) in the 5-hydroxytryptamine type 3A (5-HT3A) subunit with a heptapeptide from the prokaryotic homologue from Gloeobacter violaceus. The macroscopic electrophysiological and pharmacological characteristics of the homomeric 5-HT3A-glvM3M4 receptors were comparable to 5-HT3A wild type. The channels remained cation-selective but the 5-HT3A-glvM3M4 single channel conductance was 43.5 pS as compared with the subpicosiemens wild-type conductance. Coexpression of hRIC-3, a protein that modulates expression of 5-HT3 and acetylcholine receptors, significantly attenuated 5-HT–induced currents with wild-type 5-HT3A but not 5-HT3A-glvM3M4 receptors. A similar deletion of the M3M4 loop in the anion-selective GABA-ρ1 receptor yielded functional, GABA-activated, anion-selective channels. These results imply that the M3M4 loop is not essential for receptor assembly and function and suggest that the cytoplasmic domain may fold as an independent module from the transmembrane and extracellular domains.

Pentameric ligand-gated ion channels (pLGICs), also called Cys-loop receptors in eukaryotic superfamily members, play diverse roles in neurotransmission and serve as primary targets for many therapeutic drugs. Structural studies of full-length eukaryotic pLGICs have been challenging because of glycosylation, large size, pentameric assembly, and hydrophobicity. X-ray structures of prokaryotic pLGICs, including the Gloeobacter violaceus LGIC (GLIC) and the Erwinia chrysanthemi LGIC (ELIC), and truncated eukaryotic pLGICs have significantly improved and complemented the understanding of structural details previously obtained with acetylcholine-binding protein and Torpedo nicotinic acetylcholine receptors. Prokaryotic pLGICs share their overall structural features with eukaryotic pLGICs for the ligand-binding extracellular and channel-lining transmembrane domains. The large intracellular domain (ICD) is present only in eukaryotic members and is characterized by a low level of sequence conservation and significant variability in length (50-250 amino acids), making the ICD a potential target for the modulation of specific pLGIC subunits. None of the structures includes a complete ICD. Here, we created chimeras by adding the ICD of cation-conducting (nAChR-α7) and anion-conducting (GABAρ1, Glyα1) eukaryotic homopentamer-forming pLGICs to GLIC. GLIC-ICD chimeras assemble into pentamers to form proton-gated channels, as does the parent GLIC. Additionally, the sensitivity of the chimeras toward modulation of functional maturation by chaperone protein RIC-3 is preserved as in those of the parent eukaryotic channels. For a previously described GLIC-5HT3A-ICD chimera, we now provide evidence of its successful large-scale expression and purification to homogeneity. Overall, the chimeras provide valuable tools for functional and structural studies of eukaryotic pLGIC ICDs.

For more than two decades, bupropion has been clinically prescribed for the treatment of depression (Wellbutrin®), and more recently for smoking cessation (Zyban®). Bupropion is conventionally described as a dual norepinephrine dopamine reuptake inhibitor, therefore effective as an antidepressant. However, we and others have shown that it also interacts with receptors of the Cys-loop superfamily, namely neuronal nicotinic acetylcholine receptors. In the continuum of exploring the molecular interactions of bupropion with other prominent members of the Cys-loop superfamily, we report here that bupropion additionally inhibits 5-hydroxytryptamine (serotonin) type 3A receptors (5-HT3ARs).5-HT3ARs are cation-conducting, homo-pentameric ligand-gated ion channels of the Cys-loop superfamily which are structurally and functionally distinct from other serotonin receptors that are G-proteins. 5-HT3 receptors are current targets for anti-emetics mainly used in cancer chemotherapy, as well as potential future targets for disorders including anxiety, schizophrenia, and Alzheimer's disease.Here we characterized our newly-identified interaction of bupropion with 5-HT3ARs with electrophysiological and radioligand binding studies. In oocytes, bupropion inhibited 5-HT3AR-mediated currents with an IC50 value of 87 µM (nH = 1.2). That 300 µM bupropion inhibited [3H]-5-HT or [3H]-granisetron (a competitive antagonist) binding by ∼ 20% indicated at best weak interaction with the agonist binding site. In contrast, the effect of different concentrations of bupropion on serotonin concentration-response curves suggested a non-competitive nature of interaction. Along these lines, importantly, the inhibition of serotonin-evoked currents recorded from oocytes was proportional to the length of preincubation time with bupropion.In summary, our results are indicative of a negative allosteric interaction of bupropion with the 5-HT3AR. Currently, experiments are underway to identify the site(s) of interaction for bupropion within the 5- HT3AR.

Construction of a GABAAreceptor homology model based on the acetylcholine (ACh) receptor structure is complicated by the low sequence similarity between GABAAand ACh M3 transmembrane segments that creates significant uncertainty in their alignment. We determined the orientation of the GABAAM2 and M3 transmembrane segments using disulfide cross-linking. The M2 residues α1M266 (11′) and α1T267 (12′) were mutated to cysteine in either wild type or single M3 cysteine mutant (α1V297C, α1A300C to α1A305C) backgrounds. We assayed spontaneous and induced disulfide bond formation. Reduction with DTT significantly potentiated GABA-induced currents in α1T267C-L301C and α1T267C-F304C. Copper phenanthroline-induced oxidation inhibited GABA-induced currents in these mutants and in α1T267C-A305C. Intrasubunit disulfide bonds formed between these Cys pairs, implying that the α-carbon separation was at most 5.6 Å. The reactive α1M3 residues (L301, F304, A305) lie on the same face of an α-helix. The unresponsive ones (A300, I302, E303) lie on the opposite face. In the resting state, the reactive side of α1M3 faces M2-α1T267. In conjunction with the ACh structure, our data indicate that alignment of GABAAand ACh M3 requires a single gap in the GABAAM2–M3 loop. In the presence of GABA, oxidation of α1T267C-L301C and α1T267C-F304C had no effect, but oxidation of α1T267C-A305C caused a significant increase in spontaneous channel opening. We infer that, as the channel opens, the distance and/or orientation between M2-α1T267 and M3-α1A305 changes such that the disulfide bond stabilizes the open state. This begins to define the conformational motion that M2 undergoes during channel opening.

