Characterization Of Nicotinic Acetylcholine Receptors Research Articles

Discovery of peptide ligands through docking and virtual screening at nicotinic acetylcholine receptor homology models

Venom peptide toxins such as conotoxins play a critical role in the characterization of nicotinic acetylcholine receptor (nAChR) structure and function and have potential as nervous system therapeutics as well. However, the lack of solved structures of conotoxins bound to nAChRs and the large size of these peptides are barriers to their computational docking and design. We addressed these challenges in the context of the α4β2 nAChR, a widespread ligand-gated ion channel in the brain and a target for nicotine addiction therapy, and the 19-residue conotoxin α-GID that antagonizes it. We developed a docking algorithm, ToxDock, which used ensemble-docking and extensive conformational sampling to dock α-GID and its analogs to an α4β2 nAChR homology model. Experimental testing demonstrated that a virtual screen with ToxDock correctly identified three bioactive α-GID mutants (α-GID[A10V], α-GID[V13I], and α-GID[V13Y]) and one inactive variant (α-GID[A10Q]). Two mutants, α-GID[A10V] and α-GID[V13Y], had substantially reduced potency at the human α7 nAChR relative to α-GID, a desirable feature for α-GID analogs. The general usefulness of the docking algorithm was highlighted by redocking of peptide toxins to two ion channels and a binding protein in which the peptide toxins successfully reverted back to near-native crystallographic poses after being perturbed. Our results demonstrate that ToxDock can overcome two fundamental challenges of docking large toxin peptides to ion channel homology models, as exemplified by the α-GID:α4β2 nAChR complex, and is extendable to other toxin peptides and ion channels. ToxDock is freely available at rosie.rosettacommons.org/tox_dock.

Marie T. Filbin: in memoriam.

Marie T. Filbin: in memoriam.

Electrophysiological characterization of nicotinic acetylcholine receptors in cat petrosal ganglion neurons in culture: Effects of cytisine and its bromo derivatives

Petrosal ganglion neurons are depolarized and fire action potentials in response to acetylcholine and nicotine. However, little is known about the subtype(s) of nicotinic acetylcholine receptors involved, although α4 and α7 subunits have been identified in petrosal ganglion neurons. Cytisine, an alkaloid unrelated to nicotine, and its bromo derivatives are agonists exhibiting different affinities, potencies and efficacies at nicotinic acetylcholine receptors containing α4 or α7 subunits. To characterize the receptors involved, we studied the effects of these agonists and the nicotinic acetylcholine receptor antagonists hexamethonium and α-bungarotoxin in isolated petrosal ganglion neurons. Petrosal ganglia were excised from anesthetized cats and cultured for up to 16 days. Using patch-clamp technique, we recorded whole-cell currents evoked by 5–10 s applications of acetylcholine, cytisine or its bromo derivatives. Agonists and antagonists were applied by gravity from a pipette near the neuron surface. Neurons responded to acetylcholine, cytisine, 3-bromocytisine and 5-bromocytisine with fast inward currents that desensitized during application of the stimuli and were reversibly blocked by 1 μM hexamethonium or 10 nM α-bungarotoxin. The order of potency of the agonists was 3-bromocytisine ≫ acetylcholine ≅ cytisine ≫ 5-bromocytisine, suggesting that homomeric α7 neuronal nicotinic receptors predominate in cat petrosal ganglion neurons in culture.

Characterization of nicotinic acetylcholine receptors in cultured arterial chemoreceptor cells of the cat

Neurotransmitters appear to be involved in chemotransmission of the carotid body, a major arterial chemoreceptor. Substantial data indicate that acetylcholine (ACh) is an excitatory neurotransmitter in the carotid body, regulating the excitability of afferent nerve endings and glomus cells (putative chemoreceptor cells). In this study we characterized properties of nicotinic ACh receptors (nAChRs) in cultured cat glomus cells using immunocytochemistry and whole cell patch clamp techniques. Cultured glomus cells expressed immunoreactivity for α3, α4, and β2 subunits of nAChRs. An application of ACh elicited inward current. Nicotinic AChRs of glomus cells showed high affinity for ACh. The current–voltage relationship showed strong inward rectification at positive membrane potential. α-Conotoxin MII (20 nM), dihydro-β-erythroidine (DHβE; 1 nM), and hexamethonium (300 μM) significantly inhibited ACh-induced current. These results indicate that cultured cat glomus cells possess functional nAChRs, and that their characteristics are consistent with those of α3, α4 and β2 containing nAChRs.

