Effects of Steroid Exposure on Ligand Binding and Functional Activities of Diverse Nicotinic Acetylcholine Receptor Subtypes

Abstract: Nicotinic acetylcholine receptors (nAChR) are diverse members of the ligand-gated ion channel superfamily of neurotransmitter receptors and play critical roles in chemical signaling throughout the nervous system. The present study tests whether nAChR are potential targets for steroids. Acute or short-term (5 min) preexposure to steroids such as progesterone (which acts most potently), estradiol, corticosterone, or dexamethasone inhibits function of human muscle-type (α1β1γδ) or ganglionic (α3β4) nAChR measured using ⁸⁶Rb⁺ efflux assays in TE671/RD clonal or SH-SY5Y neuroblastoma cells. Absolute (high nanomolar to intermediate micromolar range) and rank-order potencies for steroid-mediated functional inhibition are similar across nAChR subtypes but differ for some steroid derivatives. At concentrations that produce blockade of nAChR function, steroids do not affect binding of radioligands such as ¹²⁵I-labeled α-bungarotoxin or [³H]acetylcholine to muscle-type or ganglionic nAChR or to neuronal toxin-binding nAChR that contain α7 subunits (α7-nAChR). Steroid-mediated blockade of nAChR function is insurmountable by increasing agonist concentrations, and cell-impermeant progesterone:bovine serum albumin conjugates have full potency as inhibitors of ganglionic or muscle-type nAChR function. Chronic (48 h) exposure to progesterone or estradiol, but not the other steroids, also produces blockade of nAChR function, without significant effects on numbers of nAChR radioligand-binding sites. Collectively, these results suggest that steroids act noncompetitively at extracellular sites to inhibit nAChR function with unique potencies for different steroid-nAChR subtype combinations. Thus, nAChR could be among the targets mediating physiologically relevant effects of steroid action in the nervous system.     

Identification of Propofol Binding Sites in a Nicotinic Acetylcholine Receptor with a Photoreactive Propofol Analog

Propofol, a widely used intravenous general anesthetic, acts at anesthetic concentrations as a positive allosteric modulator of γ-aminobutyric acid type A receptors and at higher concentration as an inhibitor of nicotinic acetylcholine receptors (nAChRs). Here, we characterize propofol binding sites in a muscle-type nAChR by use of a photoreactive analog of propofol, 2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol (AziPm). Based upon radioligand binding assays, AziPm stabilized the Torpedo nAChR in the resting state, whereas propofol stabilized the desensitized state. nAChR-rich membranes were photolabeled with [³H]AziPm, and labeled amino acids were identified by Edman degradation. [³H]AziPm binds at three sites within the nAChR transmembrane domain: (i) an intrasubunit site in the δ subunit helix bundle, photolabeling in the nAChR desensitized state (+agonist) δM2-18′ and two residues in δM1 (δPhe-232 and δCys-236); (ii) in the ion channel, photolabeling in the nAChR resting, closed channel state (−agonist) amino acids in the M2 helices (αM2-6′, βM2-6′ and -13′, and δM2-13′) that line the channel lumen (with photolabeling reduced by >90% in the desensitized state); and (iii) at the γ-α interface, photolabeling αM2-10′. Propofol enhanced [³H]AziPm photolabeling at αM2-10′. Propofol inhibited [³H]AziPm photolabeling within the δ subunit helix bundle at lower concentrations (IC₅₀ = 40 μm) than it inhibited ion channel photolabeling (IC₅₀ = 125 μm). These results identify for the first time a single intrasubunit propofol binding site in the nAChR transmembrane domain and suggest that this is the functionally relevant inhibitory binding site.     

Effects of Chronic Nicotinic Ligand Exposure on Functional Activity of Nicotinic Acetylcholine Receptors Expressed by Cells of the PC12 Rat Pheochromocytoma or the TE671/RD Human Clonal Line