Based on theTorpedoacetylcholine receptor structure, Unwin and colleagues (Miyazawa et al., 2003; Unwin, 2005) hypothesized that the transduction of agonist binding to channel gate opening involves a “pin-into-socket” interaction between αV46 at the tip of the extracellular β1-β2loop and the transmembrane M2 segment and M2-M3 loop. We mutated to cysteine the aligned positions in the 5-HT3Aand 5-HT3Bsubunit β1-β2loops K81 and Q70, respectively. The maximal 5-HT-activated currents in receptors containing 5-HT3A/K81C or 5-HT3B/Q70C were markedly reduced compared with wild type. Desensitization of wild-type currents involved fast and slow components. Mutant currents desensitized with only the fast time constant. Reaction with several methanethiosulfonate reagents potentiated currents to wild-type levels, but reaction with other more rigid thiol-reactive reagents caused inhibition. Single-channel conductances of wild type, K81C, and K81C after modification were similar. We tested the proximity of K81C to the M2-M3 loop by mutating M2-M3 loop residues to cysteine in the K81C background. Disulfide bonds formed in 5-HT3A/K81C/A304C and 5-HT3A/K81C/I305C when coexpressed with 5-HT3B. We conclude that in the resting state, K81 is not in a hydrophobic pocket as suggested by the pin-into-socket hypothesis. K81 interacts with the extracellular end of M2 and plays a critical role in channel opening and in the return from fast desensitization. We suggest that during channel activation, β1-β2loop movement moves M2 and the M2-M3 loop so that the M2 segments rotate/translate away from the channel axis, thereby opening the lumen. Recovery from fast desensitization requires the interaction between K81 and the extracellular end of M2.

The synthetic procedures to obtain indole derivatives with different acidic functions at position 2 of the indole are reported. The synthesised and tested derivatives comprise 5-tetrazolyl, 1,3,4-oxadiazol-5-yl-2-one, and indole-2-carboxylic acid amides with 5-aminotetrazole, methanesulphonamide and trifluoromethanesulphonamide moieties. The binding affinity was evaluated using [3H]MDL 105,519 and pig cortical brain membranes. In general, compounds with acidic functions different from a carboxylic acid moiety are less potent than indole-2-carboxylic acid derivatives. Also, the 4,6-dichloro substitution pattern was compared to 5-tert-butyl derivatives and compounds not substituted in the benzene moiety of the indole, indicating that the affinity increases from 5-tert-butyl over unsubstituted to 4,6-dichloro substituted derivatives.

The neurotransmitter glutamate is thought to be crucially involved in a huge number of neurological and psychiatric disorders, such as Morbus Parkinson, Alzheimer's disease and schizophrenia. Aiming at an improved diagnostic tool for PET a new [18F]fluorine labelled NMDA receptor ligand was developed that may potentially allow the in vivo visualization of glutama-tergic neurotransmission. The 19F-analogue trans-5,7-dichloro-4-(3-{4-[4-(2-fluoroethyl)-piperazin-1-yl]-phenyl}-ureido)-1,2,3,4-tetrahydro quinoline-2-carboxylic acid was synthesised to determine the binding affinity, lipophilicity and biodistribution of the ligand. This substance exhibits a Ki of 12 nM for the glycine binding site using [3H]MDL-105,519 assays on pig cortical membranes. A logD of 1.3 was determined for this compound according to the OECD guidelines employing the HPLC method. Radiosynthesis of this ligand was achieved by labelling the precursor trans-5,7-dichloro-4-[3-(4-piperazin-1-yl-phenyl)-ureido]-1,2,3,4-tetrahydroquinoline-2-carboxylic acid methyl ester with 2-[18F]fluoroethyltosylate and subsequent cleaving of the methyl ester moiety, resulting in an overall decay corrected yield of 35% of the final product trans-5,7-dichloro-4-(3-{4-[4-(2-[18F]fluoroethyl)-piperazin-1-yl]-phenyl}-ureido)-1,2,3,4-tetrahydroquinoline-2-carboxylic acid. The biodistribution kinetics of this compound were determined with Sprague Dawley rats ex vivo for brain, liver, kidney, and bone. The ligand showed a maximum brain uptake 30 min.p.i. of about 0.1% ID/g. Copyright © 2003 John Wiley & Sons, Ltd.

The pentameric ligand-gated ion channel (pLGIC) superfamily includes the nicotinic acetylcholine (nACh), 5-HT3, ɣ-aminobutyric acid type A (GABAA), and glycine receptor. These channels are made up of five homologous subunits surrounding a central ion channel pore and can be divided into three pentameric domains. The extracellular domain (ECD) and transmembrane domain (TMD) of the channels show high levels of homology and have been well studied and structures have been published. In contrast, the intracellular domain (ICD) shows sequence and length divergence in the different receptors. Mutations in the ICD have been shown to alter receptor conductance. Gloeobacter violaceus LGIC (GLIC) is a prokaryotic pLGIC without a native ICD, which can be expressed and studied in Xenopus oocytes. To elucidate the mechanisms by which the ICD affects the channel pathway and conductance we have created chimeras of GLIC ECD and TMD with ICDs of both anion- and cation-conducting pLGICs (GABAρ1, Glyα1, nAChRα7, 5-HT3A). Functional expression of chimeras was first confirmed by applying low pH (pH 5) pulses to oocytes under two-electrode voltage clamp. We have begun to study the single channel conductance and gating kinetics of chimeric channels and confirmed that the GLIC wildtype has a single-channel conductance of 10pS. For the GLIC-5-HT3A-ICD chimera, we expect to see a decrease in conductance as we observed an increase in a construct of 5-HT3A upon removing the ICD. Amino acid engineering in both the chimeras and eukaryotic parent channels will then be used to further understanding of how the ICDs contribute to ion conducting properties.

