Nicotinic receptor modulation of primary afferent excitability with selective regulation of Aδ-mediated spinal actions.

Somatosensory input strength can be modulated by primary afferent depolarization (PAD) generated predominantly via presynaptic GABAA receptors on afferent terminals. We investigated whether ionotropic nicotinic acetylcholine receptors (nAChRs) also provide modulatory actions, focusing on myelinated afferent excitability in in vitro murine spinal cord nerve-attached models. Primary afferent stimulation-evoked synaptic transmission was recorded in the deep dorsal horn as extracellular field potentials (EFPs), whereas concurrently recorded dorsal root potentials (DRPs) were used as an indirect measure of PAD. Changes in afferent membrane excitability were simultaneously measured as direct current (DC)-shifts in membrane polarization recorded in dorsal roots or peripheral nerves. The broad nAChR antagonist d-tubocurarine (d-TC) selectively and strongly depressed Aδ-evoked synaptic EFPs (36% of control) coincident with similarly depressed A-fiber DRP (43% of control), whereas afferent electrical excitability remained unchanged. In comparison, acetylcholine (ACh) and the nAChR agonists, epibatidine and nicotine, reduced afferent excitability by generating coincident depolarizing DC-shifts in peripheral axons and intraspinally. Progressive depolarization corresponded temporally with the emergence of spontaneous axonal spiking and reductions in the DRP and all afferent-evoked synaptic actions (31%-37% of control). Loss of evoked response was long-lasting, independent of DC repolarization, and likely due to mechanisms initiated by spontaneous C-fiber activity. DC-shifts were blocked with d-TC but not GABAA receptor blockers and retained after tetrodotoxin block of voltage-gated Na+ channels. Notably, actions tested were comparable between three mouse strains, in rat, and when performed in different labs. Thus, nAChRs can regulate afferent excitability via two distinct mechanisms: by central Aδ-afferent actions, and by transient extrasynaptic axonal activation of high-threshold primary afferents.NEW & NOTEWORTHY Primary afferents express many nicotinic ACh receptor (nAChR) subtypes but whether activation is linked to presynaptic inhibition, facilitation, or more complex and selective activity modulation is unknown. Recordings of afferent-evoked responses in the lumbar spinal cord identified two nAChR-mediated modulatory actions: 1) selective control of Aδ afferent transmission and 2) robust changes in axonal excitability initiated via extrasynaptic shifts in DC polarization. This work broadens the diversity of presynaptic modulation of primary afferents by nAChRs.

The (α4)3(ß2)2 Stoichiometry of the Nicotinic Acetylcholine Receptor Predominates in the Rat Motor Cortex.

The α4ß2 nicotinic acetylcholine receptor (nAChR) is important in central nervous system physiology and in mediating several of the pharmacological effects of nicotine on cognition, attention, and affective states. It is also the likely receptor that mediates nicotine addiction. This receptor assembles in two distinct stoichiometries: (α4)2(ß2)3 and (α4)3(ß2)2, which are referred to as high-sensitivity (HS) and low-sensitivity (LS) nAChRs, respectively, based on a difference in the potency of acetylcholine to activate them. The physiologic and pharmacological differences between these two receptor subtypes have been described in heterologous expression systems. However, the presence of each stoichiometry in native tissue currently remains unknown. In this study, different ratios of rat α4 and ß2 subunit cDNA were transfected into human embryonic kidney 293 cells to create a novel model system of HS and LS α4ß2 nAChRs expressed in a mammalian cell line. The HS and LS nAChRs were characterized through pharmacological and biochemical methods. Isolation of surface proteins revealed higher amounts of α4 or ß2 subunits in the LS or HS nAChR populations, respectively. In addition, sazetidine-A displayed different efficacies in activating these two receptor stoichiometries. Using this model system, a neurophysiological "two-concentration" acetylcholine or carbachol paradigm was developed and validated to determine α4/ß2 subunit stoichiometry. This paradigm was then used in layers I-IV of slices of the rat motor cortex to determine the percent contribution of HS and LS α4ß2 receptors in this brain region. We report that the majority of α4ß2 nAChRs in this brain region possess a stoichiometry of the (α4)3(ß2)2 LS subtype.

