Nicotinic Acetylcholine Receptor Channels Research Articles

A re-examination of adult mouse nicotinic acetylcholine receptor channel activation kinetics.

1. During routine sequencing of our mouse muscle alpha subunit acetylcholine receptor channel (AChR) cDNA clones, we detected a discrepancy with the GenBank database entry (accession X03986). At nucleotides 1305-7 (residue 433, in the M4 domain) the database lists GTC which encodes a valine, while our putative 'wild-type' cDNA had the nucleotides GCC, which encodes an alanine. No other sequence differences were found. 2. PCR amplification of genomic DNA confirmed that the BALB/C mouse alpha subunit gene has a T nucleotide at position 1306, and, therefore, that the protein has a V at position 433 in the M4 segment. 3. In order to determine the functional consequences of this difference, either wild-type (V433) or mutant (A433) alpha subunits were co-expressed in HEK cells with mouse beta, epsilon and delta subunits. Single-channel currents were recorded in cell-attached patches, and rate and equilibrium constants were estimated from open and closed durations obtained from a range of ACh concentrations. No significant differences were found between the activation rate constants or equilibrium constants of the V433 and A433 variants. 4. Kinetic modelling of alphaV433 AChR suggests that the two transmitter binding sites have similar dissociation equilibrium constants for acetylcholine ( approximately 160 microM in 142 mM extracellular KCl). 5. Diliganded AChRs occupy a closed state that has a lifetime of approximately 1 ms. The rate constants for entering and leaving this state do not vary with the ACh concentration. 6. The kinetics of a mutant AChR that causes a slow channel congenital myaesthenic syndrome, alphaG153S, was re-examined. The properties of this mutant were similar with a V or an A at position alpha433.

Voltage-dependent modulation of the neuronal nicotinic acetylcholine receptor-channel by cartap

Voltage-dependent modulation of the neuronal nicotinic acetylcholine receptor-channel by cartap

Modulation of the neuronal nicotinic acetylcholine receptor-channel by the nootropic drug nefiracetam

The effects of nefiracetam (DM-9384) on the neuronal nicotinic acetylcholine (ACh) receptor-channel were studied by the whole-cell patch clamp technique using PC12 cells. Nefiracetam had a dual effect on ACh-induced currents: it augmented the currents induced by low concentrations (10–30 μM) of ACh and suppressed those induced by high concentrations (100–1000 μM) of ACh. These effects were reversible after washing with drug-free solution. The stimulating effect of nefiracetam was clearly observed at a concentration of 10 μM, and slight increases in currents were detected even at 0.1 μM or 1 μM. Nefiracetam at 100 μM suppressed the currents induced by a low concentration (10 μM) of ACh. The rate of desensitization of ACh-induced current was greatly accelerated by nefiracetam, and this effect could not be reversed by washing with drug-free solution. When added to the internal pipette solution, the protein kinase A inhibitor KT 5720 (0.6 μM), but not the protein kinase C inhibitor calphostin C (0.5 μM), abolished the nefiracetam stimulation of the ACh receptor. Pre-incubation of cells with 200 ng/ml pertussis toxin for 24 h also abolished the nefiracetam action. Thus, the nefiracetam modulation of the neuronal nicotinic ACh receptor-channel is exerted via G proteins and protein kinase A. The stimulation of the ACh receptor may be directly related to the cognitive enhancing action of nefiracetam.

