Physostigmine and acetylcholine differentially activate nicotinic receptor subpopulations in Locusta migratoria neurons

The effects of acetylcholine (ACh) and physostigmine (PHY) on thoracic ganglion neurons of Locusta migratoria were investigated using whole-cell and cell-attached voltage clamp. ACh activated whole-cell currents with variable amplitudes, time course and ion channel block between cells, suggesting differential expression of nicotinic acetylcholine receptor (nAChR) subtypes. This was supported by selective block of the peak of the currents by the α7-specific α-conotoxin ImI. PHY at 100 μM evoked smaller whole-cell currents with variable amplitudes and marginal desensitization. The PHY/ACh amplitude ratio varied between cells, and was positively related to the time constant of decay of the ACh response. EC₅₀ values for the peak amplitude of the ACh- and PHY-induced currents were 50 μM and 3 μM, respectively. Both agonists activated nAChR, indicated by equal voltage-dependence and reversal potentials and the same pharmacological properties of ACh and PHY responses. In addition, PHY and ACh induced ion channel block. Co-application and cross-desensitization experiments showed that ACh and PHY activate the same nAChR subpopulations. Both agonists activated nicotinic single channels with three conductance levels, which were equal for ACh and PHY, indicating activation of the same nAChR subtypes by both agonists. However, for all levels PHY displayed a lower open probability than ACh. Taken together, different whole-cell responses appear to originate from differential activation, desensitization and ion channel block by ACh and PHY of distinct nAChR populations.     

The alpha9/alpha10-containing nicotinic ACh receptor is directly modulated by opioid peptides,endomorphin-1, and dynorphin B,proposed efferent cotransmitters in the inner ear

Opioid peptides have been detected in the auditory and vestibular efferent neurons where they colocalize with the major neurotransmitter, acetylcholine. We investigated the function of opioids to modulate neurotransmission mediated by hair cell's alpha9/alpha10-containing nicotinic acetylcholine receptors (alpha9/alpha10nAChRs). The endogenous opioid peptides, endomorphin-1 (mu agonist) and dynorphin B (kappa agonist), but not a delta agonist [D-Pen2,D-Pen-5]enkephalin, inhibited the acetylcholine-evoked currents in frog saccular hair cells and rat inner hair cells. This inhibition was noncompetitive, voltage-independent, and was accompanied by an acceleration of the rate of current decay. Selective mu- and kappa-opioid receptor antagonists did not block the inhibition, although partial reduction by naloxone was observed. All opioid antagonists tested also reduced the acetylcholine response. Endomorphin-1 and dynorphin B inhibited the acetylcholine-evoked currents in alpha9/alpha10-expressing Xenopus oocytes. Because oocytes lack opioid receptors, it provides strong evidence for the direct interaction of opioid peptides with alpha9/alpha10nAChR. CONCLUSION: alpha9/alpha10nAChR is a target for modulation by endomorphin-1 and dynorphin B, efferent cotransmitters in the inner ear.     

Photo-induced covalent attachment of agonists as a tool to study allosteric mechanisms of nicotinic acetylcholine receptors

Muscular and neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels and contain either two or five binding sites for acetylcholine (ACh). Binding of ACh molecules on the nAChR will trigger the fast opening of the channel and subsequent slow desensitization process. Neuronal alpha7 nicotinic receptors are made up of five identical subunits and possess five binding sites for ACh; this raises the question of how many sites must be occupied before channel opening. However, the effect of each ligand binding on gating is difficult to assess because of the reversible aspect of ligand binding at each site. One solution is to photochemically tether agonists to their binding sites. Such methodology has been applied elegantly and successfully on the homotetrameric cyclic-nucleotide-gated channels to evaluate the functional effects of each ligand binding on gating (Ruiz and Karpen, 1997). We therefore decided to develop a similar approach on Torpedo and neuronal alpha7 nAChRs with the photoactivatable agonist AC5 to investigate the effect of binding site occupancy on allosteric transitions of the receptor. In the dark, AC5 (see structure below) evokes robust currents on oocytes expressing Torpedo nAChR, displaying maximal amplitude comparable to ACh, with EC50 = 1.2 microM (Mourot et al., 2005). When the voltage-clamp oocyte was exposed to UV light in the presence of 30 microM AC5 for 50 s, there was a prolonged activation of the Torpedo nAChR, not reversible by washing, but inhibited by the noncompetitive blockers tetracaine and proadifen (see structure below). Both UV light and AC5 are required for this effect. However, further studies are required to determine whether the gradual decrease of the inward current reflects a slow desensitization process. AC5 is thus a potent photoactivatable agonist of the nAChR, which is able, upon UV irradiation, to incorporate covalently into the ACh-binding sites and to prolong activation of the nAChR. By extending this methodology to patch-clamp experiments, we will be able to incorporate one or several AC5s covalently into the muscular and neuronal nAChR at the single-channel level. Such study will help us understand the observed cooperative effect of gating and will contribute decisively to the controversial concerted vs sequential models for nAChR allosteric transitions.     