Tunable pKa values and the basis of opposite charge selectivities in nicotinic-type receptors.

Among ion channels, only the nicotinic-receptor superfamily has evolved to generate both cation- and anion-selective members. Although other, structurally unrelated, neurotransmitter-gated cation channels exist, no other type of neurotransmitter-gated anion channel, and thus no other source of fast synaptic inhibitory signals, has been described so far. In addition to the seemingly straightforward electrostatic effect of the presence (in the cation-selective members) or absence (in the anion-selective ones) of a ring of pore-facing carboxylates, mutational studies have identified other features of the amino-acid sequence near the intracellular end of the pore-lining transmembrane segments (M2) that are also required to achieve the high charge selectivity shown by native channels. However, the mechanism underlying this more subtle effect has remained elusive and a subject of speculation. Here we show, using single-channel electrophysiological recordings to estimate the protonation state of native ionizable side chains, that anion-selective-type sequences favour whereas cation-selective-type sequences prevent the protonation of the conserved, buried basic residues at the intracellular entrance of the pore (the M2 0' position). We conclude that the previously unrecognized tunable charge state of the 0' ring of buried basic side chains is an essential feature of these channels' versatile charge-selectivity filter.

The subunit gene Ldα1 of nicotinic acetylcholine receptors plays important roles in the toxicity of imidacloprid and thiamethoxam against Leptinotarsa decemlineata.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ACh-gated ion channels. It is believed that nAChRs composed of different subunits may vary in their function and toxicological characteristics. Neonicotinoids are activators of nAChRs and important insecticides that are extensively used for crop protection and resistance has been developed by some pests. They are also major insecticides for the control of Leptinotarsa decemlineata, which is a destructive defoliator pest that invaded the Xinjiang region of China in the 1990s. However, little is known about the constitution or subunits of the target in this pest. In this study, the full-length cDNAs encoding four new nAChR subunits (named Ldα3, Ldα6, Ldα10, and Ldß1) were cloned from L. decemlineata. These genes encode 822-, 753-, 672-, and 759-amino acid proteins, respectively, which share typical features of insect nAChRs subunits and closely resemble the corresponding subunits of the nAChRs from Tribolium castaneum. Temporal and spatial expression analyses showed that these genes, as well as the previously identified Ldα1, Ldα2, and Ldα8 genes, are widely expressed in all developmental stages, including eggs, larvae of various instars, pupae, and adults. All genes monitored were expressed at higher levels in the head than in the thorax and abdomen, except for Ldα10. Dietary ingestion of double-stranded RNA bacterially expressed for Ldα1 (dsLdα1) significantly reduced the mRNA level of Ldα1 in treated larvae and adults by 48.0% and 78.6%, respectively. Among the non-target genes, Ldα3, Ldα9, and Ldß1 were significantly up-regulated in larvae. A toxicity bioassay showed that dsLdα1 treatment greatly decreased the sensitivity to imidacloprid and thiamethoxam in adults. The larval susceptibility to thiamethoxam but not to imidacloprid was also reduced because of the lower down-regulation of Ldα1. Thus, our results suggest that Ldα1 encodes a subunit of a functional nAChR that mediates the toxicity of imidacloprid and thiamethoxam against L. decemlineata and that the down-regulation of Ldα1 might be an important mechanism for resistance and/or tolerance of L. decemlineata to neonicotinoids.

α-Conotoxins Identify the α3ß4* Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells.

