Publications (13)94.37 Total impact
-
Article: GABA-induced intersubunit conformational movement in the GABAA receptor alpha 1M1-beta 2M3 transmembrane subunit interface: experimental basis for homology modeling of an intravenous anesthetic binding site.
[show abstract] [hide abstract]
ABSTRACT: The molecular basis of general anesthetic interactions with GABA(A) receptors is uncertain. An accurate homology model would facilitate studies of anesthetic action. Construction of a GABA(A) model based on the 4 A resolution acetylcholine receptor structure is complicated by alignment uncertainty between the acetylcholine and GABA(A) receptor M3 and M4 transmembrane segments. Using disulfide crosslinking we previously established the orientation of M2 and M3 within a single GABA(A) subunit. The resultant model predicts that the betaM3 residue beta2M286, implicated in anesthetic binding, faces the adjacent alpha1-M1 segment and not into the beta2 subunit interior as some models have suggested. To assess the proximity of beta2M286 to the alpha1-M1 segment we expressed beta2M286C and gamma2 with 10 consecutive alpha1-M1 cysteine (Cys) mutants, alpha1I223C to alpha1L232C, in and flanking the extracellular end of alpha1-M1. In activated states, beta2M286C formed disulfide bonds with alpha1Y225C and alpha1Q229C based on electrophysiological assays and dimers on Western blots, but not with other alpha1-M1 mutants. beta2F289, one helical turn below beta2M286, formed disulfide bonds with alpha1I228C, alpha1Q229C and alpha1L232C in activated states. The intervening residues, beta2G287C and beta2C288, did not form disulfide bonds with alpha1-M1 Cys mutants. We conclude that the beta2-M3 residues beta2M286 and beta2F289 face the intersubunit interface in close proximity to alpha1-M1 and that channel gating induces a structural rearrangement in the transmembrane subunit interface that reduces the betaM3 to alphaM1 separation by approximately 7 A. This supports the hypothesis that some intravenous anesthetics bind in the betaM3-alphaM1 subunit interface consistent with azi-etomidate photoaffinity labeling.Journal of Neuroscience 04/2009; 29(10):3083-92. · 7.11 Impact Factor -
Article: Probing protein packing surrounding the residues in and flanking the nicotinic acetylcholine receptor M2M3 loop.
[show abstract] [hide abstract]
ABSTRACT: Nicotinic acetylcholine receptors (nAChR) are cation-selective, ligand-gated ion channels of the cysteine (Cys)-loop gene superfamily. The recent crystal structure of a bacterial homolog from Erwinia chrysanthemi (ELIC) agrees with previous structures of the N-terminal domain of AChBP (acetylcholine-binding protein) and of the electron-microscopy-derived Torpedo nAChR structure. However, the ELIC transmembrane domain is significantly more tightly packed than the corresponding region of the Torpedo nAChR. We investigated the tightness of protein packing surrounding the extracellular end of the M2 transmembrane segment and around the loop connecting the M2 and M3 segments using the substituted cysteine accessibility method. The M2 20' to 27' residues were highly water accessible and the variation in reaction rates were consistent with this region being alpha-helical. At all positions tested, the presence of ACh changed methanethiosulfonate ethylammonium (MTSEA) modification rates by <10-fold. In the presence of ACh, reaction rates for residues in the last extracellular alpha-helical turn of M2 and in the M2M3 loop increased, whereas rates in the penultimate alpha-helical turn of M2 decreased. Only three of eight M2M3 loop residues were accessible to MTSEA in both the presence and absence of ACh. We infer that the protein packing around the M2M3 loop is tight, consistent with its location at the interdomain interface where it is involved in the transduction of ligand binding in the extracellular domain to gating in the transmembrane domain. Our data indicate that the Torpedo nAChR transmembrane domain structure is a better model than the ELIC structure for eukaryotic Cys-loop receptors.Journal of Neuroscience 02/2009; 29(6):1626-35. · 7.11 Impact Factor -
Article: The proton-coupled folate transporter: impact on pemetrexed transport and on antifolates activities compared with the reduced folate carrier.
