George G. Lunt

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.

Stereoselective Nicotine‐Induced Release of Dopamine from Striatal Synaptosomes: Concentration Dependence and Repetitive Stimulation

Abstract: Using a sensitive perfusion system we have studied the nicotine‐induced release of [3H]dopamine ([3H]DA) from striatal synaptosomes. Nicotine‐evoked release was concentration dependent with an EC50 of 3.8 μM. The response to 1 μM nicotine was comparable to that to 16 mM K+ 10 μM veratridine evoked a larger response. All three samuli were Ca2+ dependent but only the response to veratridine was blocked by tetrodotoxin. Repetitive stimulations by 1 μM (–)‐nicotine (100 μl) at 30‐min intervals resulted in similar levels of [3H]DA release; higher concentrations of (–)‐nicotine resulted in an attenuation of the response particularly following the third stimulation. This may reflect desensitisation or tachyphylaxis of the presynaptic nicotinic receptor. The action of nicotine was markedly stereoselective: a 100‐fold higher concentration of (+)‐nicotine was necessary to evoke the same level of response as 1 μM (–)‐nicotine. It is proposed that these presynaptic nicotinic receptors on striatal terminals are equivalent to high‐affinity nicotine binding sites described in mammalian brain.

Methyllycaconitine: a selective probe for neuronal α-bungarotoxin binding sites

The ability of methyllycaconitine (MLA) to inhibit the binding of [125I]α‐bungarotoxin to rat brain membranes, frog and human muscle extracts and the human muscle cell line TE671 has been measured. MLA showed a markedly higher affinity for the rat brain site (K i 1.4 × 10−9 M) than for the muscle receptors (K i; 10−5‐10−6 M). Structure modelling techniques were used to fit the structure of MLA to a nicotinic pharmacophore model. MLA is the first low molecular weight ligand to be shown to discriminate between muscle nicotinic receptors and their α‐bungarotoxinbinding counterpart in the brain, and as such may be a useful structural probe for pursuing the structural and functional properties of the neuronal protein.

Chapter 15 Presynaptic modulation of transmitter release by nicotinic receptors

Publisher Summary Nicotine acts on many transmitter systems in different parts of the brain to promote transmitter release, and these modulatory actions may underlie some of the psychopharmacological and behavioral effects of nicotine. Nicotine provoked the release of [ 3 H]dopamine from synaptosomes isolated from rat striata and preincubated with radiolabelled transmitter. Nicotine-evoked release was concentration-dependent over the range 10 -8 to 10 -3 M, and the half maximal response was observed with 3.8 μM nicotine. The presynaptic nicotinic receptor on dopaminergic nerve terminals and [ 3 H]nicotine binding sites may be equivalent. There are moderate numbers of [ 3 H]nicotine binding sites in the rat striatum compared with low levels of α-[ 125 I]bungarotoxin binding sites. Nicotinic modulation of transmitter release from hippocampal synaptosomes is discussed. From the data available concerning the presynaptic modulation of transmitter release by nicotine and other agonists, a model is proposed to account this phenomenon.

Methyllycaconitine and (+)-anatoxin-a differentiate between nicotinic receptors in vertebrate and invertebrate nervous systems

Specific high‐affinity binding sites for 125I‐α‐bungarotoxin and (−)‐[3H]nicotine have been measured in rat brain and locust (Schistocerca gregaria) ganglia. The binding sites for 125I‐α‐bungarotoxin had similar K d values of 1.5 × 10−9 and 0.8 × 10−9 M for rat and locust preparations, respectively; the corresponding values for the (−)‐[3H]nicotine‐binding site were 9.3 × 10−9 and 1.7 × 10−7 M. Methyllycaconitine (MLA) potently inhibited 125I‐α‐bungarotoxin binding in both rat and locust. MLA was a less effective inhibitor of (−)‐[3H]nicotine binding whereas (+)‐anatoxin‐a was a very potent inhibitor at this site in the rat but not in the locust. These data suggest that (+)‐anatoxin‐a is a useful probe for the high‐affinity nicotine‐binding receptor in vertebrate brain, whereas MLA is a preferential probe for the subclass of receptor that binds α‐bungarotoxin.

Sequence and functional expression of a single alpha subunit of an insect nicotinic acetylcholine receptor.