Modulation of alpha2beta4 neuronal nicotinic acetylcholine receptors by zinc

A study was made of the modulation of nicotinic acetylcholine receptors by the divalent cation zinc. Rat neuronal nicotinic receptors (alpha2beta4) were expressed in Xenopus oocytes and membrane currents evoked by acetylcholine (ACh currents) were recorded using a two microelectrode voltage clamp. In non-injected oocytes, or in oocytes expressing alpha2beta4 receptors, Zn2+ by itself (1 microM-4 mM) generated only very small membrane currents. In contrast, in oocytes expressing alpha2beta4 receptors, Zn2+ greatly and reversibly increased the ACh current, without affecting considerably its time course. The ACh current potentiation by Zn2+ was weakly dependent on the membrane potential (2.33+/-0.10 times the control current at -100 mV vs 2.04+/-0.06 at -60 mV, suggesting that Zn2+ interacts with the receptor in the vestibule of the ion channel or at an external domain of the protein. The inward rectification of control and Zn2+-potentiated ACh-currents was similar. We conclude that Zn2+ positively and reversibly modulates neuronal nicotinic receptors in a practically voltage-independent manner and without affecting their rate of desensitization. These results will help to understand better the roles played by Zn2+ in brain functions.     

Mutated Nicotinic Receptors Responsible for Autosomal Dominant Nocturnal Frontal Lobe Epilepsy are More Sensitive to Carbamazepine

PURPOSE: The recent linkage between a genetically transmissible form of epilepsy (ADNFLE) and mutations within the alpha4 subunit, one component of the major brain neuronal nicotinic acetylcholine receptor (nAChR), raises the question of the role of this receptor in epileptogenesis. Although acting by different mechanisms, the two genetic alterations so far identified both render the nAChR less efficient. In view of the high sensitivity of ADNFLE to carbamazepine (CBZ), we studied the effects of this drug and of valproate (VPA) on the human alpha4beta2 nAChR and its mutations. METHODS: The alpha4beta2 nAChRs from control and mutant alpha4 subunits were reconstituted in Xenopus oocytes and investigated by using a dual-electrode voltage clamp technique. Acetylcholine (ACh)-evoked currents recorded in the absence or presence of antiepileptic drugs (AEDs) were studied to analyze the mode of action of these compounds. RESULTS: ACh-evoked currents at the human alpha4beta2 nAChR were readily and reversibly inhibited by approximately 100 microM CBZ. This compound was found to be a noncompetitive inhibitor of the nAChR, which probably acts by entering the channel and causing a blockade by steric hindrance. Dose-response inhibition curves determined on the control receptor and on ADNFLE-mutant receptors showed a greater sensitivity of the mutants to CBZ, with median inhibitory concentrations (IC50s) in the range of the antiepileptic plasma levels of CBZ. In contrast, VPA had nearly no effect on control and mutant nAChRs. CONCLUSIONS: CBZ inhibits the neuronal alpha4beta2 nAChRs at pharmacologic concentrations, with ADNFLE mutants displaying about threefold higher sensitivity to this compound. The increased sensitivity of these mutant receptors supports the hypothesis that the antiepileptic activity of CBZ can, at least to some extent, be attributed to the nAChR inhibition.     

