Development of a neuronal pressure sensor.

Neuronal sensory systems are capable of performing very complex signal processing functions. Reconstruction of such sensory systems in vitro should enable whole-cell biological sensors to be generated that possess inherent signal processing capabilities. In this paper, the results of preliminary investigations to produce a mechanosensory neuronal network are presented. An in vitro network of rat dorsal root ganglion neurons has been produced on a microelectrode plate revealing an interesting rhythmical pattern of spontaneous discharges. This periodic activity has been shown to be disrupted following the application of a static pressure to the cell culture. These results indicate that neuronal networks represent a practical system that may be used for the development of intelligent, whole-cell, biological sensors.

Immunocytochemical localization of a putative inhibitory amino acid receptor subunit in the parasitic nematodes Haemonchus contortus and Ascaris suum.

A rabbit antiserum was raised against a synthetic peptide corresponding to a region near the N-terminus of the Haemonchus contortus inhibitory amino acid receptor subunit, HG1. The antiserum recognized a recombinant form of the N-terminal domain of the subunit on Western blots and reacted with the ventral nerve cord of H. contortus in immunofluorescence experiments. Immunofluorescence was also detected in specific head neurons of H. contortus: these were tentatively identified as ring motor- and inter-neurons, plus a possible sensory neuron equivalent to the AQR cell of Caenorhabditis elegans. In the roundworm Ascaris suum, immunoreactivity was limited to the muscle arms, the post-synaptic component of the neuromuscular junction. The possible ligand of receptors containing the HG1 subunit is discussed in the light of this expression pattern.

Alternative splicing of a Caenorhabditis elegans gene produces two novel inhibitory amino acid receptor subunits with identical ligand binding domains but different ion channels.

Two full-length cDNAs, gbr-2A and gbr-2B, encoding inhibitory amino acid receptor subunits have been amplified and cloned from Caenorhabditis elegans mRNA. The 5' 732 bp of the two cDNAs, encoding 237 amino acids, are identical. The 3' 758 bp of the gbr-2B cDNA are present within the 3' untranslated region of the gbr-2A clone. As a result, the two cDNAs are predicted to encode subunits which share a common extracellular N-terminal sequence of 237 amino acids, but different, though closely related, C-terminal sequences which include four predicted membrane-spanning regions. A search of the EMBL database revealed that the sequences of the two subunits are most closely related to the alpha-subunit of the C. elegans avermectin receptor. Northern blot analysis showed the presence of two related mRNAs of approximately 2.2 and 1.5 kb in a developmentally mixed population of C. elegans. The genomic DNA sequence confirms that both mRNAs were transcribed from the same gene, gbr-2, suggesting that the closely related 3' sequences have arisen as a result of a partial gene duplication event. We propose that C. elegans is utilising alternative splicing to generate receptor subunits with identical extracellular, ligand-binding domains but different transmembrane, channel forming domains.

Reporter gene constructs suggest that the Caenorhabditis elegans avermectin receptor beta-subunit is expressed solely in the pharynx.

Gene promoter/LacZ reporter constructs were made in order to analyse the expression of the beta-subunit of the Caenorhabditis elegans glutamate-gated Cl- channel (Glu-Cl) receptor. Southern blot analysis of the C. elegans cosmid C35E8 identified a 4kbp EcoRI fragment which contained the 5' portion of the Glu-Cl beta coding sequence together with 5' flanking sequences. This was subcloned and used as the template for polymerase chain reaction (PCR) amplification of a DNA fragment encoding the first 24 amino acid residues of Glu-Cl beta together with 1.4 kbp of 5' genomic sequence. The fragment was subcloned into the LacZ expression vector pPD22.11 to form a translational reporter fusion. After injection of the construct into worms, six stably transformed lines were established and assayed for beta-galactosidase activity. Stained nuclei were observed in the pharyngeal metacorpus in adults and in all larval stages, and stained nuclei were seen in many embryos undergoing morphogenesis. Additional stained nuclei towards the terminal bulb of the pharynx were observed in larval stages. These results provide further evidence that the Glu-Cl receptor mediates the glutamatergic inhibition of pharyngeal muscle via the M3 motor neurone and point to inhibition of pharyngeal pumping as a major mode of action for avermectins.

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.

