beta 2 subunit contribution to 4/7 alpha-conotoxin binding to the nicotinic acetylcholine receptor

The structures of acetylcholine-binding protein ( AChBP) and nicotinic acetylcholine receptor ( nAChR) homology models have been used to interpret data from mutagenesis experiments at the nAChR. However, little is known about AChBP-derived structures as predictive tools. Molecular surface analysis of nAChR models has revealed a conserved cleft as the likely binding site for the 4/7 alpha-conotoxins. Here, we used an alpha 3 beta 2 model to identify beta 2 subunit residues in this cleft and investigated their influence on the binding of alpha-conotoxins MII, PnIA, and GID to the alpha 3 beta 2 nAChR by two-electrode voltage clamp analysis. Although a beta 2-L119Q mutation strongly reduced the affinity of all three alpha-conotoxins, beta 2-F117A, beta 2-V109A, and beta 2-V109G mutations selectively enhanced the binding of MII and GID. An increased activity of alpha-conotoxins GID and MII was also observed when the beta 2-F117A mutant was combined with the alpha 4 instead of the alpha 3 subunit. Investigation of A10L-PnIA indicated that high affinity binding to beta 2-F117A, beta 2-V109A, and beta 2-V109G mutants was conferred by amino acids with a long side chain in position 10 (PnIA numbering). Docking simulations of 4/7 alpha-conotoxin binding to the alpha 3 beta 2 model supported a direct interaction between mutated nAChR residues and alpha-conotoxin residues 6, 7, and 10. Taken together, these data provide evidence that the beta subunit contributes to alpha-conotoxin binding and selectivity and demonstrate that a small cleft leading to the agonist binding site is targeted by alpha-conotoxins to block the nAChR.

Selective modulation of neuronal nicotinic acetylcholine receptor channel subunits by G(o)-protein subunits

protein modulation of neuronal nicotinic acetylcholine receptor ( nAChR) channels in rat intrinsic cardiac ganglia was examined using dialyzed whole-cell and excised membrane patch-recording configurations. Cell dialysis with GTP gamma S increased the agonist affinity of nAChRs, resulting in a potentiation of nicotine-evoked whole-cell currents at low concentrations. ACh- and nicotine-evoked current amplitudes were increased approximately twofold in the presence of GTP gamma S. In inside-out membrane patches, the open probability (NPo) of nAChR-mediated unitary currents was reversibly increased fourfold after bath application of 0.2mM GTP gamma S relative to control but was unchanged in the presence of GDP gamma S. The modulation of nAChR-mediated whole- cell currents was agonist specific; currents evoked by the cholinergic agonists ACh, nicotine, and 1,1-dimethyl-4-phenylpiperazinium iodide, but not cytisine or choline, were potentiated in the presence of GTP gamma S. The direct interaction between G-protein subunits and nAChRs was examined by bath application of either G(o)alpha or G beta gamma subunits to inside-out membrane patches and in glutathione S-transferase pull-down and coimmunoprecipitation experiments. Bath application of 50 nM G beta gamma increased the open probability of ACh- activated single-channel currents fivefold, whereas G(o)alpha( 50 nM) produced no significant increase in NPo. Neuronal nAChR subunits alpha 3-alpha 5 and alpha 2 exhibited a positive interaction with G(o)alpha and G beta gamma, whereas beta 4 and alpha 7 failed to interact with either of the G-protein subunits. These results provide evidence for a direct interaction between nAChR and G-protein subunits, underlying the increased open probability of ACh-activated single-channel currents and potentiation of nAChR-mediated whole-cell currents in parasympathetic neurons of rat intrinsic cardiac ganglia.

Intravenous anaesthetics inhibit nicotinic acetylcholine receptor-mediated currents and Ca2+ transients in rat intracardiac ganglion neurons