Neural nicotinic acetylcholine responses in sensory neurons from postnatal rat

The whole-cell configuration of the patch-clamp technique was used to study nicotinic acetylcholine (ACh) responses in freshly dissociated dorsal root ganglion (DRG) cells from postnatal rat. At negative holding potentials with physiological solutions in the bath and the pipette, ACh (20 microM), nicotine (5 microM) or DMPP (20 microM) activated inward currents in 51% of the cells. Average current density was higher in 1-month-old compared to newborn animals. Nicotinic agonist-induced currents were unaffected by atropine (10 microM) but reversibly blocked by hexamethonium (20 microM). Although labeling with fluorescent alpha-bungarotoxin (BGT) demonstrated the presence of toxin binding sites on DRG cells, DMPP-induced inward currents were unaffected by micromolar BGT. Neuronal bungarotoxin (100 nM), in contrast, led to a largely irreversible block of the nicotinic responses. These results show that postnatal DRG cells express functional nicotinic acetylcholine receptors (nAChR) of a neuronal type.     

Blockage of nicotinic acetylcholine receptors by 5-hydroxytryptamine

The action of 5-hydroxytryptamine (5HT) on nicotinic acetylcholine receptor (nAChR) channels was investigated in mouse myotubes, human cloned TE671/RD cells, and Xenopus laevis oocytes. The decay of the ACh-activated whole-cell currents was reversibly accelerated in the presence of 5HT (10^?5 to 10^?3 M), in a dose-dependent manner. 5HT also reduced the size and accelerated the decay of currents elicited by ACh in Xenopus oocytes injected with mRNA extracted from C2 myotubes or Torpedo electroplaques, or oocytes injected with cloned mouse muscle AChR subunit mRNAs. The effect of 5HT was promptly reversed after washout, or by depolarizing the oocyte beyond ?10 mV. In patch-clamp recordings from myotubes, bath-application of 5HT did not exert an indirect influence on the ACh-activated channels within the patch membrane. In contrast, when the patch membrane was exposed to 5HT (10^?6 M), ACh unit responses appeared as bursts of short pulses. It is concluded that the regulation of ACh responses by 5HT results from a fast noncompetitive blocking action of nAChR-channels. These results show that ligand-gated channels, activated by their specific neurotransmitter, may be regulated by a different neurotransmitter through a direct action on the receptor molecule. © 1993 Wiley-Liss, Inc.     

Mechanism of block of nicotinic acetylcholine receptor channels by purified IgG from seropositive patients with myasthenia gravis

OBJECTIVE: To clarify the mechanism of block of nicotinic receptor channels by myasthenic antibodies. BACKGROUND: Nicotinic acetylcholine receptor (nAChR) channel currents are functionally blocked by purified immunoglobulin G (IgG) of patients with myasthenia gravis (MG). METHODS: The molecular mechanism of block of IgG fractions containing antibodies to nAChR channels was tested with the patch-clamp technique in combination with a system for ultrafast solution exchange. For the experiments, outside-out patches from cultured mouse myotubes that express embryonic-type nAChR channels at their surface were used. RESULTS: Incubation of outside-out patches with purified IgG from four myasthenic patients blocked nAChR channel currents activated by the application of 1.0 mM ACh reversibly. The peak current amplitude and the time course of block of nAChR channels decreased with increasing concentrations of IgG. The block became at least partly irreversible if incubation time of outside-out patches exceeded 2 minutes. For the block of nAChR channel currents with a-bungarotoxin, a similar mechanism of block was found. CONCLUSIONS: The reversibility of functional block of nAChR channel currents by myasthenic IgG depended strongly on the incubation time of the receptors with antibodies. Interaction of myasthenic antibodies with nicotinic receptors may proceed in several stages from a low-affinity reversible to a high-affinity irreversible binding.     

Block of recombinant nicotinic receptor channels by IgG from myasthenic patients

In myasthenia gravis (MG), specific antibodies directed against nicotinic acetylcholine receptor (nAChR) channels are produced. Recently, a distinct blockade of native embryonic-type nAChR channels by purified IgG from MG patients has been demonstrated. In the present study, we investigated the interaction of myasthenic IgG with recombinant embryonic- or adult-type nAChR channels expressed on human embryonic kidney (HEK) 293 cells. For the experiments, the patch clamp technique was applied in combination with an ultra-fast application system for agonists. Repetitive 20-ms pulses of 1 mM acetylcholine (ACh) were applied with or without purified IgG added to the perfusion system. The purified IgG from MG patients blocked currents through both, embryonic- and adult-type nAChR channels to a similar extent. A block by myasthenic IgG of adult-type nAChR channels, which are present exclusively at the postsynaptic membrane of the neuromuscular synapse, was shown to occur. The reversible pharmacological blockade of adult-type nAChR channels by myasthenic IgG may be one factor responsible for rapid fluctuations of weakness or rapid recovery of muscle strength after plasmapheresis in MG.     

