Adenosine A<Subscript>2A</Subscript> receptors in Parkinson’s disease treatment

Latest results on the action of adenosine A_2A receptor antagonists indicate their potential therapeutic usefulness in the treatment of Parkinson’s disease. Basal ganglia possess high levels of adenosine A_2A receptors, mainly on the external surfaces of neurons located at the indirect tracts between the striatum, globus pallidus, and substantia nigra. Experiments with animal models of Parkinson’s disease indicate that adenosine A_2A receptors are strongly involved in the regulation of the central nervous system. Co-localization of adenosine A_2A and dopaminergic D2 receptors in striatum creates a milieu for antagonistic interaction between adenosine and dopamine. The experimental data prove that the best improvement of mobility in patients with Parkinson’s disease could be achieved with simultaneous activation of dopaminergic D2 receptors and inhibition of adenosine A_2A receptors. In animal models of Parkinson’s disease, the use of selective antagonists of adenosine A_2A receptors, such as istradefylline, led to the reversibility of movement dysfunction. These compounds might improve mobility during both monotherapy and co-administration with L-DOPA and dopamine receptor agonists. The use of adenosine A_2A receptor antagonists in combination therapy enables the reduction of the L-DOPA doses, as well as a reduction of side effects. In combination therapy, the adenosine A_2A receptor antagonists might be used in both moderate and advanced stages of Parkinson’s disease. The long-lasting administration of adenosine A_2A receptor antagonists does not decrease the patient response and does not cause side effects typical of L-DOPA therapy. It was demonstrated in various animal models that inhibition of adenosine A_2A receptors not only decreases the movement disturbance, but also reveals a neuroprotective activity, which might impede or stop the progression of the disease. Recently, clinical trials were completed on the use of istradefylline (KW-6002), an inhibitor of adenosine A_2A receptors, as an anti-Parkinson drug.     

Non-neuronal nicotinic acetylcholine receptors: Cholinergic regulation of the immune processes

Nicotinic acetylcholine receptors (nAChRs) were initially discovered and studied as mediators of fast synaptic transmission in neuromuscular junctions and autonomic ganglia. Later on, they were found in the brain and in many nonexcitable tissues where they regulate vital cellular functions and the activity of other receptors. Primary immune organs, the bone marrow and thymus, are innervated with cholinergic nerves, which mediate the control of lymphopoiesis provided by the autonomic nervous system. In addition, lymphocytes are able to produce endogenous acetylcholine that can regulate the immune processes in an auto/paracrine way. Correspondingly, both T and B lymphocytes express functional nAChRs involved in the regulation of development and activation of these cells. This review describes the structure and roles of nAChRs in the immune system with regard to its potential regulation by the autonomic nervous system, as well as by self sources of endogenous agonists. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 307–314, July–October, 2007.     

RIC-3 and nicotinic acetylcholine receptors: biogenesis, properties, and diversity

Nicotinic acetylcholine receptors (nAChRs) belong to a diverse and widely expressed family of ion channels. These receptors are pentamers assembled from multiple combinations of subunits, with different subunit compositions producing receptors having different properties and functions. The diverse functions of nAChRs include an essential role in excitation of skeletal muscles and many modulatory roles throughout the central nervous system. Nicotinic receptors are also implicated in a number of brain pathologies such as epilepsy, schizophrenia, and Alzheimer's disease. Thus, it is important to understand the cellular mechanisms controlling both the numbers and the properties of surface expressed nAChRs. Genetic analysis in Caenorhabditis elegans identified a number of proteins specifically needed for biogenesis of nAChRs. Among these proteins is RIC-3, a member of a family of proteins having conserved structure and function. RIC-3 influences both surface expression and properties of nAChRs and its effects are subtype specific. Here we suggest that receptor-specific chaperones such as RIC-3 may play important roles in controlling receptor diversity by selectively regulating surface expression of nAChRs having specific subunit compositions.     

