A computational investigation on the role of glycosylation in the binding of alpha1 nicotinic acetylcholine receptor with two alpha-neurotoxins

Based on the crystal structure of the extracellular domain (ECD) of the mouse nicotinic acetylcholine receptor (nAChR) alpha1 subunit bound to α-bungarotoxin (α-Btx) we have generated in silico models of the human nAChR α1 bound to α-Btx and α-cobratoxin (α-Cbtx), both in the presence and in the absence of the N-linked carbohydrate chain. To gain further insight into the structural role of glycosylation molecular dynamics (MD) simulations were carried out in explicit solvent so as to compare the conformational dynamics of the binding interface between nAChR α1 and the two toxins. An interesting observation during the course of the MD simulations is the strengthening of the receptor-toxin interaction in the presence of the carbohydrate chain, mediated through a shift in the position of the sugars towards the bound toxin. Critical protein-sugar interactions implicate residues Ser187 and Trp184 of nAChR and Thr6, Ser9, and Thr15 of α-Btx, as well as Thr6 and Pro7 of α-Cbtx. Analysis of the predicted residue-specific intermolecular interactions is intended to inspire biophysical studies on the functional role of glycosylation in the gating mechanism.     

神经元烟碱受体在全身麻醉机制中的作用

作为配体－门控离子通道超家族成员的神经元烟碱受体分布于中枢和外周神经系统,包括多种亚型,具有广泛的生理作用,可以成为多种疾病的药物治疗靶点。它在全身麻醉原理中的作用也被日益重视。部分全身麻醉药物（挥发性吸入、气体吸入麻醉药、硫喷妥钠、氯胺酮等）在低于临床麻醉剂量时能够明显抑制该受体功能,神经元烟碱受体可能参与了这些药物的临床作用机制。     

The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity

Gymnodimines (GYMs) are phycotoxins exhibiting unusual structural features including a spirocyclic imine ring system and a trisubstituted tetrahydrofuran embedded within a 16-membered macrocycle. The toxic potential and the mechanism of action of GYM-A, highly purified from contaminated clams, have been assessed. GYM-A in isolated mouse phrenic hemidiaphragm preparations produced a concentration- and time-dependent block of twitch responses evoked by nerve stimulation, without affecting directly elicited muscle twitches, suggesting that it may block the muscle nicotinic acetylcholine (ACh) receptor (nAChR). This was confirmed by the blockade of miniature endplate potentials and the recording of subthreshold endplate potentials in GYM-A paralyzed frog and mouse isolated neuromuscular preparations. Patch-clamp recordings in Xenopus skeletal myocytes revealed that nicotinic currents evoked by constant iontophoretical ACh pulses were blocked by GYM-A in a reversible manner. GYM-A also blocked, in a voltage-independent manner, homomeric human alpha7 nAChR expressed in Xenopus oocytes. Competition-binding assays confirmed that GYM-A is a powerful ligand interacting with muscle-type nAChR, heteropentameric alpha3beta2, alpha4beta2, and chimeric alpha7-5HT(3) neuronal nAChRs. Our data show for the first time that GYM-A broadly targets nAChRs with high affinity explaining the basis of its neurotoxicity, and also pave the way for designing specific tests for accurate GYM-A detection in shellfish samples.     

Identification of two Lynx proteins in Nilaparvata lugens and the modulation on insect nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission in the insect brain and are targets for neonicotinoid insecticides. Some proteins, other than nAChRs themselves, might play important roles in insect nAChRs function in vivo and in vitro , such as the chaperone, regulator and modulator. Here we report the identification of two nAChR modulators (Nl-lynx1 and Nl-lynx2) in the brown planthopper, Nilaparvata lugens . Analysis of amino acid sequences of Nl-lynx1 and Nl-lynx2 reveals that they are two members of the Ly-6/neurotoxin superfamily, with a cysteine-rich consensus signature motif. Nl-lynx1 and Nl-lynx2 only increased agonist-evoked macroscopic currents of hybrid receptors Nlα1/β2 expressed in Xenopus oocytes, but not change the agonist sensitivity and desensitization properties. For example, Nl-lynx1 increased I _max of acetylcholine and imidacloprid to 3.56-fold and 1.72-fold of that of Nlα1/β2 alone, and these folds for Nl-lynx2 were 3.25 and 1.51. When the previously identified Nlα1^Y151S mutation was included (Nlα1^Y151S/β2), the effects of Nl-lynx1 and Nl-lynx2 on imidacloprid responses, but not acetylcholine response, were different from that in Nlα1/β2. The increased folds in imidacloprid responses by Nl-lynx1 and Nl-lynx2 were much higher in Nlα1^Y151S/β2 (3.25-fold and 2.86-fold) than in Nlα1/β2 (1.72-fold and 1.51-fold), which indicated Nl-lynx1 and Nl-lynx2 might also serve as an influencing factor in target-site insensitivity in N. lugens . These findings indicate that nAChRs chaperone, regulator and modulator may be of importance in assessing the likely impact of the target-site mutations such as Y151S upon neonicotinoid insecticide resistance.     