Arachidonic acid induces a long-lasting facilitation of hippocampal synaptic transmission by modulating PKC activity and nicotinic ACh receptors.

The present study was conducted to understand the effect of arachidonic acid on nicotinic acetylcholine (ACh) receptor-mediated synaptic plasticity. Arachidonic acid persistently (>/=1 h) potentiated currents through neuronal nicotinic ACh receptors (alpha7 and alpha4beta2) expressed in Xenopus oocytes, and the effect was blocked by the selective protein kinase C (PKC) inhibitors, such as GF109203X, PKCI, and co-expressed active PKC inhibitor peptide. This free fatty acid markedly increased nicotine-sensitive glutamate release from hippocampal slices and enhanced the rate of nicotine-sensitive miniature excitatory postsynaptic currents without affecting the amplitude in cultured hippocampal CA1 neurons under the influence of PKC. Furthermore, arachidonic acid induced a long-lasting (>/=3 h) facilitation of hippocampal CA1 synaptic transmission in slices, and the effect was blocked by nicotinic ACh receptor antagonists, alpha-bungarotoxin and mecamylamine. The facilitation, whereas independent of N-methyl-D-aspartate (NMDA) receptors, shares a common mechanism with long-term potentiation (LTP) induced by tetanic stimulation. The results of the present study thus suggest that arachidonic acid sustains enhanced activity of nicotinic ACh receptors by interacting with a PKC pathway, thereby increasing glutamate release from presynaptic terminals, and then leading to an 'LTP-like' facilitation of hippocampal synaptic transmission.     

Presynaptic nicotinic acetylcholine receptors as a functional target of nefiracetam in inducing a long-lasting facilitation of hippocampal neurotransmission

Nefiracetam (1-10 microM), a nootropic (or cognition-enhancing) agent, persistently potentiated currents through Torpedo acetylcholine (ACh) receptors expressed in Xenopus oocytes as a result of interacting with a protein kinase C pathway and the ensuing protein kinase C phosphorylation of the receptors. A similar effect was found in neuronal nicotinic ACh receptors (alpha4beta2 and alpha7). In contrast, the other nootropic agents such as piracetam and aniracetam had no potentiating action on the receptors. A sustained enhancement in the activity of nicotinic ACh receptors induced by nefiracetam caused a marked increase in the glutamate release, leading to a long-term potentiation-like facilitation of hippocampal synaptic transmissions. One of the consistent neuropathologic features of the Alzheimer brain is a loss of nicotinic ACh receptors. This fact, together with the results of our study, raises the possibility that the loss of nicotinic ACh receptors may be a key factor in the decline of cognitive function observed in Alzheimer disease and that agents targeting neuronal nicotinic ACh receptors like nefiracetam could, therefore, be of great therapeutic importance.     

MK-801 inhibition of nicotinic acetylcholine receptor channels.

MK-801 is a potent inhibitor of the NMDA subtype of glutamate receptors. Single-channel and macroscopic currents indicate that MK-801 also inhibits nicotinic acetylcholine receptors (nAChRs). MK-801 does not significantly increase desensitization of the nAChRs or compete for the ACh binding site. Although there is a slight inhibition of the closed nAChR, the main action of MK-801 is to enter and block the open channel. The voltage dependence for block is consistent with a single binding site within the channel that is 50% of the way through the membrane field. The IC50 for block is 3 microM at -70 mV for currents induced by 0.5 microM ACh. The data from both single-channel and macroscopic currents can be used to estimate a Kd (0) of 7 microM, which is about 40 times higher than the Kd (0) for MK-801 binding to the NMDA receptor. The relative potency of tricyclic compounds like MK-801 for various neurotransmitter systems points out that the pharmacologic action of these drugs could involve complicated interactions in vivo.     

