Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor (original) (raw)

Epibatidine Binds with Unique Site and State Selectivity to Muscle Nicotinic Acetylcholine Receptors

Journal of Biological Chemistry, 1998

Ligand binding sites in fetal (␣ 2 ␤␥␦) and adult (␣ 2 ␤␦⑀) muscle acetylcholine receptors are formed by ␣␦, ␣␥, or ␣⑀ subunit pairs. Each type of binding site shows unique ligand selectivity due to different contributions by the ␦, ␥, or ⑀ subunits. The present study compares epibatidine and carbamylcholine binding in terms of their site and state selectivities for muscle receptors expressed in human embryonic kidney 293 cells. Measurements of binding to ␣␥, ␣␦, and ␣⑀ intracellular complexes reveal opposite site selectivities between epibatidine and carbamylcholine; for epibatidine the rank order of affinities is ␣⑀ > ␣␥ > ␣␦, whereas for carbamylcholine the rank order is ␣␦ Х ␣⑀ > ␣␥. Because the relative affinities of intracellular complexes resemble those of receptors in the closed activable state, the results suggest that epibatidine binds with unique site selectivity in activating the muscle receptor. Measurements of binding at equilibrium show that both enantiomers of epibatidine bind to adult and fetal receptors with shallow but monophasic binding curves. However, when receptors are fully desensitized, epibatidine binds in a biphasic manner, with dissociation constants of the two components differing by more than 170-fold. Studies of subunitomitted receptors (␣ 2 ␤␦ 2 , ␣ 2 ␤␥ 2 , and ␣ 2 ␤⑀ 2) reveal that in the desensitized state, the ␣␦ interface forms the low affinity epibatidine site, whereas the ␣␥ and ␣⑀ interfaces form high affinity sites. In contrast to epibatidine, carbamylcholine shows little site selectivity for desensitized fetal or adult receptors. Thus epibatidine is a potentially valuable probe of acetylcholine receptor binding site structure and of elements that confer state-dependent selectivities of the binding sites.

The unusual nature of epibatidine responses at the α4β2 nicotinic acetylcholine receptor

Neuropharmacology, 2000

The identification of an equatorial frog toxin, epibatidine, as a potent non-morphinic analgesic, selective for neuronal nicotinic acetylcholine receptors, provoked a marked renewal in our understanding of pain and its mechanisms. In this work we have examined the effects of epibatidine at the major brain rat α4β2 nicotinic acetylcholine receptor expressed in a cell line. Fast drug applications obtained with a modified liquid filament system were used for the analyses of the currents evoked by acetylcholine, nicotine and epibatidine. Characterized by a slow onset and offset, epibatidine responses were of smaller amplitude to those evoked by acetylcholine or nicotine. About a thousand times more sensitive to epibatidine than acetylcholine, the α4β2 receptor also displayed a more pronounced apparent desensitization to this compound. Finally, overnight exposure to 1 nM epibatidine failed to produce the functional upregulation observed with nicotine. These data indicate that, at the rat α4β2 receptor, epibatidine acts as a partial agonist causing a pronounced inhibition of agonist evoked currents at concentrations that do not activate the receptors.

High-Affinity Epibatidine Binding of Functional, Human alpha7-Nicotinic Acetylcholine Receptors Stably and Heterologously Expressed de Novo in Human SH-EP1 Cells

Journal of Pharmacology and Experimental Therapeutics, 2005

Human nicotinic acetylcholine receptor (nAChR) ␣7 subunits were stably and heterologously expressed in native nAChR-null SH-EP1 human epithelial cells. Immunofluorescence staining shows ␣7 subunit protein expression in virtually every transfected cell. Microautoradiographic analysis identifies 125 Ilabeled ␣-bungarotoxin (I-Bgt) binding sites corresponding to human ␣7 (h␣7)-nAChRs on the surface of most cells. I-Bgt binds to h␣7-nAChRs in membrane fractions with a typical apparent K D value of ϳ5 nM and B max value of ϳ1 pmol/mg membrane protein, and 62% of these sites are expressed on the cell surface. Function of heterologously expressed h␣7-nAChRs is evident as rapid, transient inward current responses to (Ϫ)-nicotine. Nicotine treatment of transfected cells produces dose-and time-dependent increases (up to ϳ100%) in numbers of I-Bgt binding sites. Epibatidine is a useful ligand for Portions of this work were presented previously [Peng J-H, Leonard SS, and Lukas RJ (1998) Heterologous expression of epibatidine-and ␣-bungarotoxinbinding human ␣7-nicotinic acetylcholine receptor in a native receptor-null human epithelial cell line. Soc Neurosci Abstr 24:831].