[show abstract] [hide abstract]
ABSTRACT: The reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT) are ubiquitously expressed in normal and malignant mammalian tissues and in human solid tumor cell lines. This article addresses the extent to which PCFT contributes to transport of pemetrexed and to the activities of this and other antifolates relative to RFC at physiological pH. Either RFC or PCFT cDNA was stably transfected into a transporter-null HeLa cell variant to achieve activities similar to their endogenous function in wild-type HeLa cells. PCFT and RFC produced comparable increases in pemetrexed activity in growth medium with 5-formyltetrahydrofolate. However, PCFT had little or no effect on the activities of methotrexate, N-(5-[N-(3,4-dihydro-2-methyl-4-oxyquinazolin-6-ylmethyl)-N-methyl-amino]-2-thenoyl)-l-glutamic acid (raltitrexed, Tomudex; ZD1694), or N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-l-ornithine (PT523) in comparison with RFC irrespective of the folate growth source. PCFT, expressed at high levels in Xenopus laevis oocytes and in transporter-competent HepG2 cells, exhibited a high affinity for pemetrexed, with an influx K(m) value of 0.2 to 0.8 muM at pH 5.5. PCFT increased the growth inhibitory activity of pemetrexed, but not that of the other antifolates in HepG2 cells grown with 5-formyltetrahydrofolate at physiological pH. These findings illustrate the unique role that PCFT plays in the transport and pharmacological activity of pemetrexed. Because of the ubiquitous expression of PCFT in human tumors, and the ability of PCFT to sustain pemetrexed activity even in the absence of RFC, tumor cells are unlikely to become resistant to pemetrexed as a result of impaired transport because of the redundancy of these genetically distinct routes.Molecular pharmacology 09/2008; 74(3):854-62. · 4.53 Impact Factor -
Article: Synthesis of GABAA Receptor Agonists and Evaluation of their α-Subunit Selectivity and Orientation in the GABA Binding Site
[show abstract] [hide abstract]
ABSTRACT: Drugs used to treat various disorders target GABAA receptors. To develop α 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 [3H]muscimol binding and in patch-clamp experiments with heterologously expressed GABAA αiβ3γ2 receptors (i = 1−6). The effects of 5-aminomethyl-3H-[1,3,4]oxadiazol-2-one 5d were comparable to GABA for all α subunit isoforms. 5-piperidin-4-yl-3H-[1,3,4]oxadiazol-2-one 5a and 5-piperidin-4-yl-3H-[1,3,4]oxadiazol-2-thione 6a were weak agonists at α2-, α3-, and α5-containing receptors. When coapplied with GABA, they were antagonistic in α2-, α4-, and α6-containing receptors and potentiated α3-containing receptors. 6a protected GABA binding site cysteine-substitution mutants α1F64C and α1S68C from reacting with methanethiosulfonate-ethylsulfonate. 6a specifically covalently modified the α1R66C thiol, in the GABA binding site, through its oxadiazolethione sulfur. These results demonstrate the feasibility of synthesizing α subtype selective GABA mimetic drugs.Journal of Medicinal Chemistry 07/2008; 51(15). · 4.80 Impact Factor -
Article: Modular design of Cys-loop ligand-gated ion channels: functional 5-HT3 and GABA rho1 receptors lacking the large cytoplasmic M3M4 loop.
[show abstract] [hide abstract]
ABSTRACT: 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 alpha-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-HT(3A)) subunit with a heptapeptide from the prokaryotic homologue from Gloeobacter violaceus. The macroscopic electrophysiological and pharmacological characteristics of the homomeric 5-HT(3A)-glvM3M4 receptors were comparable to 5-HT(3A) wild type. The channels remained cation-selective but the 5-HT(3A)-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-HT(3) and acetylcholine receptors, significantly attenuated 5-HT-induced currents with wild-type 5-HT(3A) but not 5-HT(3A)-glvM3M4 receptors. A similar deletion of the M3M4 loop in the anion-selective GABA-rho1 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.The Journal of General Physiology 03/2008; 131(2):137-46. · 3.84 Impact Factor -
Article: Rodent intestinal folate transporters (SLC46A1): secondary structure, functional properties, and response to dietary folate restriction.
[show abstract] [hide abstract]
ABSTRACT: 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 NH(2)- 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 [(3)H]folates was optimal at pH 5.5 and decreased with increasing pH due to an increase in K(m) and a decrease in V(max). At pH 7.0, folic acid and methotrexate influx was negligible, but there was residual (6S)5-methyltetrahydrofolate transport. Uptake of folates in PCFT-injected Xenopus oocytes was electrogenic and pH dependent. Folic acid influx K(m) values of MmPCFT and RnPCFT, assessed electrophysiologically, were 0.7 and 0.3 microM at pH 5.5 and 1.1 and 0.8 microM 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 approximately 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.AJP Cell Physiology 12/2007; 293(5):C1669-78. · 3.54 Impact Factor -
Article: Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption.