Possible Effect of Activation of α7-Nicotinic Acetylcholine Receptors in the Mitochondrial Membrane on the Development of Apoptosis

Using flow cytometry and sandwich-immunoenzyme assay, we showed that nicotinic acetylcholine receptors with a subunit α7 (nAChRs α7) expressed in the outer mitochondrial membrane are involved in the control of mitochondria-dependent apoptosis. Pre-incubation of the mitochondria with an nAChRs α7 agonist, choline, decreased dissipation of the membrane potential of these organelles induced by the action of 0.5 mM hydrogen peroxide (H₂O₂) but did not influence the analogous effect of a high Ca²⁺ concentration (90 μM). Agonists of nAChRs α7 (choline, acetylcholine, and PNU 282987), or an inhibitor of voltage-dependent anion channels, DIDS, prevented the release of cytochrome c from the intermembrane mitochondrial space under the action of H₂O₂. In contrast, an antagonist of nAChRs α7, methyllycaconitine, promoted the release of cytochrome c and prevented the effects of agonists. The obtained data confirm the active involvement of nAChRs α7 and voltage-dependent anion channels in the process of formation of mitochondrial pores. In this case, agonists of mitochondrial nAChRs α7 subunits exert an antiapoptotic effect, while antagonists of mitochondrial nAChRs α7 subunits manifest a proapoptotic action.     

Molecular modeling and structural analysis of nAChR variants uncovers the mechanism of resistance to snake toxins

Nicotinic acetylcholine receptors (nAChRs) are neuromuscular proteins responsible for muscle contraction upon binding with chemical stimulant acetylcholine (ACh). The α-neurotoxins of snake mimic the structure of ACh and attacks nAChRs, which block the flow of ACh and leads to numbness and paralysis. The toxin-binding site of alpha subunit in the nAChRs is highly conserved throughout chordate lineages with few exceptions in resistance organisms. In this study, we have analyzed the sequence and structures of toxin-binding/resistant nAChRs and their interaction stability with toxins through molecular docking and molecular dynamics simulation (MDS). We have reported the potential glycosylation residues within the toxin-binding cleft adding sugar moieties through N-linked glycosylation in resistant organisms. Residue variations at key positions alter the secondary structure of binding cleft, which might interfere with toxin binding and it could be one of the possible explanations for the resistance to snake venoms. Analysis of nAChR-α-neurotoxin complexes has confirmed the key interacting residues. In addition, drastic variation in the binding stability of Mongoose nAChR-α-Bungarotoxin (α-BTX) and human nAChR-α-BTX complexes were found at specific phase of MDS. Our findings suggest that specific mutations in the binding site of toxin are potentially preventing the formation of stable complex of receptor-toxin, which might lead to mechanism of resistance. This in silico study on the binding cleft of nAChR and the findings of interacting residues will assist in designing potential inhibitors as therapeutic targets.     

Study of nicotinic acetylcholine receptors on cultured antennal lobe neurones from adult honeybee brains

In insects, acetylcholine (ACh) is the main neurotransmitter, and nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission. In the honeybee, nAChRs are expressed in diverse structures including the primary olfactory centres of the brain, the antennal lobes (AL) and the mushroom bodies. Whole-cell, voltage-clamp recordings were used to characterize the nAChRs present on cultured AL cells from adult honeybee, Apis mellifera. In 90% of the cells, applications of ACh induced fast inward currents that desensitized slowly. The classical nicotinic agonists nicotine and imidacloprid elicited respectively 45 and 43% of the maximum ACh-induced currents. The ACh-elicited currents were blocked by nicotinic antagonists methyllycaconitine, dihydroxy-β-erythroidine and α-bungarotoxin. The nAChRs on adult AL cells are cation permeable channels. Our data indicate the existence of functional nAChRs on adult AL cells that differ from nAChRs on pupal Kenyon cells from mushroom bodies by their pharmacological profile and ionic permeability, suggesting that these receptors could be implicated in different functions.     