Long-lasting occupancy of central nicotinic acetylcholine receptors after smoking: a PET study in monkeys

The aim of this study was to compare the degree of occupancy of central nicotinic acetylcholine receptors (nAChR) in isoflurane anaesthetized baboon brain following inhalation of tobacco smoke (one cigarette containing 0.9 mg nicotine) or i.v. nicotine (0.6 mg i.v.). [18F]Fluoro-A-85380 and positron emission tomography (PET) were used to assess the distribution volumes (DV) of the radiotracer in selected brain areas using a one-compartment model. Eighty minutes after nicotine i.v., DV was reduced by 50 and 66% in the thalamus and putamen, respectively. Six hours after nicotine, a reduction in DV (27% in the thalamus) was still observed. Eighty minutes after inhalation of tobacco smoke, DV was decreased by 52 and 65% in the thalamus and putamen, respectively. Previous PET experiments have demonstrated a short-lasting interaction of [11C]nicotine with nAChRs. Thus, we hypothesized that a metabolite of nicotine with high affinity and long half-live (several hours) could bind at nAChRs. Eighty minutes after a high dose of nornicotine (0.5 mg i.v.), DV was reduced by 53 and 31% in thalamus and putamen, respectively. No significant effect was observed following 0.15 mg nornicotine. Therefore, nornicotine could contribute to the long-lasting occupancy of central nAChRs after smoking.     

Tyrosine kinase inhibitors alter composition of nicotinic receptors on neurons

Protein tyrosine kinase (PTK) inhibitors were used to examine the roles of tyrosine phosphorylation in synaptic function. We show here that two different PTK inhibitors, herbimycin A and lavendustin A, both selectively downregulate a subpopulation of nicotinic acetylcholine receptors (AChRs) on chick ciliary ganglion neurons in culture. The downregulation requires a number of hours to occur and involves only those receptors containing the α3, α5, and β4 gene products. Not affected are AChRs that additionally contain the β2 gene product or AChRs that are made up of the α7 gene product. The downregulation preferentially targets receptors destined for the cell surface and has little effect on the large pool of intracellular receptors. The receptor loss is not additive with that seen in the presence of either cycloheximide or tunicamycin, two compounds that the block appearance of new receptors. The downregulation induced by herbimycin A in surface receptors is accompanied by a specific decrement in the amount of α3 protein in the cells. The results indicate that PTKs, either by phosphorylating AChR gene products directly or by acting through intermediary proteins, regulate the size and composition of the AChR pool maintained on the cell surface. Receptor regulation by PTKs may provide a mechanism for long-term control of synaptic signaling between neurons. © 1996 John Wiley & Sons, Inc.     

Multiple inhibitory actions of lidocaine on Torpedo nicotinic acetylcholine receptors transplanted to Xenopus oocytes

Lidocaine is a local anaesthetic that blocks sodium channels, but also inhibits several ligand-gated ion-channels. The aim of this work was to unravel the mechanisms by which lidocaine blocks Torpedo nicotinic receptors transplanted to Xenopus oocytes. Acetylcholine-elicited currents were reversibly blocked by lidocaine, in a concentration dependent manner. At doses lower than the IC(50) , lidocaine blocked nicotinic receptors only at negative potentials, indicating an open-channel blockade; the binding site within the channel was at about 30% of the way through the electrical field across the membrane. In the presence of higher lidocaine doses, nicotinic receptors were blocked both at positive and negative potentials, acetylcholine dose-response curve shifted to the right and lidocaine pre-application, before its co-application with acetylcholine, enhanced the current inhibition, indicating all together that lidocaine also blocked resting receptors; besides, it increased the current decay rate. When lidocaine, at low doses, was co-applied with 2-(triethylammonio)-N-(2,6-dimethylphenyl) acetamide bromide, edrophonium or 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide, which are quaternary-ammonium molecules that also blocked nicotinic receptors, there was an additive inhibitory effect, indicating that these molecules bound to different sites within the channel pore. These results prove that lidocaine blocks nicotinic receptors by several independent mechanisms and evidence the diverse and complex modulation of this receptor by structurally related molecules.     