Nefiracetam facilitates hippocampal neurotransmission by a mechanism independent of the piracetam and aniracetam action

Nefiracetam, a nootropic (cognition-enhancing) agent, facilitated neurotransmission in the dentate gyrus of rat hippocampal slices in a dose-dependent manner at concentrations ranged from 1 nM to 1 μM, being evident at 60-min washing-out of the drug. The facilitatory action was blocked by the nicotinic acetylcholine (ACh) receptor antagonists, -bungarotoxin and mecamylamine. A similar facilitation was induced by the other nootropic agents, piracetam and aniracetam, but the facilitation was not inhibited by nicotinic ACh receptor antagonists and it did not occlude the potentiation induced by nefiracetam. In the Xenopus oocyte expression systems, nefiracetam potentiated currents through a variety of neuronal nicotinic ACh receptors (3β2, 3β4, 4β2, 4β4, and 7) to a different extent. In contrast, neither piracetam nor aniracetam had any potentiating action on 7 receptor currents. While aniracetam delayed the decay time of currents through the -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, GluR1, -2, -3, expressed in oocytes, nefiracetam or piracetam had no effect on the currents. Nefiracetam, thus, appears to facilitate hippocampal neurotransmission by functionally targeting nicotinic ACh receptors, independently of the action of piracetam and aniracetam.     

Caenorhabditis elegans nicotinic acetylcholine receptors are required for nociception

Polymodal nociceptors sense and integrate information on injurious mechanical, thermal, and chemical stimuli. Chemical signals either activate nociceptors or modulate their responses to other stimuli. One chemical known to activate or modulate responses of nociceptors is acetylcholine (ACh). Across evolution nociceptors express subunits of the nicotinic acetylcholine receptor (nAChR) family, a family of ACh-gated ion channels. The roles of ACh and nAChRs in nociceptor function are, however, poorly understood. Caenorhabditis elegans polymodal nociceptors, PVD, express nAChR subunits on their sensory arbor. Here we show that mutations reducing ACh synthesis and mutations in nAChR subunits lead to defects in PVD function and morphology. A likely cause for these defects is a reduction in cytosolic calcium measured in ACh and nAChR mutants. Indeed, overexpression of a calcium pump in PVD mimics defects in PVD function and morphology found in nAChR mutants. Our results demonstrate, for the first time, a central role for nAChRs and ACh in nociceptor function and suggest that calcium permeating via nAChRs facilitates activity of several signaling pathways within this neuron.     

A new Chrna4 mutation with low penetrance in nocturnal frontal lobe epilepsy

PURPOSE: To identify and characterize the mutation(s) causing nocturnal frontal lobe epilepsy in a German extended family. METHODS: Neuronal nicotinic acetylcholine receptor (nAChR) subunit genes were screened by direct sequencing. Once a CHRNA4 mutation was identified, its biophysical and pharmacologic properties were characterized by expression experiments in Xenopus oocytes. RESULTS: We report a new CHRNA4 mutation, causing a alpha4-T265I amino acid exchange at the extracellular end of the second transmembrane domain (TM). Functional studies of alpha4-T265I revealed an increased ACh sensitivity of the mutated receptors. alpha4-T265I is associated with an unusual low penetrance of the epilepsy phenotype. Sequencing of the TM1-TM3 parts of the 1 known nAChR subunits did not support a two-locus model involving a second nAChR sequence variation. CONCLUSIONS: nAChR mutations found in familial epilepsy are not always associated with an autosomal dominant mode of inheritance. alpha4-T265I is the first nAChR allele showing a markedly reduced penetrance consistent with a major gene effect. The low penetrance of the mutation is probably caused by unknown genetic or environmental factors or both.     