A role for Leu247 residue within transmembrane domain 2 in ginsenoside-mediated α7 nicotinic acetylcholine receptor regulation

Nicotinic acetylcholine receptors (nAChRs) play important roles in nervous system functions and are involved in a variety of diseases. We previously demonstrated that ginsenosides, the active ingredients of Panax ginseng, inhibit subsets of nAChR channel currents, but not α7, expressed in Xenopus laevis oocytes. Mutation of the highly conserved Leu247 to Thr247 in the transmembrane domain 2 (TM2) channel pore region of α7 nAChR induces alterations in channel gating properties and converts α7 nAChR antagonists into agonists. In the present study, we assessed how point mutations in the Leu247 residue leading to various amino acids affect 20(S)-ginsenoside Rg₃ (Rg₃) activity against the α7 nAChR. Mutation of L247 to L247A, L247D, L247E, L247I, L247S, and L247T, but not L247K, rendered mutant receptors sensitive to Rg₃. We further characterized Rg₃ regulation of L247T receptors. We found that Rg₃ inhibition of mutant α7 nAChR channel currents was reversible and concentration-dependent. Rg₃ inhibition was strongly voltage-dependent and noncompetitive manner. These results indicate that the interaction between Rg₃ and mutant receptors might differ from its interaction with the wild-type receptor. To identify differences in Rg₃ interactions between wild-type and L247T receptors, we utilized docked modeling. This modeling revealed that Rg₃ forms hydrogen bonds with amino acids, such as Ser240 of subunit I and Thr244 of subunit II and V at the channel pore, whereas Rg₃ localizes at the interface of the two wild-type receptor subunits. These results indicate that mutation of Leu247 to Thr247 induces conformational changes in the wild-type receptor and provides a binding pocket for Rg₃ at the channel pore.     

Computational approaches to understand alpha-conotoxin interactions at neuronal nicotinic receptors.

Recent and increasing use of computational tools in the field of nicotinic receptors has led to the publication of several models of ligand-receptor interactions. These models are all based on the crystal structure at 2.7 A resolution of a protein related to the extracellular N-terminus of nicotinic acetylcholine receptors (nAChRs), the acetylcholine binding protein. In the absence of any X-ray or NMR information on nAChRs, this new structure has provided a reliable alternative to study the nAChR structure. We are now able to build homology models of the binding domain of any nAChR subtype and fit in different ligands using docking programs. This strategy has already been performed successfully for the docking of several nAChR agonists and antagonists. This minireview focuses on the interaction of alpha-conotoxins with neuronal nicotinic receptors in light of our new understanding of the receptor structure. Computational tools are expected to reveal the molecular recognition mechanisms that govern the interaction between alpha-conotoxins and neuronal nAChRs at the molecular level. An accurate determination of their binding modes on the neuronal nAChR may allow the rational design of alpha-conotoxin-based ligands with novel nAChR selectivity.     

Identification of a negative allosteric site on human α4β2 and α3β4 neuronal nicotinic acetylcholine receptors

Acetylcholine-based neurotransmission is regulated by cationic, ligand-gated ion channels called nicotinic acetylcholine receptors (nAChRs). These receptors have been linked to numerous neurological diseases and disorders such as Alzheimer's disease, Parkinson's disease, and nicotine addiction. Recently, a class of compounds has been discovered that antagonize nAChR function in an allosteric fashion. Models of human α4β2 and α3β4 nicotinic acetylcholine receptor (nAChR) extracellular domains have been developed to computationally explore the binding of these compounds, including the dynamics and free energy changes associated with ligand binding. Through a blind docking study to multiple receptor conformations, the models were used to determine a putative binding mode for the negative allosteric modulators. This mode, in close proximity to the agonist binding site, is presented in addition to a hypothetical mode of antagonism that involves obstruction of C loop closure. Molecular dynamics simulations and MM-PBSA free energy of binding calculations were used as computational validation of the predicted binding mode, while functional assays on wild-type and mutated receptors provided experimental support. Based on the proposed binding mode, two residues on the β2 subunit were independently mutated to the corresponding residues found on the β4 subunit. The T58K mutation resulted in an eight-fold decrease in the potency of KAB-18, a compound that exhibits preferential antagonism for human α4β2 over α3β4 nAChRs, while the F118L mutation resulted in a loss of inhibitory activity for KAB-18 at concentrations up to 100 μM. These results demonstrate the selectivity of KAB-18 for human α4β2 nAChRs and validate the methods used for identifying the nAChR modulator binding site. Exploitation of this site may lead to the development of more potent and subtype-selective nAChR antagonists which may be used in the treatment of a number of neurological diseases and disorders.     