Electrostatic resistance to alpha-neurotoxins conferred by charge reversal mutations in nicotinic acetylcholine receptors

The evolution of venom resistance through coevolutionary chemical arms races has arisen multiple times throughout animalia. Prior documentation of resistance to snake venom α-neurotoxins consists of the N-glycosylation motif or the hypothesized introduction of arginine at positions 187 at the α-1 nicotinic acetylcholine receptor orthosteric site. However, no further studies have investigated the possibility of other potential forms of resistance. Using a biolayer interferometry assay, we first confirm that the previously hypothesized resistance conferred by arginine at position 187 in the honey badger does reduce binding to α-neurotoxins, which has never been functionally tested. We further discovered a novel form of α-neurotoxin resistance conferred by charge reversal mutations, whereby a negatively charged amino acid is replaced by the positively charged amino acid lysine. As venom α-neurotoxins have evolved strong positive charges on their surface to facilitate binding to the negatively charged α-1 orthosteric site, these mutations result in a positive charge/positive charge interaction electrostatically repelling the α-neurotoxins. Such a novel mechanism for resistance has gone completely undiscovered, yet this form of resistance has convergently evolved at least 10 times within snakes. These coevolutionary innovations seem to have arisen through convergent phenotypes to ultimately evolve a similar biophysical mechanism of resistance across snakes.     

Competitive potentiation of acetylcholine effects on neuronal nicotinic receptors by acetylcholinesterase-inhibiting drugs

The effects of the acetylcholinesterase inhibitors physostigmine and tacrine on alpha4beta2 and alpha4beta4 subtypes of neuronal nicotinic acetylcholine (ACh) receptors, expressed in Xenopus laevis oocytes, have been investigated. In voltage-clamp experiments low concentrations of physostigmine and tacrine potentiate ion currents induced by low concentrations of ACh, whereas at high concentrations they inhibit ACh-induced ion currents. These dual effects result in bell-shaped concentration-effect curves. Physostigmine and tacrine, by themselves, do not act as nicotinic receptor againsts. The larger potentiation is observed with 10 microM: physostigmine on alpha4beta4 nicotinic receptors and amounts to 70% at 1 microM: ACh. The mechanism underlying the effects of physostigmine on alpha4beta4 ACh receptors has been investigated in detail. Potentiation of ACh-induced ion current by low concentrations of physostigmine is surmounted at elevated concentrations of ACh, indicating that this is a competitive effect. Conversely, inhibition of ACh-induced ion current by high concentrations of physostigmine is not surmounted at high concentrations of ACh, and this effect appears mainly due to noncompetitive, voltage-dependent ion channel block. Radioligand binding experiments demonstrating displacement of the nicotinic receptor agonist (125)I-epibatidine from its recognition sites on alpha4beta4 ACh receptors by physostigmine confirm that physostigmine is a competitive ligand at these receptors. A two-site equilibrium receptor occupation model, combined with noncompetitive ion channel block, accounts for the dual effects of physostigmine and tacrine on ACh-induced ion currents. It is concluded that these acetylcholinesterase-inhibiting drugs interact with the ACh recognition sites and are coagonists of ACh on alpha4-containing nicotinic ACh receptors.     