Folate cofactors, Vitamin B9, play crucial roles in more than a hundred one-carbon metabolism reactions in mammalian cells. Humans cannot synthesize folates de novo and absorption through the diet is the only source of this vitamin. The proton-coupled folate transporter (PCFT) mediates this uptake in the upper small intestine by a pH-dependent process. PCFT also transports folates into the central nervous system. Point mutations in the PCFT gene cause Hereditary Folate Malabsorption (HFM), with associated hematological and neurological defects due to impaired folate transport. Certain solid tumor cell lines express high levels of PCFT mRNA. Importantly, the overall expression of PCFT is limited to only certain tissues in humans. Therefore, PCFT is a molecular target for specific delivery of anti-folate chemotherapeutic agents to tumor cells. Unfortunately, the success rate of anti-folate agents to reach clinical use is very low due to their side-effects. A structural model of PCFT can aid the development of specific anti-folate agents for their PCFT-targeted delivery and thus minimize their side-effects. To optimize and verify an initial structural model of PCFT, we applied a wide array of experimental approaches: solvent accessibility profiling through substituted cysteine accessibility scanning, studying the helix packing, and assaying the functionality of PCFT mutants. We complemented the experimental data with theoretical approaches for structure prediction such as homology modeling/threading, ligand docking and an extensive review of other secondary transporters and folate transport proteins. With these combined approaches, we have developed a structural model of PCFT that accurately reflects our experimental observations. This model forms a basis to understand the impacts of HFM mutations, and to develop folate analogues for folate deficiency intervention. Additionally, it will aid the design of PCFT structure-based anti-folate agents for the treatment of cancer.

International audience; Although the activity of the nicotinic acetylcholine receptor (nAChR) is exquisitely sensitive to its membrane environment, the underlying mechanisms remain poorly defined. The homologous prokaryotic pentameric ligand-gated ion channel, Gloebacter ligand-gated ion channel (GLIC), represents an excellent model for probing the molecular basis of nAChR sensitivity because of its high structural homology, relative ease of expression, and amenability to crystallographic analysis. We show here that membrane-reconstituted GLIC exhibits structural and biophysical properties similar to those of the membrane-reconstituted nAChR, although GLIC is substantially more thermally stable. GLIC, however, does not possess the same exquisite lipid sensitivity. In particular, GLIC does not exhibit the same propensity to adopt an uncoupled conformation where agonist binding is uncoupled from channel gating. Structural comparisons provide insight into the chemical features that may predispose the nAChR to the formation of an uncoupled state.

The proton-coupled folate transporter (PCFT) was recently identified as the major uptake route for dietary folates in humans. The three-dimensional structure of PCFT and its detailed interplay with function remain to be determined. We screened the water-accessible extracellular surface of HsPCFT using the substituted-cysteine accessibility method, to investigate the boundaries between the water-accessible surface and inaccessible buried protein segments. Single-cysteines, engineered individually at 40 positions in a functional cysteine-less HsPCFT background construct, were probed for plasma-membrane expression in Xenopus oocytes with a bilayer-impermeant primary-amine-reactive biotinylating agent (sulfosuccinimidyl 6-(biotinamido) hexanoate), and additionally for water-accessibility of the respective engineered cysteine with the sulfhydryl-selective biotinylating agent 2-((biotinoyl)amino)ethyl methanethiosulfonate. The ratio between Cys-selective over amine-selective labeling was further used to evaluate three-dimensional models of HsPCFT generated by homology / threading modeling. The closest homologues of HsPCFT with a known experimentally-determined three-dimensional structure are all members of one of the largest membrane protein super-families, the major facilitator superfamily (MFS). The low sequence identity - 14% or less – between HsPCFT and these templates necessitates experiment-based evaluation and model refinement of homology / threading models. With the present set of single-cysteine accessibilities, the models based on GlpT and PepTSt are most promising for further refinement.

Cationic pentameric ligand-gated ion channels (pLGICs) of the Cys-loop superfamily function as receptors for endogenous (e.g. acetylcholine, serotonin) and/or exogenous (e.g. nicotine) ligands. These receptors are the targets for a number of current clinical drugs used to treat nicotine addiction, Alzheimer's disease, Parkinson's disease, epilepsy, pain perception, anxiety, and depression. In order to develop new, more effective drugs and improve current treatments, research is imperative to identify and characterize the different structural elements that mediate each aspect of receptor function. From an early electron density map of a pLGIC of the Cys-loop superfamily to the most recent high-resolution crystal structures of mammalian Cys-loop receptors, their structural information has revealed that the overall architecture of these pLGICs is highly-conserved. The intracellular domain (ICD) presents the notable exception in that the functions, lengths, amino acids sequences, and likely also structural folds are vastly different between subunits. Based on its diversity, the ICD presents a target for developing subtype-selective drugs with the hope of fewer side effects than current drugs, all of which target the highly-conserved extracellular or transmembrane domains.We developed a protein expression and purification strategy to produce the ICD of a cationic pLGIC in sufficient quantity and quality. Chimeras were generated by adding the ICD of the α7 nicotinic acetylcholine receptor to a soluble protein, and conditions for optimal expression in E. coli were identified. A two-step purification process consisting of affinity chromatography followed by size-exclusion chromatography yielded 5 mg of purified protein per liter of bacterial culture. Thus, our results are consistent with the successful large-scale expression and purification of the α7-ICD chimera that is now amenable to structural studies.