[show abstract] [hide abstract]
ABSTRACT: Folates 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.Cell 01/2007; 127(5):917-28. · 32.40 Impact Factor -
Article: State-dependent cross-linking of the M2 and M3 segments: functional basis for the alignment of GABAA and acetylcholine receptor M3 segments.
[show abstract] [hide abstract]
ABSTRACT: Construction of a GABAA receptor homology model based on the acetylcholine (ACh) receptor structure is complicated by the low sequence similarity between GABAA and ACh M3 transmembrane segments that creates significant uncertainty in their alignment. We determined the orientation of the GABAA M2 and M3 transmembrane segments using disulfide cross-linking. The M2 residues alpha1M266 (11') and alpha1T267 (12') were mutated to cysteine in either wild type or single M3 cysteine mutant (alpha1V297C, alpha1A300C to alpha1A305C) backgrounds. We assayed spontaneous and induced disulfide bond formation. Reduction with DTT significantly potentiated GABA-induced currents in alpha1T267C-L301C and alpha1T267C-F304C. Copper phenanthroline-induced oxidation inhibited GABA-induced currents in these mutants and in alpha1T267C-A305C. Intrasubunit disulfide bonds formed between these Cys pairs, implying that the alpha-carbon separation was at most 5.6 A. The reactive alpha1M3 residues (L301, F304, A305) lie on the same face of an alpha-helix. The unresponsive ones (A300, I302, E303) lie on the opposite face. In the resting state, the reactive side of alpha1M3 faces M2-alpha1T267. In conjunction with the ACh structure, our data indicate that alignment of GABAA and ACh M3 requires a single gap in the GABAA M2-M3 loop. In the presence of GABA, oxidation of alpha1T267C-L301C and alpha1T267C-F304C had no effect, but oxidation of alpha1T267C-A305C caused a significant increase in spontaneous channel opening. We infer that, as the channel opens, the distance and/or orientation between M2-alpha1T267 and M3-alpha1A305 changes such that the disulfide bond stabilizes the open state. This begins to define the conformational motion that M2 undergoes during channel opening.Journal of Neuroscience 05/2006; 26(17):4492-9. · 7.11 Impact Factor -
Article: A role for the beta 1-beta 2 loop in the gating of 5-HT3 receptors.
[show abstract] [hide abstract]
ABSTRACT: Based on the Torpedo acetylcholine 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 alphaV46 at the tip of the extracellular beta1-beta2 loop and the transmembrane M2 segment and M2-M3 loop. We mutated to cysteine the aligned positions in the 5-HT3A and 5-HT3B subunit beta1-beta2 loops 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, beta1-beta2 loop 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.Journal of Neuroscience 11/2005; 25(41):9358-66. · 7.11 Impact Factor -
Article: Advances in DNA-ligands with groove binding, intercalating and/or alkylating activity: chemistry, DNA-binding and biology.
[show abstract] [hide abstract]
ABSTRACT: 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.Current Medicinal Chemistry 02/2005; 12(24):2805-47. · 4.86 Impact Factor -
Article: Variations of acidic functions at position 2 and substituents at positions 4, 5 and 6 of the indole moiety and their effect on NMDA-glycine site affinity.
[show abstract] [hide abstract]
ABSTRACT: 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.European Journal of Medicinal Chemistry 11/2003; 38(10):855-65. · 3.35 Impact Factor -
Article: Hydantoin-substituted 4,6-dichloroindole-2-carboxylic acids as ligands with high affinity for the glycine binding site of the NMDA receptor.
[show abstract] [hide abstract]
ABSTRACT: 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.Journal of Medicinal Chemistry 02/2003; 46(1):64-73. · 5.25 Impact Factor -
Article: Antagonists and agonists at the glycine site of the NMDA receptor for therapeutic interventions.
[show abstract] [hide abstract]
ABSTRACT: 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.European Journal of Medicinal Chemistry 38(7-8):661-70. · 3.35 Impact Factor
Top co-authors
Top Journals
Institutions
-
2007–2009
-
Albert Einstein College of Medicine
- • Department of Physiology & Biophysics
- • Department of Medicine
New York City, NY, USA
-
-
2003–2008
-
Johannes Gutenberg-Universität Mainz
- Department of Pharmacology
Mainz, Rhineland-Palatinate, Germany
-
-
2005–2006
-
Yeshiva University
- • Albert Einstein College of Medicine
- • Department of Physiology & Biophysics
New York City, NY, USA
-