Studies were conducted to ascertain the temporal and dose-dependent effects of nicotinic ligand exposure on functional activity of different nicotinic acetylcholine receptor (nAChR) subtypes, as expressed by cells of the PC12 rat pheochromocytoma (ganglia-type nAChR) or the TE671/RD human (muscle-type nAChR) clonal line. Chronic (3-72-h) agonist (nicotine or carbamylcholine) treatment of cells led to a complete (TE671) or nearly complete (PC12) loss of functional nAChR responses, which is referred to as "functional inactivation." Some inactivation of nAChR function was also observed for the nicotinic ligands d-tubocurarine (d-TC), mecamylamine, and decamethonium. Half-maximal inactivation of nAChR function was observed within 3 min for TE671 cells and within 10 min for PC12 cells treated with inactivating ligands. Functional inactivation occurred with dose dependencies that could not always be reconciled with those obtained for acute agonist activation of nAChR function or for acute inhibition of those responses by d-TC, decamethonium, or mecamylamine. Treatment of TE671 or PC12 cells with the nicotinic antagonist pancuronium or alcuronium alone had no effect on levels of expression of functional nAChRs. However, evidence was obtained that either of these antagonists protected TE671 cell muscle-type nAChRs or PC12 cell ganglia-type nAChRs from functional inactivation on long-term treatment with agonists. Recovery of TE671 cell nAChR function following treatment with carbamylcholine, nicotine, or d-TC occurred with half-times of 1-3 days whether cells were maintained in situ or harvested and replated after removal of ligand. By contrast, 50% recovery of functional nAChRs on PC12 cells occurred within 2-6 h after drug removal. In either case the time course for recovery from nAChR functional inactivation is much slower than recovery from nAChR "functional desensitization," which is a reversible process that occurs on shorter-term (0-5-min) agonist exposure of cells. These results indicate that ganglia-type and muscle-type nAChRs are similar in their sensitivities to functional inactivation by nicotinic ligands but differ in their rates of recovery from and onset of those effects. The ability of drugs such as the agonists d-TC, decamethonium, and mecamylamine to induce functional inactivation may relate to their activities as partial/full agonists, channel blockers, and/or allosteric regulators. Effects of drugs such as pancuronium and alcuronium are likely to reflect simple competitive inhibition of primary ligand binding at functional activation sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of the Muscarinic Acetylcholine Receptor: Effects of Phosphorylating Conditions on Agonist and Antagonist Binding

Incubation of rat brain synaptic membranes under phosphorylating conditions (i.e., in the presence of Mg2+, ATP, and cyclic AMP) leads to a loss in muscarinic acetylcholine receptors, detectable as specific binding of the muscarinic antagonist L-[3H]quinuclidinyl benzilate. A role for protein phosphorylation in this receptor loss is indicated by the finding that 5'-adenylyl imidodiphosphate, a nonhydrolysable analogue of ATP, does not support receptor loss. Furthermore, receptor loss is inhibited by adenosine and 2-deoxyadenosine, both of which inhibit protein kinase activity. The loss of muscarinic receptors is calmodulin dependent, and it has been demonstrated here that this requirement is probably at the level of calmodulin-dependent phosphorylation. An investigation of the effects of phosphorylation on the binding of the agonist carbachol to synaptic membranes from the cortex and cerebellum demonstrated that phosphorylation altered the relative proportions of the super-high-, high-, and low-affinity binding sites. The results were consistent with an apparent conversion of high- into super-high-affinity sites. In the presence of 5'-guanylyl imidodiphosphate, agonist binding demonstrated the properties expected of a population of largely low-affinity sites. This conversion of super-high- and high-affinity sites into low-affinity sites by 5'-guanylyl imidodiphosphate was partially inhibited by phosphorylation.     

Cloning and Functional Characterisation of Two Novel Nicotinic Acetylcholine Receptor α Subunits from the Insect Pest Myzus persicae

Abstract: Nicotinic acetylcholine receptors play a major role in excitatory neurotransmission in insect CNSs and constitute an important target for insecticides. Here, we report the isolation and functional characterisation of two cDNAs encoding nicotinic acetylcholine receptor α subunits from a major insect pest, the peach-potato aphid Myzus persicae . These two subunits, termed Mpα1 and Mpα2, are respective structural homologues of the Drosophila Dα2/ Schistocerca gregaria αL1 α-subunit pair and the Drosophila ALS α subunit. Xenopus oocyte expression confirmed that each Myzus subunit can form functional acetylcholine- or nicotine-gated channels. However, some electrophysiological and pharmacological properties of the Myzus subunits were distinct from those encoded by the corresponding Drosophila subunits. Coexpression of the Myzus subunits with the chick β2 subunit revealed other differences from the Drosophila system, as only very limited potentiation of agonist-induced currents was observed with Mpα2 and none with Mpα1. Available data therefore indicate that structurally homologous insect nicotinic acetylcholine receptor α subunits from different species can exhibit distinctive physiological and pharmacological characteristics.     