The 115 amino acids spanning intracellular domain (ICD) links the third (M3) and fourth (M4) transmembrane segments within each subunit of the 5-HT3A receptor, a cationic homo-pentameric ligand-gated ion channel (pLGIC). The advent of increasingly sophisticated structural studies has unraveled, in part, the three-dimensional structure of eukaryotic as well as prokaryotic pLGICs at atomic resolution. Intriguingly, within all these structures, the ICD has commonly not been completely resolved. To illustrate the lack of completeness, a recent high-resolution 5-HT3A structure, albeit providing novel structural insights, is deprived of 61 amino acids from its ICD resulting in channel activity differing remarkably from wild-type channels. Prokaryotic pLGICs lack the ICD, and instead contain a very short M3-M4 linker. In eukaryotic pLGICs, the ICD exemplifies the diversity of domain architecture, unlike the other two domains - extracellular and transmembrane - which are highly-conserved. Given the expanding functional implications of ICDs in subunit folding, assembly, targeting, posttranslational modifications, reverse allosteric conductance, and protein-protein interactions, it is of great importance to resolve the structures of full-length ICDs in order to bridge this knowledge gap.To attain this set goal, we produced and purified a soluble chimera containing the ICD of the mouse 5-HT3A receptor. Molecular weight determination by both size exclusion chromatography and dynamic light scattering indicates stable pentameric assembly of the chimera in solution. This surprising finding was further corroborated when the chimera was cross-linked with glutaraldehyde vapors and then resolved either by SDS-PAGE or ‘on-chip electrophoresis’, revealing a ladder of discrete covalently-linked oligomers. The unexpected observation that the ICD alone assembles into stable pentamers in solution and its extreme diversity with regard to both length and amino acid-composition is further suggestive of its orphaned role in receptor oligomerization and potentially the specificity thereof.

Folate vitamins are essential for DNA replication and cellular proliferation. However, mammalian organisms are devoid of de novo folate biosynthesis and thus rely on dietary sources to meet their metabolic needs. The proton coupled folate transporter (PCFT/SLC46A1) has been recently identified as the molecular entity of the carrier mediated intestinal folate uptake pathway for folic acids from food sources. PCFT is also involved in the absorption of chemotherapeutically used antifolates. Currently, there is limited information about the structure and function of PCFT. Hydropathy analysis suggests that there are 10-12 transmembrane segments. Further, using the Substituted Cysteine Accessibility Method (SCAM) evidence was provided for a 12 transmembrane segment topology. Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) is a technique for separation of protein complexes in a native state with high resolution. We expressed PCFT in Xenopus laevis oocytes. Oocyte plasma membranes were polymerized to the vitelline membrane using ludox colloidal silica solution and polyacrylic acid, isolated by centrifugation, and plasma membrane proteins subsequently solubilized with digitonin and separated by BN-PAGE. The separation characteristics of native PCFT were compared to a molecular ruler produced by partial dissociation of homopentameric 5-hydroxytryptamine type 3A (5HT3A) receptors. Under native conditions, 5HT3A subunits largely migrated as a pentamer and PCFT only as a monomer. Treatment with denaturing agents generated a ladder of five bands for 5HT3A subunits, which consisted of monomer, dimer, trimer, tetramer and pentamer. Addition of crosslinking agents resulted in migration of 5HT3A subunits as a pentamer, even in the presence of denaturing agents. In contrast, crosslinking agents did not induce oligomeric assemblies of PCFT. These results indicate that functional plasma-membrane bound PCFT is a monomeric protein.

Drugs used to treat various disorders target GABA A receptors. To develop alpha subunit selective compounds, we synthesized 5-(4-piperidyl)-3-isoxazolol (4-PIOL) derivatives. The 3-isoxazolol moiety was substituted by 1,3,5-oxadiazol-2-one, 1,3,5-oxadiazol-2-thione, and substituted 1,2,4-triazol-3-ol heterocycles with modifications to the basic piperidine substituent as well as substituents without basic nitrogen. Compounds were screened by [(3)H]muscimol binding and in patch-clamp experiments with heterologously expressed GABA A alpha ibeta 3gamma 2 receptors (i = 1-6). The effects of 5-aminomethyl-3 H-[1,3,4]oxadiazol-2-one 5d were comparable to GABA for all alpha subunit isoforms. 5-piperidin-4-yl-3 H-[1,3,4]oxadiazol-2-one 5a and 5-piperidin-4-yl-3 H-[1,3,4]oxadiazol-2-thione 6a were weak agonists at alpha 2-, alpha 3-, and alpha 5-containing receptors. When coapplied with GABA, they were antagonistic in alpha 2-, alpha 4-, and alpha 6-containing receptors and potentiated alpha 3-containing receptors. 6a protected GABA binding site cysteine-substitution mutants alpha 1F64C and alpha 1S68C from reacting with methanethiosulfonate-ethylsulfonate. 6a specifically covalently modified the alpha 1R66C thiol, in the GABA binding site, through its oxadiazolethione sulfur. These results demonstrate the feasibility of synthesizing alpha subtype selective GABA mimetic drugs.

This laboratory recently identified a human gene that encodes a novel folate transporter [ Homo sapiens proton-coupled folate transporter ( HsPCFT); SLC46A1] required for intestinal folate absorption. This study focused on mouse ( Mus musculus) PCFT ( MmPCFT) and rat ( Rattus norvegicus) PCFT ( RnPCFT) and addresses their secondary structure, specificity, tissue expression, and regulation by dietary folates. Both rodent PCFT proteins traffic to the cell membrane with the NH2- and COOH-termini accessible to antibodies targeted to these domains only in permeabilized HeLa cells. This, together with computer-based topological analyses, is consistent with a model in which rodent PCFT proteins likely contain 12 transmembrane domains. Transport of [3H]folates was optimal at pH 5.5 and decreased with increasing pH due to an increase in Kmand a decrease in Vmax. At pH 7.0, folic acid and methotrexate influx was negligible, but there was residual (6 S)5-methyltetrahydrofolate transport. Uptake of folates in PCFT-injected Xenopus oocytes was electrogenic and pH dependent. Folic acid influx Kmvalues of MmPCFT and RnPCFT, assessed electrophysiologically, were 0.7 and 0.3 μM at pH 5.5 and 1.1 and 0.8 μM at pH 6.5, respectively. Rodent PCFTs were highly specific for monoglutamyl but not polyglutamyl methotrexate. MmPCFT mRNA was highly expressed in the duodenum, proximal jejunum, liver, and kidney with lesser expression in the brain and other tissues. MmPCFT protein was localized to the apical brush-border membrane of the duodenum and proximal jejunum. MmPCFT mRNA levels increased ∼13-fold in the proximal small intestine in mice fed a folate-deficient vesus folate-replete diet, consistent with the critical role that PCFT plays in intestinal folate absorption.