Acetylcholine nicotinic receptors: finding the putative binding site of allosteric modulators using the “blind docking” approach

Allosteric potentiation of acetylcholine nicotinic receptors is considered to be one of the most promising approaches for the treatment of Alzheimer’s disease. However, the exact localization of the allosteric binding site and the potentiation mechanism at the molecular level are presently unknown. We have performed the “blind docking” of three known allosteric modulators (galanthamine, codeine and eserine) with the Acetylcholine Binding Protein and models of human α7, α3β4 and α4β2 nicotinic receptors, created by homology modeling. Three putative binding sites were identified in the channel pore, each one showing different affinities for the ligands. One of these sites is localized opposite to the agonist binding site and is probably implicated in the potentiation process. On the basis of these results, a possible mechanism for nicotinic acetylcholine receptor (nAChRs) activation is proposed. The present findings may represent an important advance for understanding the allosteric modulation mechanism of nAChRs. Electronic supplementary material Supplementary material is available for this article at     

Amino acid residues that confer high selectivity of the alpha6 nicotinic acetylcholine receptor subunit to alpha-conotoxin MII[S4A,E11A,L15A

Nicotinic acetylcholine receptors (nAChRs) containing alpha3 and beta2 subunits are found in autonomic ganglia and mediate ganglionic transmission. The closely related alpha6 nAChR subtype is found in the central nervous system where changes in its level of expression are observed in Parkinson's disease. To obtain a ligand that discriminates between these two receptors, we designed and synthesized a novel analog ofalpha-conotoxin MII, MII[S4A,E11A,L15A], and tested it on nAChRs expressed in Xenopus oocytes. The peptide blocked chimeric alpha6/alpha3beta2beta3 nAChRs with an IC(50) of 1.2 nm; in contrast, its IC(50) on the closely related alpha3beta2 as well as non-alpha6 nAChRs was three orders of magnitude higher. We identified the residues in the receptors that are responsible for their differential sensitivity to the peptide. We constructed chimeras with increasingly longer fragments of the N-terminal ligand binding domain of the alpha3 subunit inserted into the homologous positions of the alpha6 subunit, and these were used to determine that the region downstream of the first 140 amino acids was involved. Further mutagenesis of this region revealed that the alpha6 subunit residues Glu-152, Asp-184, and Thr-195 were critical, and replacement of these three residues with their homologs from the alpha3 subunit increased the IC(50) of the peptide by >1000-fold. Conversely, when these key residues inalpha3 were replaced with those fromalpha6, the IC(50) decreased by almost 150-fold. Similar effects were seen with other alpha6-selective conotoxins, suggesting the general importance of thesealpha6 residues in conferring selective binding.     