Characterization of nicotinic acetylcholine receptors (nAChRs) in myenteric neurons from guinea pig intestine

Characterization of nicotinic acetylcholine receptors (nAChRs) in myenteric neurons from guinea pig intestine

Characterization of nicotinic acetylcholine receptors (nAChRs) in myenteric neurons from guinea pig intestine

Characterization of nicotinic acetylcholine receptors (nAChRs) in myenteric neurons from guinea pig intestine

Synthesis and characterization of binding of 5-[76Br]bromo-3-[[2(S)-azetidinyl]methoxy]pyridine, a novel nicotinic acetylcholine receptor ligand, in rat brain.

5-[76Br]Bromo-3-[[2(S)-azetidinyl]methoxy]pyridine ([76Br]BAP), a novel nicotinic acetylcholine receptor ligand, was synthesized using [76Br]bromide in an oxidative bromodestannylation of the corresponding trimethylstannyl compound. The radiochemical yield was 25%, and the specific radioactivity was on the order of 1 Ci/micromol. The binding properties of [76Br]BAP were characterized in vitro and in vivo in rat brain, and positron emission tomography (PET) experiments were performed in two rhesus monkeys. In association experiments on membranes of the cortex and thalamus, >90% of maximal specific [76Br]BAP binding was obtained after 60 min. The dissociation half-life of [76Br]BAP was 51 +/- 6 min in cortical membranes and 56 +/- 3 min in thalamic membranes. Saturation experiments with [76Br]BAP revealed one population of binding sites with dissociation constant (K(D)) values of 36 +/- 9 and 30 +/- 9 pM in membranes of cortex and thalamus, respectively. The maximal binding site density (Bmax) values were 90 +/- 17 and 207 +/- 33 fmol/mg in membranes of cortex and thalamus, respectively. Scatchard plots were nonlinear, and the Hill coefficients were <1, suggesting the presence of a lower-affinity binding site. In vitro autoradiography studies showed that binding of [76Br]BAP was high in the thalamus and presubiculum, moderate in the cortex and striatum, and low in the cerebellum and hippocampus. A similar pattern of [76Br]BAP accumulation was observed by ex vivo autoradiography. In vivo, binding of [76Br]BAP in whole rat brain was blocked by preinjection of (S)(-)-nicotine (0.3 mg/kg) by 27, 52, 68, and 91% at survival times of 10, 25, 40, 120, and 300 min, respectively. In a preliminary PET study in rhesus monkeys, the highest [76Br]BAP uptake was found in the thalamus, and radioactivity was displaceable by approximately 60% with cytisine and by 50% with (S)(-)-nicotine. The data of this study indicate that [76Br]BAP is a promising radioligand for the characterization of nicotinic acetylcholine receptors in vivo.

In vivo positron emission tomography studies on the novel nicotinic receptor agonist [ 11C]MPA compared with [ 11C]ABT-418 and ( S)(−)[ 11C]nicotine in Rhesus monkeys

The novel 11C-labeled nicotinic agonist ( R, S)-1-[ 11C]methyl-2(3-pyridyl)azetidine ([ 11C]MPA) was evaluated as a positron emission tomography (PET) ligand for in vivo characterization of nicotinic acetylcholine receptors in the brain of Rhesus monkeys in comparison with the nicotinic ligands ( S)-3-methyl-5-(1-[ 11C]methyl-2-pyrrolidinyl)isoxazol ([ 11C]ABT-418) and ( S)(−)[ 11C]nicotine. The nicotinic receptor agonist [ 11C]MPA demonstrated rapid uptake into the brain to a similar extent as ( S)(−) [ 11C]nicotine and [ 11C]ABT-418. When unlabeled ( S)(−)nicotine (0.02 mg/kg) was administered 5 min before the radioactive tracers, the uptake of [ 11C]MPA was decreased by 25% in the thalamus, 19% in the temporal cortex, and 11% in the cerebellum, whereas an increase was found for the uptake of ( S)(−)[ 11C]nicotine and [ 11C]ABT-418. This finding indicates specific binding of [ 11C]MPA to nicotinic receptors in the brain in a simple classical displacement study. [ 11C]MPA seems to be a more promising radiotracer than ( S)(−)[ 11C]nicotine or [ 11C]ABT-418 for PET studies to characterize nicotinic receptors in the brain.

