Molecular modelling of the interactions of carbamazepine and a nicotinic receptor involved in the autosomal dominant nocturnal frontal lobe epilepsy.

1. The normal and a mutant (S248F) human neuronal alpha4beta2 nicotinic receptors, and their interaction with the channel blocker carbamazepine (CBZ) have been modelled. The mutant, responsible for the autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), has an enhanced sensitivity to and a slower recovery from desensitization, a lower conductance, short open times, reduced calcium permeability, and is 3 fold more sensitive to CBZ, a drug used in the treatment of partial epilepsies. 2. Mutant channel properties are explained by the physicochemical properties of the two Phe248 side chains, including size and cation-pi interaction, and their dynamic behaviour. A defective mechanism of dehydration might be responsible for the reduced calcium influx. 3. Phe248 residues are the main component of CBZ binding sites in the mutant, while this is not true for Ser248 in the normal receptor. 4. A higher number of blocking binding sites and a predicted higher affinity found for CBZ in the mutant account for its differential sensitivity to CBZ. 5. Aromatic-aromatic interactions between CBZ and the two Phe248 account for the difference in affinity, which is at least 12 times higher for the mutant, depending on the method used for calculating K(i). 6. Normal vs mutant differences in K(i), enhanced by the higher number of blocking binding sites in the mutant, seem excessive compared to the differential sensitivities to CBZ experimentally found. The negative cooperativity suggested by a predicted overlapping of blocking and non-blocking binding sites gives an explanation, as overlapping is higher in the mutant. 7. For both types of receptors we found that the carbamyl group of the best blocking conformers of CBZ forms hydrogen bonds with serine residues, which may explain the fundamental role of that moiety for this molecule to act as antiepileptic drug.

Understanding channel blocking in the nicotinic acetylcholine receptor.

Ion-channel blockers are molecules that obstruct the path used by ions to cross the membrane through a protein channel. Many of these are local anesthetics, toxins or drugs of abuse, and the knowledge of their mechanism of action at the atomic level is an important step towards the development of new compounds on a structural basis. A molecular model of the transmembrane region of the nicotinic acetylcholine receptor, an important brain and muscle fast signaling protein, was used as a target for docking several channel blockers by means of an automatic docking method. The combination of the independent docking method and molecular models (of the receptor and blockers) reproduced or explained quite accurately experimental data (photoaffinity labeling, site-directed mutagenesis, binding assays). This represents a strong support for the validity of the predictions made for those molecules for which no experimental data is available and also for the models and methods on which are based.

Prediction of the secondary structure of the nicotinic acetylcholine receptor nontransmembrane regions.

A consensus prediction for the secondary structure of the muscle nicotinic acetylcholine receptor (alpha, beta, gamma, and delta subunits) extracellular regions is presented. This protein is a member of the ligand-gated ion channel superfamily, which also encompasses the 5HT3, GABAA, and glycine receptors. The strategy used here is based on the application of six different prediction methods to an alignment of 118 sequences of this superfamily. A consensus prediction was finally produced for each of the four different subunits of the muscle nicotinic receptor nonmembrane regions. The predicted percentages, with respect to the total receptor length, and averaged for the four subunits are as follows: alpha-helix 29.7%, beta-sheet 24.9%, and turn + coil 21.7%. When adding to these values the estimations of the secondary structure reported for the transmembrane region only, the results are in agreement with those obtained experimentally by Yager et al. and Méthot et al. The deviations with respect to these experimental estimations are alpha-helix +2.8%, beta-sheet -4/-5% and turn + coil +3/+2%, respectively. Considering the predictions made for individual subunits, the best approximation was obtained for the alpha subunit, with deviations of -0.2% for alpha-helix, -2.5/-1.5% for beta-sheet, and +0.9/+1.9% for turn + coil. The prediction was used to infer the residues involved in forming three helices that presumably flank the ligand-binding pocket and to propose mechanism for transferring the information of the ligand binding to the ion channel.

Molecular modelling of the nicotinic acetylcholine receptor transmembrane region in the open state.

A model of the nicotinic acetylcholine receptor transmembrane region has been constructed which may represent the channel in its open-state. The positions of helices flanking the ion channel match those observed by electron microscopy and previously reported by others. Residues labelled, mutated or by other means known to have a strong influence on ion flux are each accessible from the lumen of the modelled channel. The model provides new insights into our current understanding of the ion channel structure, and suggests some novel explanations for the results of labelling and mutation studies such as those involving ion channel blockers and residue-dependent changes in ion selectivity.

Screening structural-functional relationships of neuropharmacologically active organic compounds at the nicotinic acetylcholine receptor.

The mechanisms of action and pharmacological effects on the nicotinic cholinoceptor of a large database of organic compounds were analyzed using a new computational procedure. This procedure is a screening method based on comparison of the molecular structures (shape and charge) of the putative active organic compounds. The resulting predictions can be used as an exploratory tool in the design of experiments aimed at testing the effects of several compounds on a target macromolecule. Unlike a conventional database search for structural similarities, the present method is able to circumscribe objectively the results to the most statistically significant molecules.

A mixed helix-beta-sheet model of the transmembrane region of the nicotinic acetylcholine receptor.