Photoaffinity Labeling of the 5-HydroxytryptamineIA Receptor in Rat Hippocampus

1-[2-(4-Azidophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (p-azido-PAPP) inhibits [3H]5-hydroxytryptamine [( 3H]5-HT) binding to 5-HT1A and 5-HT1B sites in rat brain with equilibrium dissociation constants (KD) of 0.9 nM and 230 nM, respectively. [3H]p-Azido-PAPP was synthesized and its reversible and irreversible binding properties to the hippocampal 5-HT1A site characterized. [3H]p-Azido-PAPP labeled a single class of sites in rat hippocampal membranes with a KD of 1 nM and a maximal binding density of 370 fmol/mg protein. The pharmacological profile of [3H]p-azido-PAPP binding was consistent with the radioligand's selective interaction with the 5-HT1A receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes preincubated with [3H]p-azido-PAPP and irradiated showed a major band of incorporation of radioactivity at approximately 55,000 daltons. This incorporation could be blocked when membranes were incubated with 1 microM of several agents that have high affinity for 5-HT1A sites [5-HT, 8-hydroxy-2-(di-n-propylamino)tetraline, TVX Q 7821, spiperone, buspirone, d-lysergic acid diethylamide, metergoline]. The results indicate that on photolysis [3H]p-azido-PAPP irreversibly labels a polypeptide that is, or is a subunit of, the 5-HT1A receptor in rat hippocampus.     

Functional Properties of the Subtype of Insulin Receptor Found on Neurons

In this report, we have examined the structure, regulation, and function of insulin receptors in cultured neurons from fetal chicken brain. The apparent molecular weight of the alpha-subunit of neuronal insulin receptors, analyzed by photoaffinity labeling and sodium dodecyl sulfate gel electrophoresis under reducing conditions, was 115,000. The number of insulin receptors in the cultures increased from day 2 to day 4 during a period of extensive process formation. After 5 days in culture, there were approximately 40,000 high-affinity insulin receptors per neuron. When neurons were photoaffinity labeled at 16 degrees C and then warmed to 37 degrees C for 30 min, approximately 40% of the cell-surface receptors were recovered in the intracellular, trypsin-insensitive pool. Chronic exposure of neurons to insulin (100 ng/ml) resulted in a time-dependent loss of neuronal insulin receptors with a maximal decrease of 50% after 24 h. Insulin had no effect on glucose transport, glucose oxidation, or glycogen synthase activity in neurons. On the other hand, insulin supported the growth and differentiation of a fraction of neurons isolated from chick forebrain. We conclude that (1) cultured neurons from fetal chicken brain express the same subtype of insulin receptor previously identified in adult rat and human brain, (2) the neuronal subtype of insulin receptor undergoes internalization and down-regulation in response to insulin, and (3) neuronal insulin receptors do not acutely regulate glucose metabolism but mediate growth in neurons.     

Photoaffinity Labeling of Benzodiazepine Receptors in Rat Brain with Flunitrazepam Alters the Affinity of Benzodiazepine Receptor Agonist But Not Antagonist Binding

Abstract: Recently, it was proposed that β-carbolines interact with a subset of benzodiazepine (BZD) binding sites in mouse brain. This postulate was based upon evidence showing changes in binding properties of the BZD receptor following photoaffinity labeling of membranes with flunitrazepam (FLU). Under conditions in which 80% of specific [³H]diazepam binding was lost in photolabeled membranes, specific [³H]propyl β-carboline-3-carboxylate ([³H]PCC) binding was spared. In this study, the binding of the BZD antagonists [³H]PCC, [³H]Ro15 1788 and [³H]CGS 8216 was examined in rat brain membranes following photoaffinity labeling with FLU. No significant changes in the apparent K_D and small reductions in the B_max of ³H antagonist binding were observed. However, in the same membranes, up to 89% of specific [³H]FLU binding was lost. When [³H]PCC (0.05 nM) was used to label the receptors in control and photolabeled membranes, the ability of BZD receptor agonists to inhibit [³H]PCC binding was greatly diminished in the photolabeled membranes. In contrast, the potency of BZD antagonists remained the same in both control and treated membranes. Based upon PCC/[³H]Ro15 1788 competition experiments, the ability of PCC to discriminate between BZD receptor subtypes was unaffected by photoaffinity labeling of cortical membranes. Overall, these findings suggest that β-carbolines do not interact with a subset of BZD binding sites per se, but may be a consequence of the differential interaction of BZD agonists and antagonists with BZD binding sites that have been photoaffinity labeled with FLU. A possible mechanism underlying this phenomenon is discussed. The ability of photolabeled membranes to differentiate between BZD agonists and antagonists provides a potential screen for agonist and antagonist activity in compounds that interact with the BZD receptor.     