Direct actions of organophosphate anticholinesterases on nicotinic and muscarinic acetylcholine receptors

Four nerve agents and one therapeutic organophosphate (OP) anticholinesterase (anti-ChE) bind to acetylcholine (ACh) receptors, inhibit or modulate binding of radioactive ligands to these receptors, and modify events regulated by them. The affinity of nicotinic (n) ACh receptors of Torpedo electric organs and most muscarinic (m) ACh receptors of rat brain and N1E-115 neuroblastoma cultures for the OP compounds was usually two to three orders of magnitude lower than concentrations required to inhibit 50% (IC-50) of ACh-esterase activity. However, a small population of m-ACh receptors had an affinity as high as that of ACh-esterase for the OP compound. This population is identified by its high-affinity [³H]-cis-methyldioxolane ([³H]-CD) binding. Although sarin, soman, and tabun had no effect, (O-ethyl S[2-(diisopropylamino)ethyl)] methyl phosphonothionate (VX) and echothiophate inhibited competitivel the binding of receptors. However, VX was more potent than echothiophate in inhibiting this binding and 50-fold more potent in inhibiting carbamylcholine (carb)-stimulated [³H]-cGMP synthesis in N1E-115 neuroblastoma cells—both acting as m receptor antagonist. All five OPs inhibited [³H]-CD binding, with IC-50s of 3, 10, 40, 100, and 800 nM for VX, soman, sarin, echothiophate, and tabun, respectively. The OP anticholinesterases also bound to allosteric sites on the n-ACh receptor (identified by inhibition of [³H]-phencyclidine binding), but some bound as well to the receptor's recognition site (identified by inhibition of [¹²⁵I]-α-bungarotoxin binding). Soman and echothiophate in micromolar concentrations acted as partial agonists of the n-ACh receptor and induced receptor desensitization. On the other hand, VX acted as an open channel blocker of the activated receptor and also enhanced receptor desensitization. It is suggested that the toxicity of OP anticholinesterases may include their action on n-ACh as well as m-ACh receptors if their concentrations in circulation rise above micromolar levels. At nanomolar concentrations their toxicity is due mainly to their inhibition of ACh-esterase. However, at these low concentrations, many OP anticholinesterases (eg, VX and soman) may affect a small population of m-ACh receptors, which have a high affinity for CD. Such effects on m-ACh receptors may play an important role in the toxicity of certain OP compounds.     

Phorbol esters inhibit the synthesis of acetylcholine receptors in cultured muscle cells

The acetylcholine receptor (AChR) synthesis, insertion and degradation rates are regulated by numerous intracellular and extracellular agents. Recent studies have shown that Ca2+ and Ca2+ ionophores have a profound regulatory effect on the appearance of AChR clusters and AChR synthesis. These regulatory effects may be mediated through the activation of calcium and phospholipid-dependent protein kinases by agents such as phorbol esters. In this study, we have utilized 4-beta-phorbol-12-myristate-13-acetate (PMA) in order to determine whether the activation of protein kinase C exerts a regulatory effect on the expression of AChRs in cultured chick myotubes. Our results show that 4-beta-phorbol-12-myristate-13-acetate decreased intracellular AChRs and suppressed AChR synthesis without affecting the turnover rate. Control and PMA treated cells labeled with [35S] methionine and immunoprecipitated with a monoclonal antibody to the alpha subunit of AChRs (mAb35) revealed a significant decrease in radioactivity precipitated after exposure to PMA. Polyacrylamide gel electrophoresis revealed no major changes in protein patterns, or in newly synthesized proteins as determined by [35S] methionine incorporation and autoradiography. Other enzymes important in muscle metabolism were not affected by PMA treatment. Our results indicate that activation of protein kinase C results in the suppression of AChRs synthesis and dispersal of AChR clusters.     

Structural determinants for interaction of partial agonists with acetylcholine binding protein and neuronal α7 nicotinic acetylcholine receptor

The pentameric acetylcholine‐binding protein (AChBP) is a soluble surrogate of the ligand binding domain of nicotinic acetylcholine receptors. Agonists bind within a nest of aromatic side chains contributed by loops C and F on opposing faces of each subunit interface. Crystal structures of Aplysia AChBP bound with the agonist anabaseine, two partial agonists selectively activating the α7 receptor, 3‐(2,4‐dimethoxybenzylidene)‐anabaseine and its 4‐hydroxy metabolite, and an indole‐containing partial agonist, tropisetron, were solved at 2.7–1.75 Å resolution. All structures identify the Trp 147 carbonyl oxygen as the hydrogen bond acceptor for the agonist‐protonated nitrogen. In the partial agonist complexes, the benzylidene and indole substituent positions, dictated by tight interactions with loop F, preclude loop C from adopting the closed conformation seen for full agonists. Fluctuation in loop C position and duality in ligand binding orientations suggest molecular bases for partial agonism at full‐length receptors. This study, while pointing to loop F as a major determinant of receptor subtype selectivity, also identifies a new template region for designing α7‐selective partial agonists to treat cognitive deficits in mental and neurodegenerative disorders.     