High-Affinity Epibatidine Binding of Functional, Human 7-Nicotinic Acetylcholine Receptors Stably and Heterologously Expressed de Novo in Human SH-EP1 Cells

Journal of Pharmacology and Experimental Therapeutics, 2004

Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [125I]epibatidine and 5[125I]A-85380

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2009

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, hold promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [ 125 I] epibatidine and 5[ 125 I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [ 125 I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [ 125 I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [ 125 I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.

An autoradiographic survey of mouse brain nicotinic acetylcholine receptors defined by null mutants

Biochemical Pharmacology, 2011

Nine nicotinic receptor subunits are expressed in the central nervous system indicating that a variety of nicotinic acetylcholine receptors (nAChR) may be assembled. A useful method with which to identify putative nAChR is radioligand binding. In the current study the binding of [ 125 I]α-bungarotoxin, [ 125 I]α-conotoxinMII, 5[ 125 I]-3-((2S)-azetidinylmethoxy)pyridine (A-85380), and [ 125 I]epibatidine has been measured autoradiographically to provide data on many nAChR binding sites. Each binding sites was evaluated semiquantitatively for samples prepared from wild-type and α2, α4, α6, α7, β2, β4, α5 and β3 null mutant mice. Deletion of the α7 subunit completely and selectively eliminated [ 125 I]α-bungarotoxin binding. The binding of [ 125 I]αConotoxinMII was eliminated in most brain regions by deletion of either the α6 or β2 subunit and is reduced by deletion of either the α4 or β3 subunit. The binding of 5[ 125 I]A-85380 was completely eliminated by deletion of the β2 subunit and significantly reduced by deletion of the α4 subunit. Most, but not all, α4-independent sites require expression of the α6 subunit. The effect of gene deletion on total [ 125 I]epibatidine binding was very similar to that on [ 125 I]A-85380 binding. [ 125 I]Epibatidine also labels β4* nAChR, which was readily apparent for incubations conducted in the presence of 100 nM cytisine. The effects of α3 gene deletion could not be evaluated, but persistence of residual sites implies the expression of α3* nAChR. Taken together these results confirm and extend previously published evaluations of the effect of nAChR gene deletion and help to define the nAChR subtypes measurable by ligand binding.

Structural Basis for Epibatidine Selectivity at Desensitized Nicotinic Receptors

Molecular Pharmacology, 2005

The agonist binding sites of the fetal muscle nicotinic acetylcholine receptor are formed at the interfaces of ␣-subunits and neighboring ␥and ␦-subunits. When the receptor is in the nonconducting desensitized state, the ␣-␥ site binds the agonist epibatidine 200-fold more tightly than does the ␣-␦ site. To determine the structural basis for this selectivity, we constructed ␥/␦-subunit chimeras, coexpressed them with complementary wild-type subunits in HEK 293 cells, and determined epibatidine affinity of the resulting complexes. The results reveal three determinants of epibatidine selectivity: ␥104-117/␦106-␦119, ␥164-171/␦166-177, and ␥Pro190/␦Ala196. Point mutations reveal that three sequence differences within the ␥104-117/␦106-␦119 region are determinants of epibatidine selectivity: ␥Lys104/␦Tyr106, ␥Ser111/ ␦Tyr113, and ␥Tyr117/␦Tyr119. In the ␦-subunit, simultaneous mutation of these residues to their ␥ equivalent produces high affinity, ␥-like epibatidine binding. However, converting ␥ to ␦ affinity requires replacement of the ␥104-117 segment with ␦ sequence, suggesting interplay of residues in this region. The structural basis for epibatidine selectivity is explained by computational docking of epibatidine to a homology model of the ␣-␥ binding site.