Effects of Nitenpyram on the Neuronal Nicotinic Acetylcholine Receptor-Channel in Rat Phaeochromocytoma PC12 Cells

Nitenpyram, (E)-N-(6-chloro-3-pyridylmethyl)-N-ethyl-N'-methyl-2-nitro-vinylidenediamine, is known to act on the nicotinic acetylcholine receptor-channel (AChR). We examined the effects of nitenpyram on the neuronal type of the nicotinic AChR in rat clonal phaeochromocytoma PC12 cells using a patch clamp technique to clarify the mechanisms of action at the single-channel level. Nitenpyram itself induced single-channel currents mediated by the neuronal nicotinic AChR. Co-application of ACh and nitenpyram opened the channels exhibiting the two conductance states. The proportion of main conductance levels was decreased and that of subconductance levels was increased in the presence of nitenpyram. The open time and closed time distribution, but not for the burst duration, for the main conductance state currents with co-application of ACh and nitenpyram were similar to those of ACh-induced current. These changes of single-channel kinetics result in a decrease in probability of openings of main conductance state currents explaining the effects of nitenpyram on the neuronal nicotinic AChR. The effects of nitenpyram on the acetylcholine-induced current are deemed directly responsible for the toxic actions in animals.

2] Fluorescence techniques for measuring ion channel activity

Publisher Summary Changes in intracellular free calcium concentration ([Ca 2+ ] i ) play a crucial role in cellular physiology. A number of cell surface receptors and channels are known to regulate [Ca 2+ ] i through different molecular mechanisms. Therefore, the functional and pharmacologic properties of many of these cell surface receptors and ion channels can be studied effectively by measuring changes in [Ca 2+ ] i in intact cells. For drug discovery efforts, several ion channel and receptor systems have been targeted that play different roles in neuronal physiology and pathophysiology. These molecular targets include voltage- and ligand-gated ion channels: the human neuronal voltage-gated calcium channels (VGCCs), ligand-gated nicotinic acetylcholine receptor channels (NAChRs), ionotropic N -methyl-D-aspartic acid (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and kainate-type excitatory amino acid receptor (EAA) channels. All of these channels mediate elevation of [Ca 2+ ] i via Ca 2+ influx from the extracellular medium upon depolarization or activation by agonist. This chapter describes the experimental methods with particular emphasis on the validation of the assay for human VGCCs, NAChRs, and NMDA receptors.

Cell-type specific expression of ATP-sensitive potassium channels in the rat hippocampus.

1. The distribution of ATP-sensitive K+ channels (KATP channels) was investigated in four cell types in hippocampal slices prepared from 10- to 13-day-old rats: CA1 pyramidal cells, interneurones of stratum radiatum in CA1, complex glial cells of the same area and granule cells of the dentate gyrus. The neuronal cell types were identified visually and characterized by the shapes and patterns of their action potentials and by neurobiotin labelling. 2. The patch-clamp technique was used to study the sensitivity of whole-cell currents to diazoxide (0.3 mM), a KATP channel opener, and to tolbutamide (0.5 mM) or glibenclamide (20 microM), two KATP channel inhibitors. The fraction of cells in which whole-cell currents were activated by diazoxide and inhibited by tolbutamide was 26% of pyramidal cells, 89 % of interneurones, 100% of glial cells and 89% of granule cells. The reversal potential of the diazoxide-induced current was at the K+ equilibrium potential and a similar current activated spontaneously when cells were dialysed with an ATP-free pipette solution. 3. Using the single-cell RT-PCR method, the presence of mRNA encoding KATP channel subunits (Kir6.1, Kir6.2, SUR1 and SUR2) was examined in CA1 pyramidal cells and interneurones. Subunit mRNA combinations that can result in functional KATP channels (Kir6.1 together with SUR1, Kir6.2 together with SUR1 or SUR2) were detected in only 17% of the pyramidal cells. On the other hand, KATP channels may be formed in 75% of the interneurones, mainly by the combination of Kir6.2 with SUR1 (58% of all interneurones). 4. The results of these combined analyses indicate that functional KATP channels are present in principal neurones, interneurones and glial cells of the rat hippocampus, but at highly different densities in the four cell types studied.