Identifying the Binding Site of Novel Methyllycaconitine (MLA) Analogs at α4β2 Nicotinic Acetylcholine Receptors

Neuronal nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels that mediate fast synaptic transmission. Methyllycaconitine (MLA) is a selective and potent antagonist of the α7 nAChR, and its anthranilate ester side-chain is important for its activity. Here we report the influence of structure on nAChR inhibition for a series of novel MLA analogs, incorporating either an alcohol or anthranilate ester side-chain to an azabicyclic or azatricyclic core against rat α7, α4β2, and α3β4 nAChRs expressed in Xenopus oocytes. The analogs inhibited ACh (EC(50)) within an IC(50) range of 2.3-26.6 μM. Most displayed noncompetitive antagonism, but the anthranilate ester analogs exerted competitive behavior at the α7 nAChR. At α4β2 nAChRs, inhibition by the azabicyclic alcohol was voltage-dependent suggesting channel block. The channel-lining residues of α4 subunits were mutated to cysteine and the effect of azabicyclic alcohol was evaluated by competition with methanethiosulfonate ethylammonium (MTSEA) and a thiol-reactive probe in the open, closed, and desensitized states of α4β2 nAChRs. The azabicyclic alcohol was found to compete with MTSEA between residues 6' and 13' in a state-dependent manner, but the reactive probe only bonded with 13' in the open state. The data suggest that the 13' position is the dominant binding site. Ligand docking of the azabicyclic alcohol into a (α4)(3)(β2)(2) homology model of the closed channel showed that the ligand can be accommodated at this location. Thus our data reveal distinct pharmacological differences between different nAChR subtypes and also identify a specific binding site for a noncompetitive channel blocker.     

Short-term depression and long-term enhancement of ACh-gated channel currents induced by linoleic and linolenic acid

The effects of cis-unsaturated free fatty acids such as linoleic and linolenic acid on ACh-evoked currents were examined using normal and mutant nicotinic acetylcholine (ACh) receptors lacking protein kinase C (PKC) phosphorylation sites on the α and δ subunits expressed in Xenopus oocytes. These free fatty acids reduced ACh-gated channel currents during treatment and to a greater extent in Ca²⁺-free extracellular solution. After treatment, the currents were enhanced as the drug was washed out, but this effect was not observed in the absence of extracellular Ca²⁺. Linolenic acid was more potent of the current enhancement (300% of the control) than linoleic acid (190% of the control). The current enhancement induced by these free fatty acids was inhibited by the selective PKC inhibitor, GF109203X, while the current depression was not affected. Furthermore, these lipids decreased ACh-evoked currents in mutant ACh receptors to the same extent as in normal ACh receptors, but never enhanced the currents. These results indicate that linoleic and linolenic acid have biphasic actions on ACh receptor currents; a short-term depression and a long-term enhancement. The short-term depression may be due to an interaction with the ACh receptor channels, presumably at Ca²⁺ binding sites. The long-lasting enhancement appears to result from Ca²⁺-dependent PKC activation followed by PKC phosphorylation of the ACh receptors. © 1997 Elsevier Science B.V. All rights reserved.     

The anti-dementia drug nefiracetam facilitates hippocampal synaptic transmission by functionally targeting presynaptic nicotinic ACh receptors

Nefiracetam, a pyrrolidone derivative developed as an anti-dementia drug, persistently potentiated currents through neuronal nicotinic acetylcholine (ACh) receptors (alpha7, alpha4beta2) expressed in Xenopus oocytes, and the potentiation was blocked by either the selective protein kinase C (PKC) inhibitors, GF109203X and staurosporine, or co-expressed active PKC inhibitor peptide. In primary cultures of rat hippocampal neurons, nefiracetam increased the rate of nicotine-sensitive miniature excitatory postsynaptic currents, without affecting the amplitude, and the increase was inhibited by GF109203X. In addition, the drug caused a marked increase in the glutamate release from electrically stimulated guinea pig hippocampal slices, and the effect was abolished by the nicotinic ACh receptor antagonists, alpha-bungarotoxin and mecamylamine. Nefiracetam induced a long-lasting facilitation of synaptic transmission in both the CA1 area and the dentate gyrus of rat hippocampal slices, and the facilitation was inhibited by alpha-bungarotoxin and mecamylamine. Such facilitatory action was still found in the hippocampus with selective cholinergic denervation. The results of the present study, thus, suggest that nefiracetam enhances activity of nicotinic ACh receptors by interacting with a PKC pathway, thereby increasing glutamate release from presynaptic terminals, and then leading to a sustained facilitation of hippocampal neurotransmission. This may represent a cellular mechanism underlying the cognition-enhancing action of nefiracetam. The results also provide the possibility that nefiracetam could be developed as a promising therapeutic drug for senile dementia or Alzheimer's disease.