Single-residue alteration in alpha-conotoxin PnIA switches its nAChR subtype selectivity.

alpha-Conotoxins are disulfide-rich peptides that are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). Despite their small size, different alpha-conotoxins are able to discriminate among different subtypes of mammalian nAChRs. In this report, the activity of two peptides from the venom of Conus pennaceus, alpha-conotoxins PnIA and PnIB, are examined. Although the toxins differ in only two residues, PnIA preferentially blocks alpha3beta2 nAChRs, whereas PnIB prefers the alpha7 subtype. Point mutation chimeras of these alpha-conotoxins were synthesized and their activities assessed on Xenopus oocytes expressing specific nAChRs. Change of a single residue, Ala10 to Leu, in PnIA (to form PnIA [A10L]) converts the parent peptide from alpha3beta2-preferring to alpha7-preferring; furthermore, PnIA [A10L] blocks the alpha7 receptor with an IC(50) (12.6 nM) that is lower than that of either parent peptide. Kinetic analysis indicates that differences in affinity among the analogues are primarily due to differences in off-rate, with PnIA [A10L]'s interaction with alpha7 having the smallest off-rate (k(off) = 0.17 min(-)(1)). Thermodynamic analysis indicates that Leu10 enhances the peptide's interaction with alpha7, but not alpha3beta2, receptors, whereas Ser11 (in PnIA [N11S]) reduces its affinity for both alpha7 and alpha3beta2 nAChRs.     

Nicotinic acetylcholine receptors: from structure to brain function

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels and can be divided into two groups: muscle receptors, which are found at the skeletal neuromuscular junction where they mediate neuromuscular transmission, and neuronal receptors, which are found throughout the peripheral and central nervous system where they are involved in fast synaptic transmission. nAChRs are pentameric structures that are made up of combinations of individual subunits. Twelve neuronal nAChR subunits have been described, α2–α10 and β2–β4; these are differentially expressed throughout the nervous system and combine to form nAChRs with a wide range of physiological and pharmacological profiles. The nAChR has been proposed as a model of an allosteric protein in which effects arising from the binding of a ligand to a site on the protein can lead to changes in another part of the molecule. A great deal is known about the structure of the pentameric receptor. The extracellular domain contains binding sites for numerous ligands, which alter receptor behavior through allosteric mechanisms. Functional studies have revealed that nAChRs contribute to the control of resting membrane potential, modulation of synaptic transmission and mediation of fast excitatory transmission. To date, ten genes have been identified in the human genome coding for the nAChRs. nAChRs have been demonstrated to be involved in cognitive processes such as learning and memory and control of movement in normal subjects. Recent data from knockout animals has extended the understanding of nAChR function. Dysfunction of nAChR has been linked to a number of human diseases such as schizophrenia, Alzheimer's and Parkinson's diseases. nAChRs also play a significant role in nicotine addiction, which is a major public health concern. A genetically transmissible epilepsy, ADNFLE, has been associated with specific mutations in the gene coding for the α4 or β2 subunits, which leads to altered receptor properties. Electronic Publication     

In silico characterization of cytisinoids docked into an acetylcholine binding protein

Homology models of nicotinic acetylcholine receptors (nAChRs) suggest that subtype specificity is due to non-conserved residues in the complementary subunit of the ligand-binding pocket. Cytisine and its derivatives generally show a strong preference for heteromeric α4β21 nAChRs over the homomeric α7 subtype, and the structural modifications studied do not cause large changes in their nAChR subtype selectivity. In the present work we docked cytisine, N-methylcytisine, and several pyridone ring-substituted cytisinoids into the crystallographic structure of the Lymnaea stagnalis acetylcholine binding protein (AChBP) co-crystallized with nicotine (1UW6). The graphical analysis of the best poses showed that cytisinoids have weak interactions with the side chains of the non-conserved amino acids in the complementary subunit justifying the use of PDB 1UWB as a surrogate for nAChR. Furthermore, we found a high correlation (R² = 0.96) between the experimental pIC₅₀ values at α4β21 nAChR and docking energy (S) of the best cytisinoid poses within the AChBP. Due to the quality of the correlation we suggest that this equation might be used as a predictive model to propose new cytisine-derived nAChRs ligands. Our docking results also suggest that further structural modifications of these cytisinoids will not greatly alter their α4β21/α7 selectivity.     