The NO synthase inhibitors l-Name and l-NMMA, but not l-arginine, block the mammalian nicotinic acetylcholine receptor channel

(1) Nitric oxide (NO) synthase inhibitors (NOS-I) such as l-Name ( N G-nitro l-arginine methyl ester) and l-NMMA ( N G-monomethyl l-arginine) may enhance anesthesia indirectly by inhibiting the NO pathway. Moreover, NOS-I interact directly with receptor proteins. In an animal study, l-NMMA potentiated muscle relaxants. (2) The present experiments investigate the effects of l-NMMA, l-Name, and l-arginine on the nicotinic acetylcholine receptor channel (nAChR) using patch clamp techniques and a piezo-driven application system. Both NOS-I appear to directly interact with the nAChR in the open as well as in the closed conformation. l-Arginine has no effect.

Ion channel modulation as the basis for general anesthesia

(1) Modulation of the function of the GABA A and neuronal nicotinic acetylcholine receptor channels caused by general anesthetics and modulation of the GABA A receptor-channel by halothane, enflurane, isoflurane, and n-octanol was channel state-dependent. (3) Halothane modulation of the GABA A receptor was independent of subunits, but n-octanol modulation was subunit-dependent. (4) Ethanol at 30–100 μM was very potent in accelerating the desensitization of currents induced by acetylcholine. (5) The ethanol modulation was subunit- and state-dependent, occurring in the α3 β4 combination but only weakly in the α3 β2 combination. (6) In contrast, halothane at 430 μM (∼1 MAC) potently suppressed ACh-induced currents in the α3 β2 subunit combination.

Desensitization of mouse nicotinic acetylcholine receptor channels. A two-gate mechanism.

The rate constants of acetylcholine receptor channels (AChR) desensitization and recovery were estimated from the durations and frequencies of clusters of single-channel currents. Diliganded-open AChR desensitize much faster than either unliganded- or diliganded-closed AChR, which indicates that the desensitization rate constant depends on the status of the activation gate rather than the occupancy of the transmitter binding sites. The desensitization rate constant does not change with the nature of the agonist, the membrane potential, the species of permeant cation, channel block by ACh, the subunit composition (epsilon or gamma), or several mutations that are near the transmitter binding sites. The results are discussed in terms of cyclic models of AChR activation, desensitization, and recovery. In particular, a mechanism by which activation and desensitization are mediated by two distinct, but interrelated, gates in the ion permeation pathway is proposed.

Dimensions of neuronal nicotinic acetylcholine receptor channel as estimated from the analysis of the channel-blocking effects.

Acetylcholine-induced membrane currents and excitatory postsynaptic currents (EPSCs) were recorded from the neurons of rat superior cervical ganglion (SCG) using the whole-cell patch clamp and the two-electrode voltage clamp techniques, correspondingly. The EPSC decay was bi-exponential, with fast and slow components characterized by time constants 5.5 +/- 0.5 msec and 20.4 +/- 1.2 msec (mean +/- SEM; n = 23), respectively. Blocking of these currents by a series of newly synthesized bis-cationic ammonium compounds, the pentamethonium and pentaethonium derivatives, was analyzed. Blocking effects were due to a block of nicotinic acetylcholine receptor (nAChR) open channel, with mean blocker binding rate constants for the fast component three to five times higher than those for the slow component. Dimensions of a nAChR ionic channel were deduced from a relationship between blocking activity of the compounds and the size of the projections of their three-dimensional molecular models on the neuronal membrane plane. The results suggest that there are two populations of nAChRs in rat SCG neurons; while these polulations differ in the rate constants of the binding by the blocker to their open channels, they exhibit similar channel diameter, 11.8 A, at the level at which the blockers bind to the channel.