Ligands that selectively inhibit human α3ß2 and α6ß2 nicotinic acetylcholine receptor (nAChRs) and not the closely related α3ß4 and α6ß4 subtypes are lacking. Current α-conotoxins (α-Ctxs) that discriminate among these nAChR subtypes in rat fail to discriminate among the human receptor homologs. In this study, we describe the development of α-Ctx LvIA(N9R,V10A) that is 3000-fold more potent on oocyte-expressed human α3ß2 than α3ß4 and 165-fold more potent on human α6/α3ß2ß3 than α6/α3ß4 nAChRs. This analog was used in conjuction with three other α-Ctx analogs and patch-clamp electrophysiology to characterize the nAChR subtypes expressed by human adrenal chromaffin cells. LvIA(N9R,V10A) showed little effect on the acetylcholine-evoked currents in these cells at concentrations expected to inhibit nAChRs with ß2 ligand-binding sites. In contrast, the ß4-selective α-Ctx BuIA(T5A,P6O) inhibited >98% of the acetylcholine-evoked current, indicating that most of the heteromeric receptors contained ß4 ligand-binding sites. Additional studies using the α6-selective α-Ctx PeIA(A7V,S9H,V10A,N11R,E14A) indicated that the predominant heteromeric nAChR expressed by human adrenal chromaffin cells is the α3ß4* subtype (asterisk indicates the possible presence of additional subunits). This conclusion was supported by polymerase chain reaction experiments of human adrenal medulla gland and of cultured human adrenal chromaffin cells that demonstrated prominent expression of RNAs for α3, α5, α7, ß2, and ß4 subunits and a low abundance of RNAs for α2, α4, α6, and α10 subunits.

The unique α4+/-α4 agonist binding site in (α4)3(ß2)2 subtype nicotinic acetylcholine receptors permits differential agonist desensitization pharmacology versus the (α4)2(ß2)3 subtype.

Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (α4)2(ß2)3) or low-sensitivity (LS) (α4)3(ß2)2) isoforms of human α4ß2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using (86)Rb(+) efflux in a stably transfected SH-EP1-hα4ß2 human epithelial cell line, and two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS α4ß2-nAChR constructs (HSP and LSP). Unlike previously studied agonists, desensitization by the highly selective agonists A-85380 [3-(2(S)-azetidinylmethoxy)pyridine] and sazetidine-A (Saz-A) preferentially reduced α4ß2-nAChR HS-phase versus LS-phase responses. The concatenated-nAChR experiments confirmed that approximately 20% of LS-isoform acetylcholine-induced function occurs in an HS-like phase, which is abolished by Saz-A preincubation. Six mutant LSPs were generated, each targeting a conserved agonist binding residue within the LS-isoform-only α4(+)/(-)α4 interface agonist binding site. Every mutation reduced the percentage of LS-phase function, demonstrating that this site underpins LS-phase function. Oocyte-surface expression of the HSP and each of the LSP constructs was statistically indistinguishable, as measured using ß2-subunit-specific [(125)I]mAb295 labeling. However, maximum function is approximately five times greater on a "per-receptor" basis for unmodified LSP versus HSP α4ß2-nAChRs. Thus, recruitment of the α4(+)/(-)α4 site at higher agonist concentrations appears to augment otherwise-similar function mediated by the pair of α4(+)/(-)ß2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two α4ß2-nAChR isoforms, and demonstrate that HS versus LS α4ß2-nAChR activity can be selectively manipulated using pharmacological approaches. Since α4ß2 nAChRs are the predominant neuronal subtype, these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development.

International Union of Basic and Clinical Pharmacology. LXXXII: Nomenclature and Classification of Hydroxy-carboxylic Acid Receptors (GPR81, GPR109A, and GPR109B).

The G-protein-coupled receptors GPR81, GPR109A, and GPR109B share significant sequence homology and form a small group of receptors, each of which is encoded by clustered genes. In recent years, endogenous ligands for all three receptors have been described. These endogenous ligands have in common that they are hydroxy-carboxylic acid metabolites, and we therefore have proposed that this receptor family be named hydroxy-carboxylic acid (HCA) receptors. The HCA(1) receptor (GPR81) is activated by 2-hydroxy-propanoic acid (lactate), the HCA(2) receptor (GPR109A) is a receptor for the ketone body 3-hydroxy-butyric acid, and the HCA(3) receptor (GPR109B) is activated by the ß-oxidation intermediate 3-hydroxy-octanoic acid. HCA(1) and HCA(2) receptors are found in most mammalian species, whereas the HCA(3) receptor is present only in higher primates. The three receptors have in common that they are expressed in adipocytes and are coupled to G(i)-type G-proteins mediating antilipolytic effects in fat cells. HCA(2) and HCA(3) receptors are also expressed in a variety of immune cells. HCA(2) is a receptor for the antidyslipidemic drug nicotinic acid (niacin) and related compounds, and there is an increasing number of synthetic ligands mainly targeted at HCA(2) and HCA(3) receptors. The aim of this article is to give an overview on the discovery and pharmacological characterization of HCAs, and to introduce an International Union of Basic and Clinical Pharmacology (IUPHAR)-recommended nomenclature. We will also discuss open questions regarding this receptor family as well as their physiological role and therapeutic potential.