Agonists cause endocytosis of nicotinic acetylcholine receptors on cultured myotubes

Regulated trafficking of neurotransmitter receptors in excitable cells may play an important role in synaptic plasticity. In addition, agonist‐induced endocytosis of nicotinic acetylcholine receptors (nAChRs) in particular might be involved in nicotine tolerance and addiction. The existing evidence concerning regulated internalization of cell‐surface nAChRs is indirect and equivocal, however. In the present study, radioligand binding and fluorescence microscopy were used to show that agonists cause substantial endocytosis of nAChRs on cultured myotubes. Exposure to carbachol or nicotine caused a decrease in the intensity of fluorescent labeling of clusters of cell‐surface nAChRs that was blocked by low temperature. Overall, myotubes exposed to carbachol or nicotine bound 50–70% less [¹²⁵I]‐α‐bungarotoxin on the cell surface than untreated cells. The effect of carbachol was significant within 5 min, increased progressively for at least 4 h, and had a sensitivity of 100 nM or less. Exposure to carbachol caused the appearance or dramatic expansion of an intracellular pool of nAChRs, which were localized to discrete, largely perinuclear structures. A pulse‐chase labeling protocol allowed the selective labeling and localization of nAChRs that had been internalized from the cell surface. In untreated cells, very little internalization of nAChRs occurred over a period of 3 h, indicating that constitutive endocytosis of receptors over this period was minimal. Exposure to carbachol, however, caused a dramatic increase in the endocytosis of nAChRs. These results provide direct evidence that agonists, including the tobacco alkaloid nicotine, can cause substantial endocytosis of cell‐surface nAChRs. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 212–223, 2001     

A model for the assembly of nicotinic receptors based on subunit-subunit interactions

Neuronal ion-channels are complex multimeric proteins. Within a given family, the variability of their pharmacological responses depends on subunit composition and subunit arrangement. We report here that protein assembly in the pentameric nicotinic acetylcholine receptor family, the best characterized of all neuronal receptors, can be predicted using information derived from homology modeled surface to surface subunit interactions based on the atomic structure of a snail acetylcholine-binding protein. An empirical assembly model is able to establish both subunit stoichiometry and subunit arrangement of known neuronal and muscle nicotinic receptors. This contribution to the understanding of nicotinic receptor assembly and variability might be extended to other types of ion-channels.     

Functional divergence analysis of vertebrate neuronal nicotinic acetylcholine receptor subunits

Nicotinic acetylcholine receptors (nAChRs) are pentamers formed by subunits from a large multigene family and are highly variable in kinetic, electrophysiological and pharmacological properties. Due to the essential roles of nAChRs in many physiological procedures and diversity in function, identifying the function-related sites specific to each subunit is not only necessary to understand the properties of the receptors but also useful to design potential therapeutic compounds that target these macromolecules for treating a series of central neuronal disorders. By conducting a detailed function divergence analysis on nine neuronal nAChR subunits from representative vertebrate species, we revealed the existence of significant functional variation between most subunit pairs. Specifically, 44 unique residues were identified for the α7 subunit, while another 22 residues that were likely responsible for the specific features of other subunits were detected. By mapping these sites onto the 3?D structure of the human α7 subunit, a structure-function relationship profile was revealed. Our results suggested that the functional divergence related sites clustered in the ligand binding domain, the β2–β3 linker close to the N-terminal α-helix, the intracellular linkers between transmembrane domains, and the “transition zone” may have experienced altered evolutionary rates. The former two regions may be potential binding sites for the α7* subtype-specific allosteric modulators, while the latter region is likely to be subtype-specific allosteric modulations of the heteropentameric descendants such as the α4β2* nAChRs.