1 The effects of intravenous (i.v.) anaesthetics on nicotinic acetylcholine receptor (nAChR)-induced transients in intracellular free Ca2+ concentration ([Ca2+](i)) and membrane currents were investigated in neonatal rat intracardiac neurons. 2 In fura-2-loaded neurons, nAChR activation evoked a transient increase in [Ca2+](i), which was inhibited reversibly and selectively by clinically relevant concentrations of thiopental. The half-maximal concentration for thiopental inhibition of nAChR-induced [Ca2+](i) transients was 28 muM, close to the estimated clinical EC50 (clinically relevant (half-maximal) effective concentration) of thiopental. 3 In fura-2-loaded neurons, voltage clamped at -60mV to eliminate any contribution of voltage-gated Ca2+ channels, thiopental (25 muM) simultaneously inhibited nAChR-induced increases in [Ca2+](i) and peak current amplitudes. Thiopental inhibited nAChR-induced peak current amplitudes in dialysed whole-cell recordings by - 40% at - 120, -80 and -40 mV holding potential, indicating that the inhibition is voltage independent. 4 The barbiturate, pentobarbital and the dissociative anaesthetic, ketamine, used at clinical EC50 were also shown to inhibit nAChR-induced increases in [Ca2+](i) by similar to40%. 5 Thiopental (25 muM) did not inhibit caffeine-, muscarine- or ATP-evoked increases in [Ca2+](i), indicating that inhibition of Ca2+ release from internal stores via either ryanodine receptor or inositol-1,4,5-trisphosphate receptor channels is unlikely. 6 Depolarization-activated Ca2+ channel currents were unaffected in the presence of thiopental (25 muM), pentobarbital (50 muM) and ketamine (10 muM). 7 In conclusion, i.v. anaesthetics inhibit nAChR-induced currents and [Ca2+](i) transients in intracardiac neurons by binding to nAChRs and thereby may contribute to changes in heart rate and cardiac output under clinical conditions.

Toxin insights into nicotinic acetylcholine receptors

Venomous species have evolved cocktails of bioactive peptides to facilitate prey capture. Given their often exquisite potency and target selectivity, venom peptides provide unique biochemical tools for probing the function of membrane proteins at the molecular level. in the field of the nicotinic acetylcholine receptors (nAChRs), the subtype specific snake alpha-neurotoxins and cone snail alpha-conotoxins have been widely used to probe receptor structure and function in native tissues and recombinant systems. However, only recently has it been possible to generate an accurate molecular view of these nAChR-toxin interactions. Crystal structures of AChBP, a homologue of the nAChR ligand binding domain, have now been solved in complex with alpha-cobratoxin, alpha-conotoxin PnIA and alpha-conotoxin Iml. The orientation of all three toxins in the ACh binding site confirms many of the predictions obtained from mutagenesis and docking simulations on homology models of mammalian nAChR. The precise understanding of the molecular determinants of these complexes is expected to contribute to the development of more selective nAChR modulators. In this commentary, we review the structural data on nAChR-toxin interactions and discuss their implications for the design of novel ligands acting at the nAChR. (c) 2006 Elsevier Inc. All rights reserved.

Calcium channels controlling acetylcholine release from preganglionic nerve terminals in rat autonomic ganglia

Little is known about the nature of the calcium channels controlling neurotransmitter release from preganglionic parasympathetic nerve fibres. In the present study, the effects of selective calcium channel antagonists and amiloride were investigated on ganglionic neurotransmission. Conventional intracellular recording and focal extracellular recording techniques were used in rat submandibular and pelvic ganglia, respectively. Excitatory postsynaptic potentials and excitatory postsynaptic currents preceded by nerve terminal impulses were recorded as a measure of acetylcholine release from parasympathetic and sympathetic preganglionic fibres following nerve stimulation. The calcium channel antagonists omega-conotoxin GVIA (N type), nifedipine and nimodipine (L type), omega-conotoxin MVIIC and omega-agatoxin IVA (P/Q type), and Ni2+ (R type) had no functional inhibitory effects on synaptic transmission in both submandibular and pelvic ganglia. The potassium-sparing diuretic, amiloride, and its analogue, dimethyl amiloride, produced a reversible and concentration-dependent inhibition of excitatory postsynaptic potential amplitude in the rat submandibular ganglion. The amplitude and frequency of spontaneous excitatory postsynaptic potentials and the sensitivity of the postsynaptic membrane to acetylcholine were unaffected by amiloride. In the rat pelvic ganglion, amiloride produced a concentration-dependent inhibition of excitatory postsynaptic currents without causing any detectable effects on the amplitude or configuration of the nerve terminal impulse. These results indicate that neurotransmitter release from preganglionic parasympathetic and sympathetic nerve terminals is resistant to inhibition by specific calcium channel antagonists of N-, L-, P/Q- and R-type calcium channels. Amiloride acts presynaptically to inhibit evoked transmitter release, but does not prevent action potential propagation in the nerve terminals, suggesting that amiloride may block the pharmacologically distinct calcium channel type(s) on rat preganglionic nerve terminals. (C) 1999 IBRO. Published by Elsevier Science Ltd.