Photoaffinity Labeling of the Cerebral Sulfonylurea Receptor Using a Novel Radioiodinated Azidoglibenclamide Analogue

Abstract: In previous studies evidence has been presented by photoaffinity labeling that a polypeptide of 145–150 kDa represents the cerebral sulfonylurea receptor. However, covalent incorporation of [³H]glibenclamide or a ¹²⁵I-labeled glibenclamide analogue into the sulfonylurea receptor required high amounts of photoenergy and took place with low yield of photoinsertion. To provide a probe with increased photoreactivity a 4-azido-5-iodosalicyloyl analogue of glibenclamide was synthesized. Binding experiments revealed specific and reversible high-affinity binding of this novel probe to the particulate ( K _D = 0.13 n M ) and solubilized ( K _D = 0.56 n M ) sulfonylurea receptor from cerebral cortex. The novel probe showed >100-fold higher sensitivity to irradiation at 356 nm than glibenclamide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specific photoincorporation into a cerebral protein of 175 kDa and indicated an efficiency of photoincorporation of 9%. From dissociation binding curves following irradiation photoincorporation was estimated as 28% of specifically bound ligand. Photoincorporation into the 175-kDa protein following saturation binding of the novel probe to particulate sites from cerebral cortex indicated a K _D value of 0.38 n M . Inhibition of photoincorporation into this protein by glibenclamide, glipizide, and tolbutamide revealed K _D values for these sulfonylureas of 0.06 n M , 1.6 n M , and 1.2 µ M , respectively. These results show that the novel photoaffinity ligand can be used as a probe for detection and characterization of the sulfonylurea receptor and suggest that a 175-kDa protein represents the cerebral sulfonylurea receptor.     

Effects of Substance P on the Binding of Agonists to the Nicotinic Acetylcholine Receptor of Torpedo Electroplaque

Abstract: Abstract: The effect of the neuropeptide substance P on the binding of the cholinergic ligands to the nicotinic acetylcholine receptor of Torpedo electroplaque membranes was examined at a physiological concentration of NaCl (150 m M ). Substance P had no effect on the initial rate of ¹²⁵I-α-bungarotoxin binding at concentrations of <100 μ M . The peptide did not bind to the high-affinity local anesthetic site but allosterically modulated [³H]phencyclidine binding, positively in the absence of agonist and negatively in the presence of agonist. Substance P increased the apparent affinity of the cholinergic agonists carbamylcholine and acetylcholine at equilibrium. The effect of substance P on the equilibrium binding of [³H]acetylcholine was examined directly, and the peptide appeared to increase the affinity of the binding of the second molecule of agonist, with no effect on the binding of the first. This indicates that substance P can affect the cooperative interactions between agonist binding sites. Substance P appeared to increase the rate of carbamylcholine-induced desensitization; however, the data are also consistent with an allosteric mechanism that does not involve the desensitized state. To attempt to differentiate between these mechanisms, the rates of recovery were determined after exposure to peptide and/or agonist. The kinetics of recovery are consistent with stabilization of the desensitized state by substance P if the peptide remains bound long enough to allow rapid recovery to the low-affinity state. However, an allosteric modulation of agonist binding that does not involve the desensitized state cannot be ruled out.     