Communicated by Ramaswamy H. Sarma     

Design of novel α7-subtype-preferring nicotinic acetylcholine receptor agonists: Application of docking and MM-PBSA computational approaches, synthetic and pharmacological studies

In the search for nicotinic acetylcholine receptor (nAChRs) agonists with a selective affinity for the homomeric α7 channels, we carried out the virtual screening of a test set of potential nicotinic ligands, and adopted a simplified MM-PBSA approach to estimate their relative binding free energy values. By means of this procedure, previously validated by a training set of compounds, we reached a realistic compromise between computational accuracy and calculation rate, and singled out a small group of novel structurally related derivatives characterized by a promising theoretical affinity for the α7 subtype. Among them, five new compounds were synthesized and assayed in binding experiments at neuronal α7 as well as α4β2 nAChRs.     

NIDA522131, a new radioligand for imaging extrathalamic nicotinic acetylcholine receptors: in vitro and in vivo evaluation

A novel radioligand, 6-chloro-3-((2-( S )-azetidinyl)methoxy)-5-(2-fluoropyridin-4-yl)pyridine (NIDA522131), for imaging extrathalamic nicotinic acetylcholine receptors (nAChRs) was characterized in vitro and in vivo using positron emission tomography. The K_d and T_1/2 of dissociation of NIDA522131 binding measured at 37°C in vitro were 4.9 ± 0.4 pmol/L and 81 ± 5 min, respectively. The patterns of radioactivity distribution in monkey brain in vivo was similar to that of 2-[¹⁸F]fluoro-3-(2( S )-azetidinylmethoxy)pyridine (2FA), a radioligand that has been successfully used in humans, and matched the α₄β₂* nAChRs distribution. Comparison between [¹⁸F]NIDA522131 and 2FA demonstrated better in vivo binding properties of the new radioligand and substantially greater radioactivity accumulation in brain. Consistent with [¹⁸F]NIDA522131 elevated affinity for nAChRs and its increased lipophilicity, both, the total and non-displaceable distribution volumes were substantially higher than those of 2FA. Estimated binding potential values in different brain regions, characterizing the specificity of receptor binding, were 3–4 fold higher for [¹⁸F]NIDA522131 than those of 2FA. Pharmacological evaluation in mice demonstrated a toxicity that was comparable to 2FA and is in agreement with a 2300 fold higher affinity at α₄β₂* versus α₃β₄* nAChRs. These results suggest that [¹⁸F]NIDA522131 is a promising positron emission tomography radioligand for studying extrathalamic nAChR in humans.     

Inhibition by 2,4-toluene diisocyanate of the calcium signaling of neuronal nicotinic acetylcholine receptors in human neuroblastoma SH-SY5Y cells

Summary Toluene diisocyanate (TDI) is widely used as a chemical intermediate in the production of polyurethane products such as foams, coatings, and elastomers. In exposed workers, chronic inhalation of TDI has resulted in significant decreases in lung function. TDI-induced asthma is related to its disturbance of acetylcholine in most affected workers but the actions of TDI on nicotinic acetylcholine receptors (nAChR) are unclear. In order to understand the role of TDI acting on nAChR, we used human neuroblastoma SH-SY5Y cells to investigate the effects of TDI on cytosolic free calcium concentration ([Ca ) changes under the stimulation of nAChR. The results showed that TDI was capable of inhibiting the [Ca rise induced by nicotinic ligands, epibatidine, DMPP and nicotine. The inhibition was remained, even increased after chronic treatment of TDI. Our study of TDI acting on human nAChR suggests a possibility that the human nerve system plays some role in the toxicity of TDI in the pulmonary system.     

Elevation of intracellular calcium levels in neurons by nicotinic acetylcholine receptors

The recognition that intracellular free calcium serves as a ubiquitous intracellular signal has motivated efforts to elucidate mechanisms by which cells regulate calcium influx. One route of entry that may offer both spatial and temporal fine resolution for altering calcium levels is that provided by cation-permeable, ligand-gated ion channels. Biophysical measurements as well as calcium imaging techniques demonstrate that neuronal nicotinic acetylcholine receptors as a class have a high relative permeability to calcium; some subtypes equal or exceed all other known receptors in this respect. Activation of nicotinic receptors on neurons can produce substantial increases in intracellular calcium levels by direct passage of calcium through the receptor channel. When multiple classes of nicotinic receptors are expressed by the same neuron, each appears capable of increasing calcium in the cell but may differ with respect to location, temporal response, agonist sensitivity, or regulation in achieving it. As a result, nicotinic receptors must be considered strong candidates for signaling molecules through which neurons regulate a diverse array of cellular events.