O-187 - Pharmacological characterization of nicotinic acetylcholine receptor (nACh-R)-mediated acetylcholine release in rat brain - in vivo microdialysis study

O-187 - Pharmacological characterization of nicotinic acetylcholine receptor (nACh-R)-mediated acetylcholine release in rat brain - in vivo microdialysis study

Binding of native κ-neurotoxins and site-directed mutants to nicotinic acetylcholine receptors

The κ-neurotoxins are useful ligands for the pharmacological characterization of nicotinic acetylcholine receptors because they are potent antagonists at only a subgroup of these receptors containing either α3- or α4-subunits ( IC 50 ≤ 100 nM). Four of these highly homologous, 66 amino acid peptides have been purified from the venom of Bungarus multicinctus [κ-bungarotoxin (κ-Bgt), κ2-Bgt, κ3-Bgt] and Bungarus flaviceps [κ-flavitoxin (κ-Fvt)]. Two approaches were taken to examine the binding of these toxins to nicotinic receptors. First, venom-derived κ-Fvt and κ-Bgt were radioiodinated and the specific binding was measured of these toxins to overlapping synthetic peptides (16–20 amino acids in length) prepared based on the known sequence of the nicotinic receptor α3-subunit. At least two main regions of interaction between the toxins and the receptor subunit were identified, both lying in the N-terminal region of the subunit that is exposed to the extracellular space. The second approach examined the importance of several sequence positions in κ-Bgt for binding to α3-containing receptors in autonomic ganglia and α1-containing muscle receptors. This was done using site-directed mutants of κ-Bgt produced by an Escherichia coli expression system. Arg-34 and position 36 were important for binding to both receptor subtypes, while replacing Gln-26 with Trp-26 (an invariant in α-neurotoxins) increased affinity for the muscle receptor by 8-fold. The results confirm that κ-neurotoxins bind potently to the α3-subunit and bind with considerably reduced affinity ( K d ≈ 10 μM) to muscle receptors. Site-directed mutagenesis of recombinant κ-Bgt is thus an important approach for the study of structure-function relationships between κ-Bgt and nicotinic receptors.

Characterization of nicotinic acetylcholine receptors expressed in primary cultures of cerebellar granule cells

Nicotinic acetylcholine receptors (nAChRs), like other calcium permeable channel receptors, may play a crucial role during neuronal development. We have characterized nAChRs in developing mouse cerebellar granule cells in primary culture. L-[3H]Nicotine, [3H]cytisine and [125I]alpha-bungarotoxin binding experiments revealed the presence of a single class of saturable and specific high affinity binding sites for each ligand. The expression of these nicotinic binding sites followed a developmental pattern reaching a maximum during the establishment of excitatory amino acid synaptic contacts. Immunolabeling with monoclonal antibodies to nAChR subunits revealed the presence of alpha 4 and beta 2 subunits in most neurons. Moreover, some neuronal cells displayed a somatic as well as a neuritic localization for the alpha 7 subunit as shown by [125I]alpha-bungarotoxin autoradiography. The reverse transcription-polymerase chain reaction (RT-PCR) detected the presence of mRNAs for alpha 3, alpha 4, alpha 5, alpha 7, beta 2 and beta 4 nAChR subunits. Non-neuronal cells did not express nAChRs, as shown by [3H]nicotine and [125I]alpha-bungarotoxin binding, immunocytochemistry and PCR. Maximum Ca2+ influx elicited by nicotine, and partly sensitive to alpha-bungarotoxin, was observed around 10-14 days after plating. This correlated with the time period at which the highest number of nicotine binding sites was detected. Sensitivity to several NMDA receptor antagonists as well as to removal of endogenous glutamate by pyruvate transaminase treatment revealed a glutamatergic component in the nicotine stimulated calcium influx. The time-dependent specific nAChR expression and the potential association between nAChRs and NMDA receptor activation suggest that nAChRs may regulate glutamatergic activity during synaptogenesis in cerebellar granule cells.