Research update:alpha7 nicotinic acetylcholine receptor mechanisms in Alzheimer's disease

Aberrant amyloid-ß peptide (Aß) accumulation along with altered expression and function of nicotinic acetylcholine receptors (nAChRs) stand prominently in the etiology of Alzheimer's disease (AD). Since the discovery that Aß is bound to a7 nAChRs under many experimental settings, including post-mortem AD brain, much effort has been expended to understand the implications of this interaction in the disease milieu. This research update will review the current literature on the a7 nAChR-Aß interaction in vitro and in vivo, the functional consequences of this interaction from sub-cellular to cognitive levels, and discuss the implications these relationships might have for AD therapies.

Nicotinic acetylcholine receptor interaction with β-amyloid:molecular, cellular, and physiological consequences

Elevated amyloid-β peptide (Aβ) and loss of nicotinic acetylcholine receptors (nAChRs) stand prominently in the etiology of Alzheimer's disease (AD). Since the discovery of an Aβ - nAChR interaction, much effort has been expended to characterize the consequences of high versus low concentrations of Aβ on nAChRs. This review will discuss current knowledge on the subject at the molecular, cellular, and physiological levels with particular emphasis on understanding how Aβ - nAChR interaction may contribute to normal physiological processes as well as the etiology of AD. ©2010 Bentham Science Publishers Ltd.

Varenicline decreases alcohol consumption in heavy-drinking smokers

Rationale Emerging evidence suggests that the α4β2 form of the nicotinic acetylcholine receptor (nAChR) modulates the rewarding effects of alcohol. The nAChR α4β2 subunit partial agonist varenicline (Chantix™), which is approved by the Food and Drug Administration for smoking cessation, also decreases ethanol consumption in rodents (Steensland et al., Proc Natl Acad Sci U S A 104:12518–12523, 2007) and in human laboratory and open-label studies (Fucito et al., Psychopharmacology (Berl) 215:655–663, 2011; McKee et al., Biol Psychiatry 66:185–190 2009). Objectives We present a randomized, double-blind, 16-week study in heavy-drinking smokers (n = 64 randomized to treatment) who were seeking treatment for their smoking. The study was designed to determine the effects of varenicline on alcohol craving and consumption. Outcome measures included number of alcoholic drinks per week, cigarettes per week, amount of alcohol craving per week, cumulative cigarettes and alcoholic drinks consumed during the treatment period, number of abstinent days, and weekly percentage of positive ethyl glucuronide and cotinine screens. Results Varenicline significantly decreases alcohol consumption (χ 2 = 35.32, p < 0.0001) in smokers. Although varenicline has previously been associated with suicidality and depression, side effects were low in this study and declined over time in the varenicline treatment group. Conclusions Varenicline can produce a sustained decrease in alcohol consumption in individuals who also smoke. Further studies are warranted to assess varenicline efficacy in treatment-seeking alcohol abusers who do not smoke and to ascertain the relationship between varenicline effects on smoking and drinking.

The relationship between Bayesian motor unit number estimation and histological measurements of motor neurons in wild-type and SOD1 G93A mice

Objective: To assess the relationship between Bayesian MUNE and histological motor neuron counts in wild-type mice and in an animal model of ALS. Methods: We performed Bayesian MUNE paired with histological counts of motor neurons in the lumbar spinal cord of wild-type mice and transgenic SOD1 G93A mice that show progressive weakness over time. We evaluated the number of acetylcholine endplates that were innervated by a presynaptic nerve. Results: In wild-type mice, the motor unit number in the gastrocnemius muscle estimated by Bayesian MUNE was approximately half the number of motor neurons in the region of the spinal cord that contains the cell bodies of the motor neurons supplying the hindlimb crural flexor muscles. In SOD1 G93A mice, motor neuron numbers declined over time. This was associated with motor endplate denervation at the end-stage of disease. Conclusion: The number of motor neurons in the spinal cord of wild-type mice is proportional to the number of motor units estimated by Bayesian MUNE. In SOD1 G93A mice, there is a lower number of estimated motor units compared to the number of spinal cord motor neurons at the end-stage of disease, and this is associated with disruption of the neuromuscular junction. Significance: Our finding that the Bayesian MUNE method gives estimates of motor unit numbers that are proportional to the numbers of motor neurons in the spinal cord supports the clinical use of Bayesian MUNE in monitoring motor unit loss in ALS patients. © 2012 International Federation of Clinical Neurophysiology.