Ferrocenoyl derivatives of alamethicin: redox-sensitive ion channels.

The synthesis and single-channel characterization of two redox-active C-terminal derivatives of alamethicin are herein described. The reduced [Fe(II)] forms of ferrocenoyl-alamethicin (Fc-ALM) and 1'-carboxyferrocenoyl-alamethicin (cFc-ALM) are shown to form voltage-dependent ion channels at cis positive potentials in planar lipid bilayers (PLB) with conductance properties similar to those of alamethicin. In situ oxidation of Fc-ALM [to Fe(III)] in the PLB apparatus causes a time-dependent elimination of channel openings, which can be restored by an increase in the transbilayer potential. In contrast, oxidation of cFc-ALM leads to the formation of shorter-lived channels. Pretreatment of the ferrocenoyl peptides with oxidizing agent alters their single-channel properties in a qualitatively similar manner, establishing that the changes in channel properties in the presence of oxidizing agents are due specifically to ferrocenoyl oxidation. We suggest that the redox sensitivity of these ferrocene-containing ion channels may be governed by a combination of the following factors: (1) changes in hydrophobicity; (2) alteration of peptide molecular dipole; and (3) alterations in tendencies toward self-association. However, oxidation induced changes in peptide conformation cannot be ruled out. Our results provide evidence that it is possible to engineer channel-forming peptides that respond to specific changes in the chemical environment.

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.

A nicotinic acetylcholine receptor subunit from insect brain forms a non-desensitising homo-oligomeric nicotinic acetylcholine receptor when expressed in Xenopus oocytes.

The locust alpha-like nicotinic receptor subunit alpha L1 was expressed in Xenopus oocytes. Small but reproducible currents were elicited by application of high concentrations of nicotine, demonstrating that alpha L1 is capable of forming homo-oligomeric channels. Nicotine-evoked currents were blocked by alpha-bungarotoxin and methyllycaconitine. Comparison with chick alpha 7 receptors showed that the two receptors differ with respect to nicotine sensitivity and time course of evoked currents. Nicotine dose-response curves gave EC50 values of 24 and 830 microM for alpha 7 and alpha L1 respectively. Whereas alpha 7 responses showed characteristic fast onset and rapid desensitization within 3 s, alpha L1 currents displayed a slow onset and showed no tendency to desensitize during 45 s of agonist application. Thus alpha L1 is a novel nicotine subunit for the further exploration of structure-function relationships of ligand-gated ion channels. The question of the subunit composition of native insect receptors remains open.

The beta-subunit of Caenorhabditis elegans avermectin receptor responds to glycine and is encoded by chromosome 1.

A full-length cDNA encoding the beta subunit of the recently described avermectin receptor was amplified from Caenorhabditis elegans mRNA. When this cDNA was injected into Xenopus oocytes a dose-dependent response to glycine was observed, together with a smaller response to 1 mM GABA. The EC50 of the glycine response was similar to that described previously for glutamate (0.38 mM). Hybridisation of the cDNA to polytene filters identified three yeast artificial chromosome clones that gave a positive signal, Y37B3, Y38E5, and Y24C9, all of which are mapped to chromosome 1. Hybridisation to a series of cosmid clones covering this area further mapped the gene encoding this subunit to the region -2,818 to -2,824.

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.

Cloning of a putative inhibitory amino acid receptor subunit from the parasitic nematode Haemonchus contortus.

A cDNA, HGl, encoding an inhibitory amino-acid receptor subunit has been cloned from a mixed egg population of the parasitic nematode Haemonchus contortus. The predicted amino-acid sequence of the subunit shows 24% to 32% homology with other vertebrate and invertebrate GABAA and glycine receptor subunits and has all the expected motifs for a member of the ligand-gated ion channel superfamily. When expressed in Xenopus oocytes HGl gives a small response to 1 mM glycine, but not to 1 mM GABA, glutamate, taurine or L-alanine.

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.

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.

Agonist pharmacology of the neuronal alpha 7 nicotinic receptor expressed in Xenopus oocytes.