Characterization of Nicotinic Acetylcholine Receptors Expressed by Cells of the SH-SY5Y Human Neuroblastoma Clonal Line

Radioligand binding studies show that cells of the SH-SY5Y human neuroblastoma clonal line express a single class of neuronal/nicotinic α-bungarotoxin binding sites (nBgtS). These sites are defined by their ability to bind radioiodinated α-bungarotoxin (Bgt) with high affinity ( K D = 4 n M) and nicotinic ligands with lower (μ M) affinities. Radioligand binding studies also show that SH-SY5Y cells express high affinity specific binding sites for 3 H-labeled acetylcholine that could reflect interactions with two classes of sites ( K D′ - 1 n/ M, K D″ = 100 n M). These sites are distinguished from nBgtS by their insensitivity to blockade by Bgt and by their ability to bind other nicotinic agonists with high (n M) affinity. 86 Rb + efflux studies indicate that SH-SY5Y cells express functional nicotinic acetylcholine receptor (nAChR) ion channels that, like radioagonist binding sites, are insensitive to blockade by Bgt. However, there are far more functional nAChR than high affinity radioagonist binding sites. Functional nAChR have a pharmacological profile expected of ganglia-type receptors composed of nAChR alpha3 and beta4 subunits. Northern blot analysis indicates that genes corresponding to human alpha3, alpha5, beta2, and beta4 subunits are expressed by SH-SY5Y cells. We conclude that SH-SY5Y cells express at least two members of the nAChR family (nBgtS and ganglia-type nAChR) and that high affinity radioagonist binding sites are a subset of functional ganglia-type nAChR.

Characterization of nicotinic acetylcholine receptors in the brain by slice patch methods

Characterization of nicotinic acetylcholine receptors in the brain by slice patch methods

P-004 - Purification and characterization of nicotinic acetylcholine receptor in rat brain

P-004 - Purification and characterization of nicotinic acetylcholine receptor in rat brain

An isotopic rubidium ion efflux assay for the functional characterization of nicotinic acetylcholine receptors on clonal cell lines

An isotopic rubidium ion efflux assay has been developed for the functional characterization of nicotinic acetylcholine receptors on cultured neurons. This assay first involves the intracellular sequestration of isotopic potassium ion analog by the ouabain-sensitive action of a sodium-potassium ATPase. Subsequently, the release of isotopic rubidium ion through nicotinic acetylcholine receptor-coupled monovalent cation channels is activated by application of nicotinic agonists. Specificity of receptor-mediated efflux is demonstrated by its sensitivity to blockade by nicotinic, but not muscarinic, antagonists. The time course of agonist-mediated efflux, within the temperal limitations of the assay, indicates a slow inactivation of receptor function on prolonged exposure to agonist. Dose-response profiles (i) have characteristic shapes for different nicotinic agonists, (ii) are described by three operationally defined parameters, and (iii) reflect different affinities of agonists for binding sites that control receptor activation and functional inhibition. The rubidium ion efflux assay provides fewer hazards but greater sensitivity and resolution than isotopic sodium or rubidium ion influx assays for functional nicotinic receptors.

Purification and biochemical characterization of nicotinic acetylcholine receptors of human muscle.

Purification and biochemical characterization of nicotinic acetylcholine receptors of human muscle.

Purification and characterization of nicotinic acetylcholine receptors from muscle.

The nicotinic acetylcholine receptor was purified from normal and denervated rat skeletal muscle. The purification protocol included alpha-cobratoxin biospecific adsorption, ion exchange chromatography, and gel filtration steps. The highest specific activity achieved was 7.5 pmol of 125I-alpha-bungarotoxin binding sites per microgram protein. Sodium dodecyl sulfate gel electrophoresis of purified AChR revealed subunits with molecular weights of 42,000 and 66,000 daltons and a minor component with a molecular weight of 52,000 daltons. Normal muscle AChR is comprised of one toxin binding component. Upon denervation a second component appears, but both components are increased as a consequence of denervation. A dissociation constant of 1.5 x 10(-8)M was determined for d-tubocurarine from receptor from both normal and denervated muscle. A dissociation constant of 1 x 10(-7)M for acetylcholine, perhaps analogous to the high affinity acetylcholine binding observed in electric fish receptor, was determined.