Folate cofactors play crucial roles in hundreds of reactions in cells including DNA and protein synthesis. The human proton-coupled folate transporter (PCFT) is the only means of absorption of dietary folates in humans. PCFT expression and function is associated with many disorders including hereditary folate malabsorption, neural tube defects, Down syndrome, cancer, heart diseases, Alzheimer's and Parkinson's disease. Upregulation of PCFT expression in tumor cells is of significant consideration for development of PCFT-targeted chemotherapeutic agents. However, not much is known about the structure and function of PCFT, which contributes to the low clinical success rate of folate-based agents. To address this gap in the knowledge we performed extensive Cys-mutagenesis studies. We analyzed 40 residues towards the extracellular face of PCFT, 35 positions towards the cytoplasmic face of PCFT and 28 positions along the proposed folate-binding pocket of PCFT. Based on the accessibility studies of the extracellular face of PCFT we determined loop-helix boundaries of this face and identified the glycerol-3-phosphate transporter (PDB#1PW4) and tripeptide-proton symporter (PDB#4APS) as the best templates for modeling PCFT. Based on the accessibility studies of the cytoplasmic face of PCFT, we identified loop-helix boundaries of this face. Here, we also show that our accessibility studies support the hypothesis that PCFT is present predominantly in an inward-open conformation in the absence of substrate (pH 7.5 and no folic acid). We also show that the folate-binding pocket of PCFT is formed by residues present in PCFT transmembrane helices I, IV, V, X and XI. Our results are of high significance in understanding the details of folate-homeostasis mechanisms and in design of PCFT-targeted therapeutic and diagnostic agents.

Bupropion is one of the most common drugs prescribed for treating depression and aiding smoking cessation, and is generally well tolerated with the exception of a rare dose-dependent incidence of seizures. It inhibits norepinephrine and dopamine reuptake, as well as noncompetitively antagonizes neuronal nicotinic acetylcholine receptors, which are members of the Cys-loop superfamily. While investigating its effects on other key members of this superfamily, bupropion showed complex modulation of the γ-aminobutyric acid type A (GABAA) α1β2γ2 receptor function.In the brain, GABAA α1β2γ2 receptors are one of the most abundant chloride-conducting hetero-pentameric ligand-gated ion channels. They are targets for a number of drugs including anesthetics, anticonvulsants, anxiolytics, hypnotics, and muscle relaxants.To the best of our knowledge, the interaction of bupropion with the GABAA receptor has not yet been described. Therefore, the primary goal of this study was to characterize the functional interactions of bupropion with the GABAA α1β2γ2 receptor. In oocytes, bupropion-alone (0.1 mM - 3 mM) directly activated GABAA receptor-mediated currents. The comparison of dose-response curves showed that bupropion-activation was lower in potency (EC50=1.5 mM vs. 1.3 μM) and efficacy (49.5% vs. 100%) than that for GABA, consistent with partial agonism of the GABAA receptor. Interestingly, at lower concentrations (0.001 µM - 100 μM) bupropion inhibited 0.5 μM GABA-induced currents by ∼ 8 - 20%; in contrast, at higher concentrations (1 mM - 3 mM) it potentiated GABA-induced currents by ∼ 50 - 70%. Notably, in many ways bupropion mimics the actions of some of the general anesthetics (e.g. propofol) at the GABAA receptor. This warrants exploring the bupropion binding site(s) within these receptors.

It is known that DNA is a well-characterized intracellular target but its size and sequential characteristics make it an elusive target for selective drug action. Binding of low molecular weight ligands to DNA causes a variety of significant biological responses. In this context the main consideration is given to recent developments in DNA sequence selective binding agents bearing conjugated effectors because of their potential application in treatment of cancers, in diagnosis as well as in molecular biology. In the present review recent results about analogues of netropsins, distamycin A and of some lexitropsins and combilexins or related hybrid molecules with sequence reading, intercalating or alkylating activity are described and evaluated for prospective applications. Furthermore there exists DNA minor groove binder with different basic structures which does not possess the typical polyamide chain, including dimeric intercalating chromophores. Finally new results about peptide nucleic acids and related nucleic acid bases linked with polyamides are reported. In pronounced examples the structural chemistry, synthesis, DNA binding with several biophysical methods, molecular aspects, structure activity relationship, topoisomerase inhibition, antitumour and antibacterial effects are discussed in detail.

For decades neuroreceptor research has focused on the development of NMDA glycine-site antagonists, after Johnson and Ascher found out in 1987 about the co-agonistic character of this achiral amino acid at the NMDA receptor. Contrary to the inhibitory glycine receptor (glycine(A)) the glycine binding site on the NMDA receptor (glycine(B)) is strychnine-insensitive. A great diversity of diseases showing a disturbed glutamate neurotransmission have been linked to the NMDA receptor. Glycine site antagonists have been investigated for acute diseases like stroke and head trauma as well as chronic ones like dementia and chronic pain.