Comparison of diagnostic tests in myasthenia gravis

Results of 3 tests, intravenous edrophonium chloride, EMG, and acetylcholine receptor antibody testing, were compared in patients with generalised and ocular myasthenia gravis. None of the 3 tests was positive in any patient with a diagnosis other than myasthenia. However, equivocal results were obtained with edrophonium and EMG testing in some patients with myasthenia gravis and in patients with other diseases. It is concluded from this survey that antibody and edrophonium testing were equally efficient in detecting generalised myasthenia gravis. Edrophonium testing was superior in ocular myasthenia gravis. Although the yields from each test varied, all 3 tests were needed for the evaluation of some myasthenia gravis patients as each test may provide additional information.

Chemotherapy-induced nausea and vomiting : a review to inform clinical practice

Chemotherapy-induced nausea and vomiting (CINV) is a common sideeffect of cytotoxic treatment and despite the widespread use of anti-emetic medication, it continues to affect a significant proportion of patients with up to 23% and 73% of chemotherapy patients still experiencing vomiting and nausea symptoms, respectively. This is of particular concern in oncology patients as nausea and vomiting may result in malnutrition, decreased quality of life and in extreme cases, treatment stoppage. Therefore, the primary aim of this paper was to inform clinicians on the current literature regarding CINV including its effect on the patient, its pathophysiology, and current treatment options. In addition, this review will also discuss the usage of dietetic interventions as well as less utilised, novel interventions such as oral ginger extracts in the treatment of CINV. In order to address these issues, a systematic literature search was conducted using Pubmed, CINAHL, MEDLINE, Embase, and Health Source (Nursing/Academic Edition). A key finding of this review was that common dietary strategies (e.g. eating slowly, avoiding fatty foods) seem to be solely based on professional opinion as no clinical trials investigating these strategies were identified. In contrast, ginger extracts were found to possess several viable mechanisms that interact with CINV progression including 5-HT3, Substance P and acetylcholine receptor antagonism; anti-inflammatory and antioxidant properties; and gastrointestinal motility and gastric emptying modulation. In conclusion, research investigating dietetic interventions in the management of CINV is sparse and requires further investigation while novel intervention such as ginger, possess multiple mechanisms that may benefit CINV management. This review will discuss the prevalence and significance of CINV, dietetic and novel treatment options, and provide implications for clinical practise and future research.

Ginger - mechanism of action in chemotherapy-induced nausea and vomiting: A review

Despite advances in anti-emetic therapy, chemotherapy-induced nausea and vomiting (CINV) still poses a significant burden to patients undergoing chemotherapy. Nausea, in particular, is still highly prevalent in this population. Ginger has been traditionally used as a folk remedy for gastrointestinal complaints and has been suggested as a viable adjuvant treatment for nausea and vomiting in the cancer context. Substantial research has revealed ginger to possess properties that could exert multiple beneficial effects on chemotherapy patients who experience nausea and vomiting. Bioactive compounds within the rhizome of ginger, particularly the gingerol and shogaol class of compounds, interact with several pathways that are directly implicated in CINV in addition to pathways that could play secondary roles by exacerbating symptoms. These properties include 5-HT3, substance P and acetylcholine receptor antagonism; anti-inflammatory properties; and modulation of cellular redox signalling, vasopressin release, gastrointestinal motility, and gastric emptying rate. This review outlines these proposed mechanisms by discussing the results of clinical, in vitro and animal studies both within the chemotherapy context and in other relevant fields. The evidence presented in this review indicates that ginger possesses multiple properties that could be beneficial in reducing chemotherapy-induced nausea and vomiting.

The role of the glucocorticoids in developing resilience to stress and addiction

There is emerging evidence that individuals have the capacity to learn to be resilient by developing protective mechanisms that prevent them from the maladaptive effects of stress that can contribute to addiction.The emerging field of the neuroscience of resilience is beginning to uncover the circuits and molecules that protect against stress-related neuropsychiatric diseases, such as addiction. Glucocorticoids (GCs) are important regulators of basal and stress-related homeostasis in all higher organisms and influence a wide array of genes in almost every organ and tissue. GCs, therefore, are ideally situated to either promote or prevent adaptation to stress. In this review, we will focus on the role of GCs in the hypothalamic-pituitary adrenocortical axis and extra-hypothalamic regions in regulating basal and chronic stress responses. GCs interact with a large number of neurotransmitter and neuropeptide systems that are associated with the development of addiction. Additionally, the review will focus on the orexinergic and cholinergic pathways and highlight their role in stress and addiction. GCs play a key role in promoting the development of resilience or susceptibility and represent important pharmacotherapeutic targets that can reduce the impact of a maladapted stress system for the treatment of stress-induced addiction.