Nicotinic acetylcholine receptors in dorsal root ganglion neurons include the α6ß4* subtype.

The α6-containing nicotinic acetylcholine receptors (nAChRs) have recently been implicated in diseases of the central nervous system (CNS), including Parkinson's disease and substance abuse. In contrast, little is known about the role of α6* nAChRs in the peripheral nervous system (where the asterisk denotes the possible presence of additional subunits). Dorsal root ganglia (DRG) neurons are known to express nAChRs with a pharmacology consistent with an α7, α3ß4*, and α4ß2* composition. Here we present evidence that DRG neurons also express α6* nAChRs. We used RT-PCR to show the presence of α6 subunit transcripts and patch-clamp electrophysiology together with subtype-selective α-conotoxins to pharmacologically characterize the nAChRs in rat DRG neurons. α-Conotoxin BuIA (500 nM) blocked acetylcholine-gated currents (I(ACh)) by 90.3 ± 3.0%; the recovery from blockade was very slow, indicating a predominance of α(x)ß4* nAChRs. Perfusion with either 300 nM BuIA[T5A;P6O] or 200 nM MII[E11A], α-conotoxins that target the α6ß4* subtype, blocked I(ACh) by 49.3 ± 5 and 46.7 ± 8%, respectively. In these neurons, I(ACh) was relatively insensitive to 200 nM ArIB[V11L;V16D] (9.4±2.0% blockade) or 500 nM PnIA (23.0±4% blockade), α-conotoxins that target α7 and α3ß2*/α6ß2* nAChRs, respectively. We conclude that α6ß4* nAChRs are among the subtypes expressed by DRG, and to our knowledge, this is the first demonstration of α6ß4* in neurons outside the CNS.

A prokaryotic proton-gated ion channel from the nicotinic acetylcholine receptor family.

Ligand-gated ion channels (LGICs) mediate excitatory and inhibitory transmission in the nervous system. Among them, the pentameric or 'Cys-loop' receptors (pLGICs) compose a family that until recently was found in only eukaryotes. Yet a recent genome search identified putative homologues of these proteins in several bacterial species. Here we report the cloning, expression and functional identification of one of these putative homologues from the cyanobacterium Gloeobacter violaceus. It was expressed as a homo-oligomer in HEK 293 cells and Xenopus oocytes, generating a transmembrane cationic channel that is opened by extracellular protons and shows slow kinetics of activation, no desensitization and a single channel conductance of 8 pS. Electron microscopy and cross-linking experiments of the protein fused to the maltose-binding protein and expressed in Escherichia coli are consistent with a homo-pentameric organization. Sequence comparison shows that it possesses a compact structure, with the absence of the amino-terminal helix, the canonical disulphide bridge and the large cytoplasmic domain found in eukaryotic pLGICs. Therefore it embodies a minimal structure required for signal transduction. These data establish the prokaryotic origin of the family. Because Gloeobacter violaceus carries out photosynthesis and proton transport at the cytoplasmic membrane, this new proton-gated ion channel might contribute to adaptation to pH change.

[Nicotinic acetylcholine receptor in Alzheimer's disease].

Nicotinic acetylcholine receptor (nAChR) mediates pleiotropic actions in brain not only for nicotinic neurotransmission but also glutamatergic, dopaminergic, norepinephrinergic, GABAergic, and serotonergic transmissions, especially through allosteric potentiating ligand (APL) action. Because nAChR is rich in thalamus, the direct stimulation of nAChR through APL action is expected to show increasing function of cerebral cortex and limbic system through thalamic activation. In fact, a choline esterase inhibitor with this APL action such as galantamine exerts both cognitive and affective improvements which is called dual benefit for Alzheimer's disease patients.

Unraveling the high- and low-sensitivity agonist responses of nicotinic acetylcholine receptors.