Probing the Structure of the Nicotinic Acetylcholine Receptor Ion Channel with the Uncharged Photoactivable Compound [3H]Diazofluorene

The uncharged photoactivable probe 2-[3H]diazofluorene ([3H]DAF) was used to examine structural changes in the Torpedo californica nicotinic acetylcholine receptor (AChR) ion channel induced by agonists. Photoincorporation of [3H]DAF into the AChR consisted of the following two components: a nonspecific component consistent with incorporation into residues situated at the lipid-protein interface, and a specific component, inhibitable by noncompetitive antagonists and localized to the M2 hydrophobic segments of AChR subunits. The nonspecific [3H]DAF incorporation was characterized in the M4 segment of each AChR subunit. The observed distribution and periodicity of labeled residues reinforce the conclusion that the M4 segments are organized as transmembrane alpha-helices with a common "face" of each helix in contact with lipid. Within the M2 segments, in the absence of agonist [3H]DAF specifically labeled homologous residues betaVal-261 and deltaVal-269, with incorporation into deltaVal-269 at a 5-fold greater efficiency than into betaVal-261. This observation, coupled with the lack of detectable incorporation into alpha-M2 including the homologous alphaVal-255, indicates that within the resting channel [3H]DAF is bound with its photoreactive diazo group oriented toward deltaVal-269. In the presence of agonist, there is an approximately 90% reduction in the labeling of betaVal-261 and deltaVal-269 accompanied by specific incorporation into residues (betaLeu-257, betaAla-258, deltaSer-262, and deltaLeu-265) situated 1 or 2 turns of an alpha-helix closer to the cytoplasmic end of the M2 segments. The results provide a further characterization of agonist-induced rearrangements of the M2 (ion channel) domain of the AChR.

Purified IgG from seropositive and seronegative patients with mysasthenia gravis reversibly blocks currents through nicotinic acetylcholine receptor channels.

The mechanism of block of nicotinic acetylcholine receptor (nAChR) channels by purified antibodies from patients with myasthenia gravis (MG) was investigated by using an ultrafast system for solution exchange at outside-out patches. IgG of MG patients and controls was purified by using protein A-Sepharose columns. Probes from 9 seropositive MG patients and 3 seronegative MG patients were tested. As a preparation, cultured mouse myotubes expressing the embryonic-type nAChR channels were used. Twenty-millisecond pulses of 1.0 mM ACh were applied repetitively to outside-out patches. Outside-out patches were preexposed with IgG in concentrations between 0.1 and 200 mg/L during application of ACh pulses. The peak current amplitude was reduced to values between 6% and 71% of control for the 9 seropositive and 3 seronegative MG patients. The block was concentration dependent and fully reversible after washout of antibodies. Incubation with IgG from different control patients did not reduce the peak current amplitude. In addition, our findings with purified IgG from seronegative MG patients support the idea of the immunopathogenesis of this disorder and may allow the development of a diagnostic test for seronegative MG patients.

Models and simulations of ion channels and related membrane proteins

The past year has seen major advances in our understanding of ion channels, resulting from molecular dynamics simulations and modelling studies. Simulations of gramicidin have revealed that proton conduction along a water wire is limited by the dynamics of water reorientation. Plausible models are now available for a number of other channels, including alamethicin, the influenza A virus M2 protein, and the pore domains of the nicotinic acetylcholine receptor and Kv channels. Molecular dynamics simulations and continuum calculations have revealed some of the subtleties of the interactions between transmembrane helices and their lipid bilayer environment.

Weakening of ion-channel interactions of Na+ and Li+ in acetylcholine-receptor channels of frog skeletal muscle with an increase in agonist concentration.