We have modelled the transmembrane region of the alpha 7 nicotinic acetylcholine receptor as a mixed alpha-helical/beta-sheet structure. The model was mainly based on the crystal structure of a pore-forming toxin, heat-labile enterotoxin. This is a pentameric protein having a central pore or channel composed of five alpha-helices, one from each of the 5 B subunits that form this pentamer. The remainder of this structure is beta-sheet, loops and a short alpha-helix, not included in the model. The model uses this channel as a template to build the transmembrane region, from M1 to the middle of M3. The remainder of M3 and M4 were built de novo as alpha-helices. Great consideration was given to labelling data available for the transmembrane region. In general terms, the shape of the model agrees very well with that obtained independently by electron microscopic analysis and the secondary structure predicted by the model is in accord with that estimated independently by Fourier transform infrared spectroscopy. The M2 helical region of the model is only slightly kinked, contrary to what is inferred from electron microscopic analysis, but has the same overall shape and form. On the membrane face of the model, the presence of deep pockets may provide the structural basis for the distinction between annular and non-annular lipid binding sites. Also, the transmembrane region is clearly asymmetric in the direction perpendicular to the membrane, and this may have strong influence on the surrounding lipid composition of each leaflet of the cytoplasmic membrane.

Evolutionary history of the ligand-gated ion-channel superfamily of receptors.

The fast-acting ligand-gated ion channels (LGICs) constitute a group that encompasses nicotinic ACh, 5-HT3, GABAA and glycine receptors. Undoubtedly, they all share a common evolutionary ancestor, and the group can therefore be considered to be a gene superfamily. Because the members of the superfamily are all receptors, it is reasonable to suppose that their common ancestor must also have been some type of receptor, and because the receptors are made of similar subunits, the ancestor was probably homo-oligomeric. Although we failed to find a group of proteins that are related evolutionarily to this superfamily, the analysis of the evolutionary relationships within the superfamily is possible and can give rise to information about the evolution of the structure and function of present-day receptors and indeed of the nervous system itself.

The transmembrane region of the nicotinic acetylcholine receptor: is it an all-helix bundle?

The nicotinic acetylcholine receptor is the best characterised member of the Ligand-Gated-Ion-Channel family of receptors. In spite of a wealth of data from molecular cloning studies these receptors have so far eluded all attempts at crystallisation; quantitative structural data are few and are at relatively low resolution. The widely accepted current model for the topology of the receptors is that of a pentameric cylindrical bundle that spans the membrane. The disposition of the transmembrane region of the individual subunits is based on hydropathy profiles calculated from sequence data which are interpreted as indicating a common structural motif of four antiparallel alpha-helices, M1 to M4. Until very recently this model has been unquestioned even though there are few direct experimental data to support it. We have constructed models of this key functional region for the nicotinic acetylcholine receptor, building out from the ion-channel. The model of the basic ion-channel comprises a five helical M2 bundle with a left-handed twist. The remainder of the region (M1, M3, M4) was homology modelled, together with M2, as a four helix antiparallel bundle per subunit, using the crystal structure of myohaemerythrin as a template. The models strongly suggest that the four helix bundle model is inappropriate and that recent suggestions of a mixed motif of helix and sheet may better accommodate the existing data.

CEDIT: a C interface and macro facility for protein sequence alignment editing in colour with Microsoft Word 5.0 for PCs.

CEDIT, a C interface and macro facility that provides for the colour editing of protein sequence alignments (up to 2000 sequences, 5000 residues each) using Microsoft Word 5.0 for PCs is presented. CEDIT uses the ability of MS-Word 5.0 to display letters with the desired colour to easily identify conservative homologies across the sequences. A glossary file with useful macros for the sequence editing is provided, along with several utilities programs for error checking, estimating sequence similarities and homology significance. CEDIT has a menu interface and a context sensitive help.

Homologies and disparities of glutamate receptors: a critical analysis.

Based on analysis of aligned amino acid sequences the following statements are made: (i) There is evolutionary homology between the N-terminal extracellular region of ionotropic Glutamate receptors/Kainate Binding Proteins and a family of procaryote amino acid binding proteins. (ii) Homology of the N-terminal extracellular domain of the metabotropic glutamate receptors with a family of receptors with a guanylate cyclase intracellular domain appears to be valid. (iii) There is no evidence for homology between the N-terminal extracellular domain of the nicotinic Acetylcholine, GABA, Glycine and 5HT3 receptors and that of the ionotropic Glutamate receptors/Kainate Binding proteins. (iv) The proposal of homology for the N-terminal extracellular domain of metabotropic Glutamate receptors and that of ionotropic Glutamate receptors does not appear to hold.

Cytogenetics and karyosystematics of phyllotine rodents (Cricetidae, Sigmodontinae). I. Chromosome multiformity and gonosomal-autosomal translocation in Reithrodon.

The chromosomes of 14 specimens of the genus Reithrodon from three different localities of Argentina and two localities of Uruguay were studied using G- and C-banding techniques. Specimens of Uruguay showed a karyotype of 2n = 28 chromosomes having a large metacentric X, and a telocentric Y chromosome. This karyotype is very similar to that recently described in a sample from southern Brazil, differing only in the nature of the Y chromosome, which is metacentric in the Brazilian form. All specimens from Argentina showed a 2n = 34 karyotype, differing from the Brazilian karyotype by two centric fusions, an acquisition of chromosome material, and at least one pericentric inversion, and by the telocentric nature of both the X and the Y chromosomes. G- and C-banding suggest that the metacentric gonosomes in the Brazilian form resulted from a double autosomal-X-Y Robertsonian translocation. The Uruguayan cytotype is interpreted as derived from a hypothetical neo-X/Y1Y2 ancestral form by the secondary loss of the Y1 chromosome. The karyotypic differences between the Brazilian-Uruguayan and the Argentinian forms afford evidence of species differentiation. It is proposed to assign the former to Reithrodon typicus, and the later to R. auritus.