The neuronal α4ß2 nicotinic acetylcholine receptors exist as two distinct subtypes, (α4)(2)(ß2)(3) and (α4)(3)(ß2)(2), and biphasic responses to acetylcholine and other agonists have been ascribed previously to coexistence of these two receptor subtypes. We offer a novel and radical explanation for the observation of two distinct agonist sensitivities. Using different expression ratios of mammalian α4 and ß2 subunits and concatenated constructs, we demonstrate that a biphasic response is an intrinsic functional property of the (α4)(3)(ß2)(2) receptor. In addition to two high-sensitivity sites at α4ß2 interfaces, the (α4)(3)(ß2)(2) receptor contains a third low-sensitivity agonist binding site in the α4α4 interface. Occupation of this site is required for full activation and is responsible for the widened dynamic response range of this receptor subtype. By site-directed mutagenesis, we show that three residues, which differ between the α4ß2 and α4α4 sites, control agonist sensitivity. The results presented here provide a basic insight into the function of pentameric ligand-gated ion channels, which enables modulation of the receptors with hitherto unseen precision; it becomes possible to rationally design therapeutics targeting subpopulations of specific receptor subtypes.

Biochemical and functional properties of distinct nicotinic acetylcholine receptors in the superior cervical ganglion of mice with targeted deletions of nAChR subunit genes.

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in ganglia of the autonomic nervous system. Here, we determined the subunit composition of hetero-pentameric nAChRs in the mouse superior cervical ganglion (SCG), the function of distinct receptors (obtained by deletions of nAChR subunit genes) and mechanisms at the level of nAChRs that might compensate for the loss of subunits. As shown by immunoprecipitation and Western blots, wild-type (WT) mice expressed: alpha 3 beta 4 (55%), alpha 3 beta 4 alpha 5 (24%) and alpha 3 beta 4 beta 2 (21%) nAChRs. nAChRs in beta 4 knockout (KO) mice were reduced to < 15% of controls and no longer contained the alpha 5 subunit. Compound action potentials, recorded from the postganglionic (internal carotid) nerve and induced by preganglionic nerve stimulation, did not differ between alpha 5 beta 4 KO and WT mice, suggesting that the reduced number of receptors in the KO mice did not impair transganglionic transmission. Deletions of alpha 5 or beta2 did not affect the overall number of receptors and we found no evidence that the two subunits substitute for each other. In addition, dual KOs allowed us to study the functional properties of distinct alpha 3 beta4 and alpha 3 beta 2 receptors that have previously only been investigated in heterologous expression systems. The two receptors strikingly differed in the decay of macroscopic currents, the efficacy of cytisine, and their responses to the alpha-conotoxins AuIB and MII. Our data, based on biochemical and functional experiments and several mouse KO models, clarify and significantly extend previous observations on the function of nAChRs in heterologous systems and the SCG.

Diversity of vertebrate nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) are pentameric neurotransmitter receptors. They are members of the Cys-loop family of ligand-gated ion channels which also include ionotropic receptors for 5-hydroxytryptamine (5-HT), gamma-aminobutyric acid (GABA) and glycine. Nicotinic receptors are expressed in both the nervous system and at the neuromuscular junction and have been implicated in several neurological and neuromuscular disorders. In vertebrates, seventeen nAChR subunits have been identified (alpha1-alpha10, beta1-beta4, gamma, delta and epsilon) which can co-assemble to generate a diverse family of nAChR subtypes. This review will focus on vertebrate nAChRs and will provide an overview of the extent of nAChR diversity based on studies of both native and recombinant nAChRs.

Interaction of the 43 kd postsynaptic protein with all subunits of the muscle nicotinic acetylcholine receptor.

The 43 kd postsynaptic protein (43K) plays a key role in the aggregation of muscle nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane of the neuromuscular junction. By transiently coexpressing 43K and a single AChR subunit (alpha, beta, gamma, or delta) in the quail fibroblast cell line, QT-6, we show that 43K interacts with each subunit to form cell surface clusters in which 43K and receptor subunit are precisely colocalized. Although the level of cell surface expression of single subunits is much lower than that of fully assembled receptor, the clustering of both single subunits and fully assembled AChR occurs efficiently. In addition, 43K-induced clustering is specific for AChR subunits. From these results, we conclude that each pentameric AChR has five potential sites for interacting with 43K during cluster formation.