Photoaffinity Labeling of the GABAA Receptor with [3H]Muscimol

Muscimol is one of the most potent agonist ligands at the gamma-aminobutyric acidA (GABAA) receptor. Analysis of its chemical structure showed it to be a candidate for photoaffinity labeling. In practice, UV irradiation at 254 nm both changed the UV spectrum of muscimol and induced an irreversible binding of [3H]-muscimol to rat cerebellar synaptosomal membrane. After 10 min of irradiation, using 10 nM [3H]muscimol, the specific portion of this binding was 270 fmol/mg protein. (Nonspecific binding was defined as that arising in the presence of 1 mM GABA.) Specific binding increased asymptotically up to 100 nM [3H]muscimol. Irradiation of the membranes themselves did not significantly alter the KD or Bmax of reversible [3H]muscimol binding. However, irradiation of [3H]muscimol reduced its capacity subsequently to photolabel the membranes by 86 +/- 3%. Dose-dependent inhibition of binding was observed with muscimol, GABA, and bicuculline methiodide; with 10 nM [3H]muscimol maximum inhibition was 70% of total labeling and the order of potencies of these three compounds was characteristic of labeling to the GABAA receptor. Baclofen, l-glutamate, and diazepam exerted no effect at high concentrations. SDS-PAGE of the photolabeled membranes indicated specific incorporation of radioactivity into two molecular-weight species. One failed to enter the separating gel, implying a molecular weight greater than 250,000 daltons (250 kD). The molecular weight of the other was identified by fluorography to be about 52,000 daltons (52 kD).     

Architecture of the Neuronal Nicotinic Acetylcholine Receptor Ion Channel at the Binding Site of bis-Ammonium Blockers

Structure-activity relationships of 56 pentamethylenbis-ammonium compounds, the blockers of the neuronal nicotinic acetylcholine receptor (nAChR) ion channel, have been studied to estimate the cross-sectional dimensions of the channel pore. The cat superior cervical sympathetic ganglion in situ and isolated guinea pig ileum were used to evaluate the potency of the compounds to block ganglionic transmission. Minimum-energy conformations of each compound were calculated by the molecular mechanics method. A topographic model of the binding site of the blockers was proposed. It incorporates two narrowings, a large and a small one. The small narrowing is located between the large one and the cytoplasmic end of the pore. The cross-sectional dimensions of the large and small narrowings estimated from the dimensions of the blockers are 6.1 × 8.3 ? and 5.5 × 6.4 ?, respectively, the distance between the narrowings along the pore being approximately 7 ?. Most potent blockers would occlude the pore via binding to the channel at the levels of both narrowings. Less potent blockers are either too large or too small to bind to both narrowings simultaneously: large blockers would occlude the pore at the level of large narrowing, while small blockers would pass the large narrowing and occlude the pore at the level of small narrowing only. A comparison of the topographic model with a molecular five-helix bundle model of nAChR pore predicts Serine and Threonine rings to be the most probable candidates for the large and small narrowings, respectively. Received: 6 September 1995/Revised: 12 March 1996     

Further Characterization of the Avian Benzodiazepine Receptor Subunits Including Phylo-and Ontogenetic Aspects

The two avian benzodiazepine binding proteins offer an opportunity for further studies concerning their regional variation and their phylo- and ontogenetic development. Accordingly, regional variation of the benzodiazepine binding proteins is investigated further in two reptiles and chicken using photoaffinity labeling with [3H]flunitrazepam followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Whereas regional heterogeneity is pronounced in chicken, it is not readily apparent in the two reptiles. The ontogeny of the benzodiazepine binding proteins in chicken forebrain and cerebellum is remarkably similar to that previously reported in rodents. The results are discussed in light of the possible existence of the gamma-aminobutyric acid/benzodiazepine receptor as an isoreceptor complex.     

Relationship Between Dopamine Receptor Occupation by Spiperone and Acetylcholine Levels in the Rat Striatum After Long-Term Haloperidol Treatment Depends on Dopamine Innervation

The effect of chronic neuroleptic treatment on the relationship between the blockade of dopamine (DA) receptors by the neuroleptic drug spiperone and the decline in acetylcholine (ACh) levels was determined in the rat striatum in vivo. In rats, a unilateral lesion of the nigrostriatal pathway was produced with 6-hydroxydopamine. The rats were treated for 6 weeks with haloperidol (twice a day at 1 mg kg-1). Partial and complete receptor occupation was determined with radioactive spiperone (a D2 antagonist), given in various doses of different specific activity 2 h before death. ACh, choline, and radioactivity contents were measured in the same striatum. Following long-term haloperidol treatment, an increase in the maximal number of binding sites for spiperone was found. Virtually identical negative (linear) correlations between striatal ACh content and the number of receptors occupied by spiperone were found in saline- or subchronic haloperidol-treated rats when DA innervation was intact. The slope of the line describing the decrease in ACh content per occupied receptor, however, was much lower in haloperidol-treated rats than in saline-treated animals. After lesioning of the dopaminergic pathway, there was no longer a correlation between the receptor occupation and ACh levels in the striatum. These results show that receptor occupation by a neuroleptic correlates highly with function only when dopaminergic innervation is intact. Also, it appears that there is no fixed number of striatal ACh molecules per DA receptor, and, finally, that in vivo receptor detection methods distinguish differences in receptor density (as do in vitro techniques).     