A novel series of C-3 substituted 4,6-dichloroindole-2-carboxylic acids was synthesized to investigate the influence of different hydrogen-bond donor and acceptor groups at this specific position on the affinity to the glycine site of the NMDA receptor. These novel 3-indolylmethyl derivatives with ring-open (amines, sulfonamides, amides, ureas) and cyclic substituents (imidazolidin-2-ones, (thio)hydantoins) led to the discovery that compounds bearing a hydantoin substituent at the C-3 position of the indole nucleus are the most promising ones. In this series the hydantoins, ureas, and imidazolidin-2-ones were identified as very potent inhibitors of the binding of the glycine site specific ligand [(3)H]MDL 105,519 to pig cortical brain membranes. Since the hydantoins can be produced via a versatile synthetic approach, further amendments of the hydantoin-substituted compounds were conducted to elucidate the influence of aromatic and aliphatic moieties at position 3 of the hydantoin as well as of sterically hindered compounds (5-substituted hydantoins). On the basis of the pharmacological data obtained in displacement experiments with [(3)H]MDL 105,519 and the emerging structure-activity relationships, we confirm the existing pharmacophore model that suggests a hydrogen-bond acceptor and an aromatic substituent at position 3 of the indole as the key features for high affinity. Log P values indicate brain permeability and selected compounds showed anticonvulsant activity in vivo. Binding studies for the sodium channel (site 2) were also performed on some selected compounds.

Cys-loop receptors are pentameric ligand-gated ion channels that are responsible for fast excitatory and inhibitory transmission in the peripheral and central nervous systems. Each receptor has an extracellular domain (ECD) that contains the ligand-binding sites, a transmembrane domain (TMD) that forms the ion channel pore and an intracellular domain (ICD). All prokaryotic homologs of Cys-loop receptors, for example, GLIC and ELIC, lack an ICD that consists of a 50 to 270 amino acid long loop in eukaryotes. Very little is known about the structure of the ICD since none of the recently published X-ray structures of prokaryotic homologs contained an ICD, and the Torpedo nAChR cryo-EM structure resolved only 1/3rd of the ICD. We have recently constructed chimeras in which the ICD from eukaryotic serotonin (5-HT3A) receptors was inserted into GLIC (1).To extend our studies we generated GLIC chimeras with ICDs from other Cys-loop receptors (GlyR-α1, GABA-ρ1, nAChR-α7). All chimeras were expressed heterologously in Xenopus laevis oocytes to investigate the ion channel functionality by using two-electrode voltage clamp experiments. All functional chimeras were also expressed in E. coli membranes to enable overexpression and protein purification. Chimeric proteins were successfully purified by using Ni-NTA affinity and size exclusion chromatography (SEC). Limited proteolysis and spectroscopic methods (circular dichroism and fourier transform infrared spectroscopy) will be used to study conformational features and secondary structure elements of eukaryotic ICDs of the engineered GLIC-ICD chimeras. Our data will shed light on the structure and function of the intracellular domain of Cys-loop receptors, which will enable identification of potentially important drug targeting sites in intracellular domains for the treatment of neurodegenerative diseases.1. Goyal R. et al, (2011). J Biol Chem 286, 34635-42

Receptors belonging to the Cys-loop gene superfamily of neurotransmitter-gated ion channels (e.g. acetylcholine, serotonin, GABA, and glycine receptors) mediate fast synaptic transmission. These receptors are targeted by a number of clinically used drugs (e.g. antiepileptics, antipsychotics, anesthetics). Cys-loop receptors contain three domains: extracellular (ECD), transmembrane (TMD), intracellular (ICD). The ECD and TMD have been studied in great detail and their 3-D structures have been determined. The recent identification of bacterial Cys-loop receptor homologues has propelled the structural knowledge to atomic resolution. However, all prokaryotic members lack the ICD. The ICD of metazoan Cys-loop receptors is the most diverse domain with respect to both length and amino-acid composition. The ICD therefore represents an attractive target for developing subtype-selective drugs with the promise of fewer side effects than drugs that target the highly-conserved ECD and TMD. We have engineered functional chimeras with ICDs from different eukaryotic receptors in the two-domain prokaryotic homologue GLIC (one chimera set published, Goyal, Salahudeen, Jansen, JBC 2011). Based on structure predictions others have hypothesized that the ICD is mainly unstructured, but contains an α-helical segment pre-TM4. We hypothesize based on computational results that cationic and anionic receptors have different ICD secondary structures, i.e., only cationic receptors contain an α-helical portion pre-TM4. For each eukaryotic ICD we generated 12 chimeras that differ by the linkers both N- and C-terminal to the inserted ICD. Interestingly, the number of functional chimeras in each set varies drastically between different ICDs. We are testing the functional consequences of inserting various ICDs using electrophysiological studies, and investigating the structure of the ICDs tethered to the TMD and at the lipid bilayer with spectroscopic and biochemical methods.

The Proton-Coupled Folate Transporter (PCFT) encoded by the gene SLC46A1 is the major pathway for uptake of folic acid in the intestine and into the brain. Mutations in the PCFT gene lead to hereditary folate malabsorption (HFM), a folate deficiency disease characterized by impaired intestinal folate absorption and transport of folate into the central nervous system. PCFT is also involved in the uptake of antifolates such as Pemetrexed by tumor cells. At the present time our knowledge about the structure/function relationships of PCFT are very limited. Our initial goal is to identify the lipid-protein interface of PCFT by identifying transmembrane segments labeled by hydrophobic photoreactive probes like 1-Azidopyrene (1-AP) and 2-[3H]diazofluorene ([3H]DAF). These probes partition efficiently (>95%) into the lipid bilayer and upon UV-irradiation form covalent linkages with close-by amino acids. 1-AP and [3H]DAF labeled peptides can then be identified by mass spectrometry or by automated Edman degradation coupled with radio-sequencing. To carry out these experiments requires large amounts (several mg) of PCFT protein. Out of three expression systems we have evaluated (mammalian, cell-free, and bacterial), expression in BL21 E. coli is so far the most promising. From preliminary labeling experiments we have confirmed the membrane insertion of PCFT: 1) PCFT membranes were washed with NaHCO3 and urea, and 2) With 1-AP labeled PCFT, we identified peptide fragments of PCFT using mass spectrometry. Future experiments will involve: 1) screening of additional bacterial expression host strains, 2) labeling of PCFT with [3H]DAF and identification of labeled peptides as well as individual labeled amino-acids using radio-sequencing. Our results will define the PCFT lipid-protein interface and therefore will help verify 1) the α-helical secondary structure of transmembrane segments and 2) the arrangement of transmembrane segments within the membrane-spanning domain.