Exploring the pharmacological properties of insect nicotinic acetylcholine receptors.

Insect nicotinic acetylcholine (nACh) receptors are molecular targets of insecticides such as neonicotinoids that are used to control disease-carrying insects and agricultural pests. To date, several insect nACh receptor subunits have been identified, indicating different nACh receptor subtypes and pharmacological profiles. Because of the difficulty in expressing functional insect nACh receptors in heterologous systems, new research tools are needed. Studies on insects resistant to the insecticide imidacloprid and on laboratory-generated hybrid and chimaeric nACh receptors in vitro have provided information about the molecular basis of receptor diversity, neonicotinoid resistance and selectivity. Additionally, recent results indicate that the sensitivity of insect nACh receptors to imidacloprid can be modulated by intracellular phosphorylation mechanisms, which offers a new approach to studying insect nACh receptor pharmacology.

Phenotypic characterization of two Ancylostoma caninum isolates with different susceptibilities to the anthelmintic pyrantel.

The anthelmintic pyrantel plays an important role in the control of gastrointestinal helminths of humans and domestic animals. Despite the demonstration of pyrantel resistance in several helminth species over the last 20 years, the resistance mechanism remains unclear. It has been hypothesized that resistance may arise as a consequence of changes to the relative proportions of subpopulations of nicotinic acetylcholine receptors (nAchRs). To test this hypothesis, we examined the responses of two isolates of the canine hookworm Ancylostoma caninum with low-level resistance (isolate NT) and high-level resistance (isolate PR) to pyrantel to nicotinic agonist drugs reported to be selective for three nAchR subtypes. We used larval motility and conformation assays and force transduction experiments with adult worms. Pyrantel and levamisole were less potent against larvae of isolate PR than larvae of isolate NT (up to an 18-fold increase in the 50% inhibitory concentration); on the other hand, bephenium was more potent against larvae of isolate PR than larvae of isolate NT (twofold) and nicotine had the same potency against larvae of both isolates. In adults, pyrantel, levamisole, and nicotine were less potent against isolate PR than isolate NT (two- to threefold), but the potency of bephenium against the two isolates was equivalent. Our data indicate a complex pattern of nAchRs in this species and suggest that the two isolates differ in their relative sensitivities to agonists targeting different nAchRs.

Rapsyn carboxyl terminal domains mediate muscle specific kinase-induced phosphorylation of the muscle acetylcholine receptor.

At the developing vertebrate neuromuscular junction, postsynaptic localization of the acetylcholine receptor (AChR) is regulated by agrin signaling via the muscle specific kinase (MuSK) and requires an intracellular scaffolding protein called rapsyn. In addition to its structural role, rapsyn is also necessary for agrin-induced tyrosine phosphorylation of the AChR, which regulates some aspects of receptor localization. Here, we have investigated the molecular mechanism by which rapsyn mediates AChR phosphorylation at the rodent neuromuscular junction. In a heterologous COS cell system, we show that MuSK and rapsyn induced phosphorylation of beta subunit tyrosine 390 (Y390) and delta subunit Y393, as in muscle cells. Mutation of beta Y390 or delta Y393 did not inhibit MuSK/rapsyn-induced phosphorylation of the other subunit in COS cells, and mutation of beta Y390 did not inhibit agrin-induced phosphorylation of the delta subunit in Sol8 muscle cells; thus, their phosphorylation occurs independently, downstream of MuSK activation. In COS cells, we further show that MuSK-induced phosphorylation of the beta subunit was mediated by rapsyn, as MuSK plus rapsyn increased beta Y390 phosphorylation more than rapsyn alone and MuSK alone had no effect. Intriguingly, MuSK also induced tyrosine phosphorylation of rapsyn itself. We then used deletion mutants to map the rapsyn domains responsible for activation of cytoplasmic tyrosine kinases that phosphorylate the AChR subunits. We found that rapsyn C-terminal domains (amino acids 212-412) are both necessary and sufficient for activation of tyrosine kinases and induction of cellular tyrosine phosphorylation. Moreover, deletion of the rapsyn RING domain (365-412) abolished MuSK-induced tyrosine phosphorylation of the AChR beta subunit. Together, these findings suggest that rapsyn facilitates AChR phosphorylation by activating or localizing tyrosine kinases via its C-terminal domains.