We report the isolation and sequence of a cDNA clone that encodes a locust (Schistocerca gregaria) nervous system nicotinic acetylcholine receptor (AChR) subunit (alpha L1). The calculated molecular weight of the unglycosylated polypeptide, which contains in the proposed extracellular domain two adjacent cysteine residues which are characteristic of alpha (ligand binding) subunits, is 60,641 daltons. Injection into Xenopus oocytes, of RNA synthesized from this clone in vitro, results in expression of functional nicotinic receptors in the oocyte membrane. In these, nicotine opens a cation channel; the receptors are blocked by both alpha‐bungarotoxin (alpha‐Bgt) and kappa‐bungarotoxin (kappa‐Bgt). Reversible block of the expressed insect AChR by mecamylamine, d‐tubocurarine, tetraethylammonium, bicuculline and strychnine has also been observed. These data are entirely consistent with previously reported electrophysiological studies on in vivo insect nicotinic receptors and also with biochemical studies on an alpha‐Bgt affinity purified locust AChR. Thus, a functional receptor exhibiting the characteristic pharmacology of an in vivo insect nicotinic AChR can be expressed in Xenopus oocytes by injection with a single subunit RNA.

(+)‐Anatoxin‐a Is a Potent Agonist at Neuronal Nicotinic Acetylcholine Receptors

Abstract: 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 α4 and β2 subunits. A presynaptic nicotinic receptor mediating acetylcholine release from hippocampal synaptosomes was similarly sensitive to (+)‐anatoxin‐a (EC50= 140 nM). α‐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 γM, whereas α7 homooligomers reconstituted in Xenopus oocytes yielded an EC50 value of 0.58 γM for (+)‐anatoxin‐a. In these diverse preparations, (+)‐anatoxin‐a was between three and 50 times more potent than (–)‐nicotine and ˜20 times more potent than acetylcholine, making it the most efficacious nicotinic agonist thus far described.

Ligand-gated ion channels. Homology and diversity.

Outline of the Ligand-Gated Ion Channel (LGIC) Superfamily Structure and Function The Recognition Site for Agonists and Competitive Antagonists Other Features of the Extracellular Domain The Transmembrane Domain and the Ion Channel The Major Intracellular Region Quaternary Structure Evolutionary Diversity of LGICs Origins of the Superfamily Events in nACh Receptor Evolution Events in GABA A and Glycine Receptor Evolution Concluding Remarks References

Agonist pharmacology of the neuronal α7 nicotinic receptor expressed in Xenopus oocytes

The potencies and efficacies of seven agonists at chick α7 nicotinic receptors expressed in Xenopus oocytes were determined by whole cell recording. (+)‐Anatoxin‐a was the most potent agonist (EC50 = 0.58 μM) and acetylcholine was the least potent (EC50 = 320 μM). 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]α‐bungarotoxin sites show a similar agonist profile, and the identity of homo‐oligomeric α7 receptors with native α‐bungarotoxin‐sensitive neuronal nicotinic receptors is discussed.

γ‐Aminobutyric Acid Receptor Complex of Insect CNS: Characterization of a Benzodiazepine Binding Site

Abstract: The specific binding of [N‐methyl‐3H] flunita‐zepam ([3H]FNZP) to a membrane fraction from the supraoesophageal ganglion of the locust (Schistocerca gregaria) has been measured. The ligand binds reversibly with a KD of 47 nM. The binding is Ca2+‐dependent, a property not found for the equivalent binding site in vertebrate brain. The pharmacological characteristics of the locust binding site show similarities to both central and peripheral benzo‐diazepine receptors in mammals. Thus binding is enhanced by γ‐aminobutyric acid (GABA), a feature of mammalian central receptors, whereas the ligand Ro 5‐4864 was more effective in displacing [3H]FNZP than was clonazepam, which is the pattern seen in mammalian peripheral receptors. The locust benzodiazepine binding site was photo‐affnity‐labelled by [3H]FNZP, and two major proteins of Mr 45K and 59K were specifically labelled. In parallel experiments with rat brain membranes a single major protein of Mr 49K was labelled, a finding in keeping with many reports in the literature. We suggest that the FNZP binding site described here is part of the GABA receptor complex of locust ganglia. The insect receptor appears to have the same general organization as its mammalian counterpart but differs significantly in its detailed properties.