The Cys-Loop gene super-family of ligand gated ion channels includes nicotinic acetylcholine (nACh), gamma aminobutyric acid (GABAA), glycine (Gly), and serotonin (5-HT3A) receptors. Each receptor is either a homo- or heteropentamer made up of 5 identical or homologous subunits. Each subunit has an extracellular N-terminal domain, which houses the ligand binding site; a transmembrane domain which spans the membrane four times as α-helical segments (M1-M4); and a long loop between M3 and M4 that constitutes the intracellular domain. M2 lines the ion channel, whereas M1, M3 and M4 are abluminal.Muscle nAChRs consist of four different subunits with the clockwise arrangement αγαβδ when viewed from the extracellular side. We used disulphide trapping between individually engineered Cys in the αM2 segments to investigate arrangement and flexibility of the upper part of αM2. Disulfide bond formation was monitored in α2βδγ nAChR expressed in Xenopus laevis oocytes by two electrode voltage clamp experiments and Western blotting. In properly arranged Cys pairs disulfide bond formation can either occur spontaneously or it can be induced by oxidizing with copper phenanthroline (CuPhen). Cystine bond formation is reversible by reducing with dithiothreitol (DTT). To eliminate interference from the native vicinal disulphide present in the ligand binding site of the α-subunit, we utilized the background mutations αC192S-C193S. Position αE262C that was previously shown to face the channel formed DTT reducible dimers both spontaneously and upon application of the oxidizing agent CuPhen. Based on Unwin's 4-A resolution model, the formation of disulfide bonds at this channel level would require substantial movement of the channel-lining M2 segments. No dimer formation was observed in αL263C. We are investigating a series of αM2 Cys mutants to determine which positions in M2 can form disulfide bonds.

SummaryFolates are essential nutrients that are required for one-carbon biosynthetic and epigenetic processes. While folates are absorbed in the acidic milieu of the upper small intestine, the underlying absorption mechanism has not been defined. We now report the identification of a human proton-coupled, high-affinity folate transporter that recapitulates properties of folate transport and absorption in intestine and in various cell types at low pH. We demonstrate that a loss-of-function mutation in this gene is the molecular basis for hereditary folate malabsorption in a family with this disease. This transporter was previously reported to be a lower-affinity, pH-independent heme carrier protein, HCP1. However, the current study establishes that a major function of this gene product is proton-coupled folate transport required for folate homeostasis in man, and we have thus amended the name to PCFT/HCP1.

Bupropion is clinically prescribed for the treatment of depression (Wellbutrin) and for smoking cessation (Zyban). While there is consensus that the primary mechanism of action involves increased synaptic concentrations of dopamine and norepinephrine via inhibition of the respective reuptake transporters, DAT and NET, there is growing evidence that some of the therapeutic benefits of bupropion may result from non-competitive antagonism of neuronal nicotinic acetylcholine receptors (nAChRs), in particular α4β2 and α3β4 subtypes. To aid in elucidating the mechanism of bupropion action and the development of new therapeutic agents for smoking cessation and depression, our goal is to determine the specific molecular interactions between bupropion and several nAChR subtypes (Torpedo, α4β2, α3β4). We first established that bupropion dose-dependently and reversibly inhibits ACh-induced currents in Xenopus laevis oocytes injected with affinity-purified and lipid-reconstituted Torpedo nAChRs (IC50 0.34 μM). Using a photoreactive analog of bupropion ([125I]-SADU-3-72), we next established that bupropion inhibits the binding of [125I]-SADU-3-72 to the Torpedo nAChR (IC50 closed state, 3.6 μM; desensitized state, 1.2 μM) and that binding to the closed nAChR is fully inhibited by tetracaine (IC50 0.42 μM) consistent with a bupropion/[125I]-SADU-3-72 binding site within the nAChR channel. [125I]-SADU-3-72 photolabeled Torpedo nAChR subunits in an agonist-sensitive and bupropion-inhibitable (specific) manner. Finally for the closed nAChR, we identified sites of specific [125I]-SADU-3-72 labeling within δM2 (δLeu265/δM2-9) and βM2 (βLeu257/βM2-9). In the desensitized state, TCP-inhibitable (specific) labeling was identified within δM2 (δLeu265, δM2-9 with minor labeling of δSer258, δM2-2). Our results establish that bupropion binds in the middle (M2-9) of the Torpedo nAChR channel, with a slightly broader binding locus in the desensitized channel. Currently, studies are underway to identify the site(s) of [125I]-SADU-3-72 labeling in affinity-purified α4β2 and α3β4 nAChRs.