Peptide-toxin tools for probing the expression and function of fetal and adult subtypes of the nicotinic acetylcholine receptor.

Although the neuromuscular nicotinic acetylcholine receptor (nAChR) is one of the most intensively studied ion channels in the nervous system, the differential roles of fetal and adult subtypes of the nAChR under normal and pathological conditions are still incompletely defined. Until recently, no pharmacological tools distinguished between fetal and adult subtypes. Waglerin toxins (from snake venom) and alphaA(S)-conotoxins (from cone-snail venom) have provided such tools. Because these peptides were characterized by different research groups using different methods, we have: 1) more extensively tested their subtype selectivity, and 2) begun to explore how these peptides may be used in concert to elucidate expression patterns and functions of fetal and adult nAChRs. In heterologous expression systems and native tissues, Waglerin-1 and an alphaA(S)-conotoxin analog, alphaA-OIVA[K15N], are high-affinity, highly selective inhibitors of the adult and fetal muscle nAChRs, respectively. We have used the peptides and their fluorescent derivatives to explore the expression and function of the fetal and adult nAChR subtypes. While fluorescent derivatives of these peptides indicated a gradual transition from fetal to adult muscle nAChRs in mice during the first 2 weeks postnatal, we unexpectedly observed a steeper transition in functional expression in the mouse diaphragm muscle using electrophysiology. As a toolkit of pharmacological agents with complementary specificity, alphaA-OIVA[K15N] and Waglerin-1 should have further utility in determining the roles of fetal and adult nAChR subtypes in development, in mature tissues, and under pathological conditions.

Adult and periadolescent rats differ in expression of nicotinic cholinergic receptor subtypes and in the response of these subtypes to chronic nicotine exposure.

Adolescence is a time of significant brain development, and exposure to nicotine during this period is associated with higher subsequent rates of dependence. Chronic nicotine exposure alters expression of nicotinic acetylcholine receptors (nAChRs), changing the pattern of nicotine responsiveness. We used quantitative autoradiography to measure three major subtypes of nAChRs after chronic nicotine exposure by osmotic minipump in adult and periadolescent rats. Comparison of control animals at the two different ages revealed that periadolescents express consistently greater numbers of alpha4beta2* nAChRs compared to the same brain regions of adults. Similar but less pronounced increases in alpha7 nAChRs were found in control periadolescent rats compared to adults. Binding of [(125)I]alpha-conotoxin MII (largely to alpha6* nAChRs) did not systematically differ between adults and periadolescents. The response to chronic nicotine exposure also differed by age. Up-regulation of alpha4beta2* nAChRs was prominent and widespread in adult animals; in periadolescents, alpha4beta2* up-regulation also occurred, but in fewer regions and to a lesser extent. A similar pattern of response was seen with alpha7 receptors: adults were more responsive than periadolescents to nicotine-induced up-regulation. In adult animals, chronic nicotine exposure did not cause up-regulation of alpha6* nAChRs; binding was down-regulated in three regions. Unlike the other subtypes, the response of alpha6* nAChRs to chronic nicotine was greater in periadolescents, with more regions showing greater down-regulation compared to adults. These differences in receptor expression and regulation between age groups are likely to be important given the unique vulnerability of adolescents to nicotine-induced behavioral changes and susceptibility to drug abuse.

Influence of neuronal nicotinic receptors over nicotine addiction and withdrawal.

Cigarette smoking represents an enormous, global public health threat. Nearly five million premature deaths during a single year are attributable to smoking. Despite the resounding message of risks associated with smoking and numerous public health initiatives, cigarette smoking remains the most common preventable cause of disease in the United States. Fortunately, even in an adult smoker, smoking cessation can reverse many of the potential harmful effects. The symptoms associated with nicotine withdrawal represent the major obstacle to smoking cessation. This minireview examines the roles of various nicotinic receptors in the mechanisms of nicotine dependence, discusses the potential role of the habenula-interpeduncular nucleus axis in nicotine withdrawal, and highlights nicotinic receptors containing the beta4 subunit as a potential pharmacological target for smoking cessation strategies.