The potencies and efficacies of seven agonists at chick alpha 7 nicotinic receptors expressed in Xenopus oocytes were determined by whole cell recording. (+)-Anatoxin-a was the most potent agonist (EC50 = 0.58 microM) and acetylcholine was the least potent (EC50 = 320 microM). The rank order of agonist potencies was: (+)-anatoxin-a >> cytisine > (-)-nicotine > (+)-nicotine > DMPP > 1-acetyl-4-methylpiperazine methiodide > acetylcholine. DMPP evoked only very small currents: comparison of maximally effective agonist concentrations showed that DMPP was only one-fifth as efficacious as other agonists. Previously published IC50 values for rat brain [125I]alpha-bungarotoxin sites show a similar agonist profile, and the identity of homo-oligomeric alpha 7 receptors with native alpha-bungarotoxin-sensitive neuronal nicotinic receptors is discussed.

(+)-Anatoxin-a is a potent agonist at neuronal nicotinic acetylcholine receptors.

The effects of the nicotinic agonist (+)-anatoxin-a have been examined in four different preparations, representing at least two classes of neuronal nicotinic receptors. (+)-Anatoxin-a was most potent (EC50 = 48 nM) in stimulating 86Rb+ influx into M10 cells, which express the nicotinic receptor subtype comprising alpha 4 and beta 2 subunits. A presynaptic nicotinic receptor mediating acetylcholine release from hippocampal synaptosomes was similarly sensitive to (+)-anatoxin-a (EC50 = 140 nM). alpha-Bungarotoxin-sensitive neuronal nicotinic receptors, studied using patch-clamp recording techniques, required slightly higher concentrations of this alkaloid for activation: Nicotinic currents in hippocampal neurons were activated by (+)-anatoxin-a with an EC50 of 3.9 microM, whereas alpha 7 homooligomers reconstituted in Xenopus oocytes yielded an EC50 value of 0.58 microM for (+)-anatoxin-a. In these diverse preparations, (+)-anatoxin-a was between three and 50 times more potent than (-)-nicotine and approximately 20 times more potent than acetylcholine, making it the most efficacious nicotinic agonist thus far described.

BIOSITE: a program for the interactive comparison of aligned homologous protein sequences.

A program, BIOSITE, providing for the interactive visual comparison of aligned homologous amino-acid sequences is presented, including an example of its application. The program allows for two types of comparison sequence to be generated: an 'identity' sequence and a 'difference' sequence. These may be used on subsets of sequences and in further comparisons to identify candidate sites involved in a distinct functional property. The program should prove a useful tool for biologists engaged in understanding sequence--function relationships.

Inhibition of muscarinic cholinergic receptors by disulfide reducing agents and arsenicals. Differential effect on locust and rat.

Muscarinic receptors are altered by sulfhydryl reagents. Arsenic compounds, which have been used as insecticides, exert their toxic effects by combining with sulfhydryl groups. We compared the action of arsenicals and other sulfhydryl reagents on the muscarinic receptor from invertebrate and vertebrate species (locust and rat). Disulfide-reducing reagents dithiothreitol (DTT) and British Anti-Lewisite (BAL), but not arsenicals, inhibited [3H]quinuclidinyl benzilate ([3H]QNB) binding. However, after disulfide reduction, arsenicals caused a further inhibition of muscarinic binding. The effect of DTT + arsenicals was largely irreversible. The locust receptors were more sensitive to the action of both disulfide reagents either in the absence or presence of arsenicals than the rat receptors. The sulfhydryl reagent p-chloromercuric benzoate (PCMB) was more effective at inhibiting the locust receptors than the rat receptors, but addition of arsenicals did not cause further inhibition in either the locust or rat receptors. In locust, DTT + cacodylate and DTT + arsenite caused a reduction in the number of sites without modifying the affinity of [3H]QNB binding. In rat, DTT + arsenite caused a decrease in the affinity, while DTT + cacodylate caused a decrease in the affinity of [3H]QNB binding and its number of sites. Competition experiments after DTT + cacodylate showed that the IC50 and the Hill coefficient (nH) remained unchanged in the locust. In the rat, the IC50 for atropine was increased without alteration in the nH, and both parameters were increased for carbachol. These results are explained assuming that the binding site of the locust receptor has a disulfide group similar to that of the mammalian receptor, but that the hydrophobic interactions within the binding site are weaker in the locust receptor. The higher sensitivity of the insect receptor to sulfhydryl reagents could be of interest for developing methods of pest control.