Michaela Jansen

Identification of an Intestinal Folate Transporter and the Molecular Basis for Hereditary Folate Malabsorption

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.

The Proton-Coupled Folate Transporter: Impact on Pemetrexed Transport and on Antifolates Activities Compared with the Reduced Folate Carrier

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 Nα-(4-amino-4-deoxypteroyl)-Nδ-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 Km value of 0.2 to 0.8 μM 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.

Advances in DNA-Ligands with Groove Binding, Intercalating and/or Alkylating Activity: Chemistry, DNA-Binding and Biology

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.

Modular Design of Cys-loop Ligand-gated Ion Channels: Functional 5-HT3 and GABA ρ1 Receptors Lacking the Large Cytoplasmic M3M4 Loop

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.

A Role for the β1-β2 Loop in the Gating of 5-HT3 Receptors

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 αV46 at the tip of the extracellular β1-β2 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 β1-β2 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, β1-β2 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.

State-Dependent Cross-Linking of the M2 and M3 Segments: Functional Basis for the Alignment of GABAA and Acetylcholine Receptor M3 Segments

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 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 GABAA and ACh M3 requires a single gap in the GABAA M2–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.

GABA-Induced Intersubunit Conformational Movement in the GABAA Receptor α1M1-β2M3 Transmembrane Subunit Interface: Experimental Basis for Homology Modeling of an Intravenous Anesthetic Binding Site

The molecular basis of general anesthetic interactions with GABAA receptors is uncertain. An accurate homology model would facilitate studies of anesthetic action. Construction of a GABAA model based on the 4 Å resolution acetylcholine receptor structure is complicated by alignment uncertainty between the acetylcholine and GABAA receptor M3 and M4 transmembrane segments. Using disulfide crosslinking we previously established the orientation of M2 and M3 within a single GABAA subunit. The resultant model predicts that the βM3 residue β2M286, implicated in anesthetic binding, faces the adjacent α1-M1 segment and not into the β2 subunit interior as some models have suggested. To assess the proximity of β2M286 to the α1-M1 segment we expressed β2M286C and γ2 with 10 consecutive α1-M1 cysteine (Cys) mutants, α1I223C to α1L232C, in and flanking the extracellular end of α1-M1. In activated states, β2M286C formed disulfide bonds with α1Y225C and α1Q229C based on electrophysiological assays and dimers on Western blots, but not with other α1-M1 mutants. β2F289, one helical turn below β2M286, formed disulfide bonds with α1I228C, α1Q229C and α1L232C in activated states. The intervening residues, β2G287C and β2C288, did not form disulfide bonds with α1-M1 Cys mutants. We conclude that the β2-M3 residues β2M286 and β2F289 face the intersubunit interface in close proximity to α1-M1 and that channel gating induces a structural rearrangement in the transmembrane subunit interface that reduces the βM3 to αM1 separation by ∼7 Å. This supports the hypothesis that some intravenous anesthetics bind in the βM3-αM1 subunit interface consistent with azi-etomidate photoaffinity labeling.

Antagonists and agonists at the glycine site of the NMDA receptor for therapeutic interventions.

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.

Structural sensitivity of a prokaryotic pentameric ligand-gated ion channel to its membrane environment*

Background: The lipid sensitivity of the prokaryotic pentameric ligand-gated ion channel (pLGIC), GLIC, is poorly characterized. Results: GLIC is more thermally stable and does not exhibit the same propensity to adopt an uncoupled conformation as the Torpedo nAChR. Conclusion: GLIC is less sensitive to its surrounding membrane environment. Significance: The GLIC and nAChR structures suggest molecular features governing the lipid sensitivity of pLGICs. 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.

Synthesis of GABAA Receptor Agonists and Evaluation of their α-Subunit Selectivity and Orientation in the GABA Binding Site

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.