The possibility that increases in agonist concentration beyond threshold levels may force changes in the character of high-conductance open states of skeletal muscle nicotinic acetylcholine receptor channels (nAChR) was examined by seeing whether differences in several critical ionic properties of nAChR currents could be detected with changes in agonist level. Single- and bi-ionic whole-cell currents of Na+ and Li+ in voltage-clamped frog (Rana pipiens) muscle fibers were measured during local superfusion of endplates with carbamylcholine (carb) at concentrations of 54 microm (low-carb) and 270 microM (high-carb). Three ionic properties that would be affected by changes in the open-state configuration of channel subunits were tested. First, ion-saturation characteristics. Peak Na+ and Li+ currents in low-carb trials showed sublinear dependence on ion concentrations from 0 to 60 mM with Km values of 78 (Na+) and 49 (Li+) mM and a power function slope of 0. 75 on double-log plot. In contrast, the concentration dependence of Na+ and Li+ currents in high-carb tests was linear through the origin with a power function slope of 1.02. Second, Na+/Li+ selectivity. The ratio of peak Na+ and Li+ currents in low-carb tests varied from 1.86 to 2.28 for ion concentrations of from 20 to 60 mM [mean = 2.02 +/- 0.06 (SEM)] whereas the ratio for high-carb trials ranged from only 1.29 to 1.52 [mean = 1.42 +/- 0.40 (SEM)]. Third, competitive interactions of Na+ and Li+ currents. Equimolar mixtures of Na+ and Li+ in low-carb tests produced bi-ionic inward currents which were never larger than the single-ion Na+ current alone, but bi-ionic currents at the high-carb level were always greater than the single-ion Na+ current, approximating the sum of the single-ion Na+ and Li+ currents in most cases. The results are consistent with a decrease in ion-channel binding at the high-carb level and support the possibility of agonist-induced changes in the high-conductance open-state configuration of nAChR subunits which result in a weakening of constraints on cation movements through the channel.

Kinked-Helices Model of the Nicotinic Acetylcholine Receptor Ion Channel and Its Complexes with Blockers: Simulation by the Monte Carlo Minimization Method

A model of the nicotinic acetylcholine receptor ion channel was elaborated based on the data from electron microscopy, affinity labeling, cysteine scanning, mutagenesis studies, and channel blockade. A restrained Monte Carlo minimization method was used for the calculations. Five identical M2 segments (the sequence EKMTLSISVL 10LALTVFLLVI 20V) were arranged in five-helix bundles with various geometrical profiles of the pore. For each bundle, energy profiles for chlorpromazine, QX-222, pentamethonium, and other blocking drugs pulled through the pore were calculated. An optimal model obtained allows all of the blockers free access to the pore, but retards them at the rings of residues known to contribute to the corresponding binding sites. In this model, M2 helices are necessarily kinked. They come into contact with each other at the cytoplasmic end but diverge at the synaptic end, where N-termini of M1 segments may contribute to the pore. The kinks disengage α-helical H-bonds between Ala 12 and Ser 8. The uncoupled lone electron pairs of Ser 8 carbonyl oxygens protrude into the pore, forming a hydrophilic ring that may be important for the permeation of cations. A split network of H-bonds provides a flexibility to the chains Val 9-Ala 12, the numerous conformations of which form only two or three intrasegment H-bonds. The cross-ectional dimensions of the interface between the flexible chains vary essentially at the level of Leu 11. We suggest that conformational transitions in the chains Val 9-Ala 12 are responsible for the channel gating, whereas rotations of more stable α-helical parts of M2 segments may be necessary to transfer the channel in the desensitized state.

Cartap Modulation of the Nicotinic Acetylcholine Receptor-Channel of Clonal Rat Phaeochromocytoma (PC12) Cells

Cartap, the synthetic analog of nereistoxin, is known to act on the nicotinic acetylcholine receptor-channel (AChR). Effects of cartap on the neuronal nicotinic AChR of clonal rat phaeochromocytoma (PC 12) cells were studied using a single-channel patch clamp method. When cartap was co-applied with acetylcholine, the single-channel openings were shortened and occurred in bursts separated by brief closures or gaps. The current amplitudes in the presence of cartap were virtually identical to those of currents induced by ACh only. Mean open time and burst duration were significantly decreased in the presence of cartap in a dose-dependent manner. These changes of single-channel kinetics result in a significant decrease in total charge carried through the open AChR explaining the suppressive effect of cartap on nicotinic AChR. Results suggest that cartap could act as an open channel blocker at the nicotinic AChR.