Identification of a novel nicotinic binding site in mouse brain using [ 125 I]-epibatidine

British Journal of Pharmacology, 2000

Epibatidine binds to multiple nicotinic acetylcholine receptor (nAChR) subtypes with high anity. In this study, [ 125 I]-epibatidine was used to label and characterize a novel nAChR subtype found in mouse brain inferior colliculus, interpeduncular nucleus, and olfactory bulb homogenates. 2 Binding of [ 125 I]-epibatidine was saturable and apparently monophasic in each brain region (K D =71+12 pM mean+s.e.mean across regions) but inhibition of [ 125 I]-epibatidine binding (200 pM) by A85380, cytisine and (7)-nicotine was biphasic, indicating the presence of multiple binding sites. 3 The sites with lower agonist anity comprised 30.0+2.2, 58.6+0.1 and 48.7+3.3% of speci®c [ 125 I]-epibatidine (200 pM) binding in inferior colliculus, interpeduncular nucleus, and olfactory bulb homogenates, respectively. 4 The anity dierence between A85380-sensitive and-resistant binding sites was particularly marked (approximately 1000 fold). Thus A85380 was used to dierentiate agonist-sensitive and-resistant sites. 5 The pharmacological pro®les of the A85380-resistant sites in each region were assessed with inhibition binding experiments, using 14 agonists and ®ve antagonists. The pro®les were indistinguishable across regions, implying that A85380-resistant [ 125 I]-epibatidine binding sites in inferior colliculus, interpeduncular nucleus, and olfactory bulb represent a single nAChR subtype. 6 The pharmacological pro®le of the A85380-resistant sites is very dierent from that previously reported for high anity (7)-[ 3 H]-nicotine-, [ 125 I]-a-bungarotoxin-, or [ 125 I]-a-conotoxin MII-binding sites, suggesting that they represent a novel nAChR population in mouse brain.

Gene targeting demonstrates that α4 nicotinic acetylcholine receptor subunits contribute to expression of diverse [3H]epibatidine binding sites and components of biphasic 86Rb+ efflux with high and low sensitivity to stimulation by acetylcholine

Neuropharmacology, 2007

[ 3 H]Epibatidine binds to nAChR subtypes in mouse brain with higher (K D ≈0.02 nM) and lower affinity (KD≈7 nM), which can be further subdivided through inhibition by selected agonists and antagonists. These subsets are differentially affected by targeted deletion of α7, β2 or β4 subunits. Most, but not all, higher and lower affinity binding sites require β2 (Marks et al., 2006). Effects of functional α4 gene deletion are reported here. Deletion of α4 virtually eliminated cytisine-sensitive, higher-affinity [ 3 H]epibatidine binding as did β2 deletion, confirming that these sites are α4β2*-nAChR. Cytisine-resistant, higher-affinity [ 3 H]epibatidine binding sites are diverse and some of these sites require α4 expression. Lower affinity [ 3 H]epibatidine binding sites are also heterogeneous and can be subdivided into α-bungarotoxin-sensitive and-resistant components. Deleting α4 did not affect the α-bungarotoxin-sensitive component, but markedly reduced the α-bungarotoxin-resistant component. This effect was similar, but not quite identical, to the effect of β2 deletion. This provides the first evidence that lower-affinity epibatidine binding sites in the brain require expression of α4 subunits. The effects of α4 gene targeting on receptor function were measured using a 86 Rb + efflux assay. Concentration-effect curves for ACh-stimulated 86 Rb + efflux are biphasic (EC 50 values = 3.3 µM and 300 µM). Targeting α4 produced substantial gene-dose dependent reductions in both phases in whole brain and in most of the 14 brain regions assayed. These effects are very similar to those following deletion of β2. Thus, α4β2*-nAChRs mediate a significant fraction of both phases of ACh stimulated 86 Rb + efflux.

Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor (original) (raw)

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