Fluorescence Studies of the Nicotinic Acetylcholine Receptor in its Membrane Environment

Since the discovery of the fraction of immobilized lipid in contact with the nicotinic acetylcholine receptor (AChR), the lipid-belt region around this protein has become the focus of a variety of biophysical studies aimed at defining its properties. Here we summarize recent spectroscopic studies from our laboratory using Laurdan fluorescence to characterize distinct sites for lipids and to describe their effect on the AChR microenvironment.     

Elongation Factor 1A Family Regulates the Recycling of the M4 Muscarinic Acetylcholine Receptor

In this study, we tested the hypothesis that the elongation 1A (eEF1A) family regulates the cell surface density of the M4 subtype of the muscarinic acetylcholine receptors (mAChR) following agonist-induced internalization. Here, we show that mouse brains lacking eEF1A2 have no detectable changes in M4 expression or localization. We, however, did discover that eEF1A1, the other eEF1A isoform, is expressed in adult neurons contrary to previous reports. This novel finding suggested that the lack of change in M4 expression and distribution in brains lacking eEF1A2 might be due to compensatory effects of eEF1A1. Supporting this theory, we demonstrate that the overexpression of either eEF1A1 or eEF1A2 inhibits M4 recovery to the cell surface after agonist-induced internalization in PC12 cells. Furthermore, eEF1A1 or eEF1A2 had no effect on the recovery of the M1 subtype in PC12 cells. These results demonstrate the novel ability of the eEF1A family to specifically regulate the M4 mAChR.     

The Nicotinic Acetylcholine Receptor: The Founding Father of the Pentameric Ligand-gated Ion Channel Superfamily

A critical event in the history of biological chemistry was the chemical identification of the first neurotransmitter receptor, the nicotinic acetylcholine receptor. Disciplines as diverse as electrophysiology, pharmacology, and biochemistry joined together in a unified and rational manner with the common goal of successfully identifying the molecular device that converts a chemical signal into an electrical one in the nervous system. The nicotinic receptor has become the founding father of a broad family of pentameric membrane receptors, paving the way for their identification, including that of the GABA_A receptors.     

A Small Molecule Agonist THIQ as a Novel Pharmacoperone for Intracellularly Retained Melanocortin-4 Receptor Mutants

Although mutations in the melanocortin-4 receptor (MC4R) gene cause severe early-onset obesity, we still do not have effective approaches to correct the defects of these mutations. Several antagonists have been identified as pharmacoperones of the MC4R whereas no agonist of the MC4R has been reported. In the present study, we investigated the effect of a small molecule agonist of the MC4R, THIQ, on the cell surface expression and signaling of ten intracellularly retained MC4R mutants using different cell lines. We showed that THIQ increased the cell surface expression of three mutants (N62S, C84R, and C271Y) and two of them (N62S and C84R) had increased signaling in HEK293 cells. Interestingly, THIQ increased the signaling of two other mutants (P78L and P260Q) without increasing their cell surface expression in HEK293 cells. In neuronal cells, THIQ exhibited a more potent effect, correcting the cell surface expression and signaling of seven mutants (N62S, I69R, P78L, C84R, W174C, P260Q, and C271Y). Other mutants were not rescued by THIQ. We also showed that THIQ did not rescue MC4R mutants defective in ligand binding or signaling or one intracellularly retained mutant of the melanocortin-3 receptor. In summary, we demonstrated that a small molecule agonist acted as a pharmacoperone of the MC4R rescuing the cell surface expression and signaling of some intracellularly retained MC4R mutants.     

Ion Channel Topography of the Neuronal Nicotinic Acetylcholine Receptor: Pharmacochemical Approaches

Forty-three bisammonium ganglionic blockers were synthesized to study the structure of the ion channel of nicotinic acetylcholine receptor. The conformational parameters of these blockers were studied, and their effects toward the ganglionic transmission in situ on the sympathetic feline superior cervical ganglia and in vitro on the parasympathetic guinea-pig small intestine ganglia were determined. A model of the binding site for the bisammonium ganglionic blockers in the neuronal ion channel was proposed.