Folate vitamins are essential for DNA replication and cellular proliferation. However, mammalian organisms are devoid of folate biosynthesis and rely on dietary sources to meet their metabolic requirement for folate cofactors. The proton coupled folate transporter (PCFT/SLC46A1) has been recently identified as the molecular entity responsible for intestinal folate uptake displaying optimal transport activity at acidic pH. PCFT is also involved in transport of chemotherapeutically used antifolates. Currently, there is limited experimentally derived information about the structure and transmembrane topology of PCFT. Hydropathy analysis suggests 10 to 12 transmembrane segments for PCFT. The aim of our study is to construct a reliable homology model and study the structure function interplay in more detail experimentally. We have made various DNA constructs with different N- and C-terminal tags for the heterologous expression and purification of PCFT: 1) for bacterial expression of the gene, 2) for expression in oocytes and mammalian cells, 3) for cell free expression using the Membrane Max cell free expression kit. We will screen the expression systems to determine which is optimal for generating suitable quantity / quality protein that will be used for structural studies. We will engineer individual Cysteines to study the topology with the Substituted Cysteine Accessibility Model (SCAM). Accessibility will be assessed by Western Blotting (PEGylation, biotinylation). The lipid-protein interface will be investigated by hydrophobic photoaffinity labeling. Both SCAM and photoaffinity labeling will be used to study substrate pathways. We will complement these studies with functional assays (uptake and two electrode voltage clamping). Results from our structure function studies of PCFT will be used in exploring therapeutic strategies for folate malabsorption and in optimizing antifolate drug therapies.

Heterologous expression after mRNA/cDNA injection into Xenopus oocytes is a well established system for studying the structure and function of a variety of membrane proteins including ligand-gated ion channels (LGIC). In the early 1990's, the Miledi group [1] introduced a rather unique variant of this system by injecting pre-assembled nicotinic acetylcholine receptors (nAChR) into oocytes and measuring function using electrophysiological recordings. Native or affinity-purified Torpedo nAChR-membranes were injected into Xenopus oocytes and after incubation to allow receptor-containing membranes to fuse with the plasma membrane, functional receptor channels were detected using two-electrode voltage clamp recordings. Our goal is to utilize this system to assess the functionality and pharmacology of affinity-purified and lipid-reconstituted LGICs. We first determined that the optimal incubation period for the appearance of maximal functional channels following injection of native or affinity-purified and lipid-reconstituted Torpedo nAChRs was 48 hr. We next established that injection of affinity-purified Torpedo nAChRs reconstituted in lipid vesicles comprised of only phosphatidylcholine (DOPC) had minimal functionality (300 μM i.e. >EC100 ACh-induced currents at −60 mV; ∼ 13-23 nA). In contrast, affinity-purified Torpedo nAChRs reconstituted in vesicles comprised of DOPC/DOPA/CH (3:1:1) were fully functional (300 μM ACh at −60 mV; ∼ 1-3 μA) and the EC50 for ACh (∼ 50 μM) was comparable to that obtained for Torpedo nAChR mRNA injected oocytes. These results indicate that CH and anionic lipids (PA) are not only required for receptor functionality [2] but are protective of receptors during detergent solubilization and affinity-purification. Currently, studies are underway to examine the functionality of several affinity-purified and lipid-reconstituted LGICs, including α4β2 and α3β4 nAChRs, and 5-HT3A receptors.[1] Marsal et al (1995) PNAS 92, 5224-5228.[2] Hamouda et al (2006) Biochemistry 45, 4327-4337.

Eukaryotic pentameric ligand-gated ion channels (pLGIC) represent targets for a wide variety of drugs such as skeletal muscle relaxants, anti-psychotics, anti-epileptics and drugs against Parkinson's and Alzheimer's diseases. Each subunit consists of an extracellular domain (ECD), a transmembrane domain (TMD) with 4 α-helical segments, and an intracellular domain (ICD) that is 4-14 amino acids long in prokaryotes and ∼50-270 amino acids in eukaryotes. The ICD of eukaryotes has been implicated in modulating single channel conductance and kinetic properties of the channel. Additionally, it interacts with cytosolic proteins such as the resistance to inhibitors of cholinesterase protein, RIC-3, that affects plasma membrane-expression of some pLGIC. We created chimeras by introducing the ICD of cationic (5-HT3A , nACh α7) as well as anionic (Glycine α1, GABAA ρ1) eukaryotic pLGICs into the Gloeobacter violaceus pLGIC, GLIC, a well-studied prokaryotic homologue, that only contains ECD and TMD. Electrophysiological experiments after X. laevis oocyte expression (EC50 and IC50) demonstrate that for each of the four chimera sets, we have constructs that act as functional proton-gated ion channels, similar to the parent GLIC. Co-expression of cationic chimeras as well as wild-type pLGIC together with RIC-3 showed significant changes in current amplitudes, whereas RIC-3 did not affect anionic receptor chimeras or wild-type channels. We established that cationic chimeras and RIC-3, both overexpressed and purified to homogeneity from E. coli, bind to each other. Currently, we are using the chimeras to identify interacting proteins in native brain lysates. Our results clearly demonstrate a direct interaction between the ICD of cationic pLGIC and RIC-3. Specific interaction points will be identified in further studies.

Neuronal nicotinic acetylcholine receptors (nAChRs) are allosteric proteins which belong to the Cys-loop superfamily of pentameric ligand-gated ion channels. Many therapeutically relevant drugs act as positive/negative allosteric modulators of these receptors and are sensitive/selective to the conformational state of the receptor. Studies to identify the molecular interactions of an agent with a particular neuronal nAChR via a technique such as photoaffinity labeling require purified receptors that retain the ability to undergo conformational transitions between functional states. However, producing purified neuronal nAChRs that are fully functional has proven to be a challenge (see Ref 1-2). With the present study we are systematically assessing the functionality of neuronal nAChRs at different critical steps during isolation (membrane isolation, detergent solubilization, lipid reconstitution, etc). Each step is optimized with respect to both increasing the yield of protein, but importantly also to ascertain functionality. Pre-assembled nAChRs are microinjected into oocytes, a method pioneered by the Miledi group (3), and following fusion of receptors with the oocyte plasma-membrane, two-electrode voltage-clamp recordings are used to monitor receptor functionality.[1] Hamouda et al (2007) Biochemistry 46(48), 13837-46.[2] daCosta et al (2011) Biochem Biophys Res Commun 407(3), 456-60.[3] Marsal et al (1995) PNAS 92, 5224-28.