Modelling and Simulation of Ion Channels: Applications to the Nicotinic Acetylcholine Receptor

Molecular dynamics simulations with experimentally derived restraints have been used to develop atomic models of M2 helix bundles forming the pore-lining domains of the nicotinic acetylcholine receptor and related ligand-gated ion channels. M2 helix bundles have been used in microscopic simulations of the dynamics and energetics of water and ions within an ion channel. Translational and rotational motion of water are restricted within the pore, and water dipoles are aligned relative to the pore axis by the surrounding helix dipoles. Potential energy profiles for translation of a Na+ion along the pore suggest that the protein and water components of the interaction energy exert an opposing effect on the ion, resulting in a relatively flat profile which favors cation permeation. Empirical conductance calculations based on a pore radius profile suggest that the M2 helix model is consistent with a single channel conductance of ca. 50 pS. Continuum electrostatics calculations indicate that a ring of glutamate residues at the cytoplasmic mouth of the α7 nicotinic receptor M2 helix bundle may not be fully ionized. A simplified model of the remainder of the channel protein when added to the M2 helix bundle plays a significant role in enhancing the ion selectivity of the channel.

P-282 - Metabolites of arachidonic acid via cyclooxygenase pathway accelerate desensitization of nicotinic acetylcholine receptor channel in mouse skeletal muscles

P-282 - Metabolites of arachidonic acid via cyclooxygenase pathway accelerate desensitization of nicotinic acetylcholine receptor channel in mouse skeletal muscles

Modulation of the Neuronal Nicotinic Acetylcholine Receptor Channel by Imidacloprid and Cartap

Imidacloprid, a nitromethylene heterocycle insecticide, and cartap, a synthetic analog of nereistoxin, are known to act on the nicotinic acetylcholine receptor channel. The effects of imidacloprid and cartap on the nicotinic acetylcholine receptor channel of clonal rat phaeochromocytoma cells were studied using the single-channel patch-clamp technique. Acetylcholine at a concentration of 10 μM induced currents predominantly in the main conductance state, whereas imidacloprid at 10 μM induced currents predominantly in the subconductance state. No current was evoked by cartap at concentrations ranging from 0.1 to 300 μM. Coapplication of acetylcholine and imidacloprid generated currents in both main conductance state and subconductance state. Coapplication of acetylcholine and cartap induced the main conductance state currents, forming bursts. The distributions of open time and burst duration of currents induced by acetylcholine, imidacloprid, acetylcholine plus imidacloprid, and acetylcholine plus cartap showed multiexponential components. Cartap, when coapplied with acetylcholine, markedly shortened the open time reflecting the appearance of bursts. These results indicate that imidacloprid and cartap modulate the nicotinic acetylcholine receptor channel with different mechanisms of action. Imidacloprid acts as a partial agonist generating predominantly subconductance state currents, whereas cartap acts as an open channel blocker at the nicotinic acetylcholine receptor channel.

Direct actions of anticholinesterases on the neuronal nicotinic acetylcholine receptor channels

Recent studies have suggested that anticholinesterases including organophosphates and carbamates act directly on the nicotinic acetylcholine receptor (AChR) channel. We performed whole-cell and single-channel patch-clamp experiments to elucidate the mechanism of action of anticholinesterases on the nicotinic AChR in rat clonal phaeochromocytoma (PC12) cells. Neostigmine and carbaryl showed a biphasic effect; enhancement and suppression of carbachol-induced whole-cell currents. The currents induced by 100 μM carbachol was enhanced by the first co-application with 10 or 100 μM neostigmine, and the current was eventually suppressed below the control level during repeated co-applications. The decay phase of current was accelerated by neostigmine. Carbaryl at 0.1 μM greatly potentiated the carbachol-induced current, and at higher concentrations (0.3–3 μM), current was suppressed. In single-channel experiments, these compounds increased the short closures or gaps during channel opening without changing the single-channel conductance. Mean open time and burst duration were decreased in the presence of neostigmine and carbaryl. These results indicate that neostigmine and carbaryl directly block the nicotinic AChR channel.