Nicotinic Acetylcholine Receptor (nAChR) Dependent Chorda Tympani Taste Nerve Responses to Nicotine,Ethanol and Acetylcholine

Nicotine elicits bitter taste by activating TRPM5-dependent and TRPM5-independent but neuronal nAChR-dependent pathways. The nAChRs represent common targets at which acetylcholine, nicotine and ethanol functionally interact in the central nervous system. Here, we investigated if the nAChRs also represent a common pathway through which the bitter taste of nicotine, ethanol and acetylcholine is transduced. To this end, chorda tympani (CT) taste nerve responses were monitored in rats, wild-type mice and TRPM5 knockout (KO) mice following lingual stimulation with nicotine free base, ethanol, and acetylcholine, in the absence and presence of nAChR agonists and antagonists. The nAChR modulators: mecamylamine, dihydro-β-erythroidine, and CP-601932 (a partial agonist of the α3β4* nAChR), inhibited CT responses to nicotine, ethanol, and acetylcholine. CT responses to nicotine and ethanol were also inhibited by topical lingual application of 8-chlorophenylthio (CPT)-cAMP and loading taste cells with [Ca²⁺]_i by topical lingual application of ionomycin + CaCl₂. In contrast, CT responses to nicotine were enhanced when TRC [Ca²⁺]_i was reduced by topical lingual application of BAPTA-AM. In patch-clamp experiments, only a subset of isolated rat fungiform taste cells exposed to nicotine responded with an increase in mecamylamine-sensitive inward currents. We conclude that nAChRs expressed in a subset of taste cells serve as common receptors for the detection of the TRPM5-independent bitter taste of nicotine, acetylcholine and ethanol.     

Nicotine-induced Ca<Superscript>2+</Superscript>-myristoyl Switch of Neuronal Ca<Superscript>2+</Superscript> Sensor VILIP-1 in Hippocampal Neurons: A Possible Crosstalk Mechanism for Nicotinic Receptors

Visinin-like protein (VILIP-1) belongs to the neuronal Ca²⁺ sensor family of EF-hand Ca²⁺-binding proteins that regulate a variety of Ca²⁺-dependent signal transduction processes in neurons. It is an interaction partner of α4β2 nicotinic acetylcholine receptor (nAChR) and increases surface expression level and agonist sensitivity of the receptor in oocytes. Nicotine stimulation of nicotinic receptors has been reported to lead to an increase in intracellular Ca²⁺ concentration by Ca²⁺-permeable nAChRs, which in turn might lead to activation of VILIP-1, by a mechanism described as the Ca²⁺-myristoyl switch. It has been postulated that this will lead to co-localization of the proteins at cell membranes, where VILIP-1 can influence functional activity of α4-containing nAChRs. In order to test this hypothesis we have investigated whether a nicotine-induced and reversible Ca²⁺-myristoyl switch of VILIP-1 exists in primary hippocampal neurons and whether pharmacological agents, such as antagonist specific for distinct nAChRs, can interfere with the Ca²⁺-dependent membrane localization of VILIP-1. Here we report, that only α7- but not α4-containing nAChRs are able to elicit a Ca²⁺-dependent and reversible membrane-translocation of VILIP-1 in interneurons as revealed by employing the specific receptor antagonists dihydro-beta-erythroidine and methylallylaconitine. The nAChRs are associated with processes of synaptic plasticity in hippocampal neurons and they have been implicated in the pathology of CNS disorders, including Alzheimer’s disease and schizophrenia. VILIP-1 might provide a novel functional crosstalk between α4- and α7-containing nAChRs.     

Dynamic structure based pharmacophore modeling of the Acetylcholinesterase reveals several potential inhibitors

Acetylcholinesterase is a critical enzyme that regulates neurotransmission by catalyzing the breakdown of neurotransmitter acetylcholine in synapses of the nervous system. It is an important target for therapeutic drugs that treat Alzheimer’s disease. Since, the degree of flexibility of the side chains of the residues in the active-site gorge of Acetylcholinesterase is diverse it results in different bound ligand conformations. The side-chain conformations of Ser293, Tyr341, Leu76, and Val73 are flexible, while the side-chain conformations of Tyr72, Tyr 124, Ser125, Phe295, and Arg296 appear to be fixed. In this study, multi-conformation dynamic pharmacophore models from the donepezyl-binding pocket based on highly populated structures chosen from molecular dynamics simulations were used for screening compounds that can properly bind acetylcholinesterase. Based on these structures, three pharmacophore models were generated. Consequently, 14 hits were retrieved as final candidates by utilizing virtual screening of ZINC database and molecular docking.     

The α7 nicotinic acetylcholine receptor in neuronal plasticity

A growing body of evidence indicates that neuronal nicotinic acetylcholine receptors (nAChRs), in addition to promoting fast cholinergic transmission, may modulate other neuronal activities within the central nervous system (CNS). In particular, the α7 nAChR is highly permeable to Ca²⁺ and may serve a distinct role in regulating neuronal plasticity. By elevating intracellular Ca²⁺ levels in discrete neuronal locations, these ligand-gated ion channels may influence numerous physiological processes in developing and adult CNS. In this article, we review evidence that both pre- and postsynaptic α7 nAChRs modulate transmitter release in the brain and periphery through Ca²⁺-dependent mechanisms. The possible role of α7 nAChRs in regulating neuronal growth and differentiation in developing CNS is also evaluated. We consider an interaction between cholinergic and glutamatergic transmission and propose a hypothesis on the possible coregulation of intracellular Ca²⁺ byN-methyl-d-aspartate (NMDA) receptors and α7 nAChRs. Finally, the clinical significance of alterations in the normal function of α7 nAChRs is discussed as it pertains to prenatal nicotine exposure, schizophrenia, and epilepsy.     

Nicotinic receptor signaling in nonexcitable cells

The finding that neuronal nicotinic acetylcholine receptors (nAChRs) are present in non-neuronal cells both within and outside the nervous system raises some interesting issues. The mechanisms underlying receptor signaling and its downstream consequences in these cells remain to be elucidated. Factors controlling the release of acetylcholine and the extent of its diffusion are likely to be different for these cells than for traditional neuronal synapses. Recent advances on the physiologic functions of some of these cell types have provided a better insight into possible functional roles for nAChRs in nonexcitable cells. The presence of nAChRs on these cells also implies a broader scope for the actions of nicotine that needs to be considered from a clinical viewpoint. Revealing the potential physiologic roles for nAChRs on nonexcitable cells is likely to provide a more complete understanding of cholinergic signaling.     

Two neuronal nicotinic acetylcholine receptors, alpha4beta4 and alpha7, show differential agonist binding modes

Nicotinic acetylcholine receptors (nAChRs) are pentameric, neurotransmitter-gated ion channels responsible for rapid excitatory neurotransmission in the central and peripheral nervous systems, resulting in skeletal muscle tone and various cognitive effects in the brain. These complex proteins are activated by the endogenous neurotransmitter ACh as well as by nicotine and structurally related agonists. Activation and modulation of nAChRs has been implicated in the pathology of multiple neurological disorders, and as such, these proteins are established therapeutic targets. Here we use unnatural amino acid mutagenesis to examine the ligand binding mechanisms of two homologous neuronal nAChRs: the α4β4 and α7 receptors. Despite sequence identity among the residues that form the core of the agonist-binding site, we find that the α4β4 and α7 nAChRs employ different agonist-receptor binding interactions in this region. The α4β4 receptor utilizes a strong cation-π interaction to a conserved tryptophan (TrpB) of the receptor for both ACh and nicotine, and nicotine participates in a strong hydrogen bond with a backbone carbonyl contributed by TrpB. Interestingly, we find that the α7 receptor also employs a cation-π interaction for ligand recognition, but the site has moved to a different aromatic amino acid of the agonist-binding site depending on the agonist. ACh participates in a cation-π interaction with TyrA, whereas epibatidine participates in a cation-π interaction with TyrC2.