Acetylcholine Receptor Channel Research Articles

Delimiting the binding site for quaternary ammonium lidocaine derivatives in the acetylcholine receptor channel.

The triethylammonium QX-314 and the trimethylammonium QX-222 are lidocaine derivatives that act as open-channel blockers of the acetylcholine (ACh) receptor. When bound, these blockers should occlude some of the residues lining the channel. Eight residues in the second membrane-spanning segment (M2) of the mouse-muscle alpha subunit were mutated one at a time to cysteine and expressed together with wild-type beta, gamma, and delta subunits in Xenopus oocytes. The rate constant for the reaction of each substituted cysteine with 2-aminoethyl methanethiosulfonate (MTSEA) was determined from the time course of the irreversible effect of MTSEA on the ACh-induced current. The reactions were carried out in the presence and absence of ACh and in the presence and absence of QX-314 and QX-222. These blockers had no effect on the reactions in the absence of ACh. In the presence of ACh, both blockers retarded the reaction of extracellularly applied MTSEA with cysteine substituted for residues from alphaVal255, one third of the distance in from the extracellular end of M2, to alphaGlu241, flanking the intracellular end of M2, but not with cysteine substituted for alphaLeu258 or alphaGlu262, at the extracellular end of M2. The reactions of MTSEA with cysteines substituted for alphaLeu258 and alphaGlu262 were considerably faster in the presence of ACh than in its absence. That QX-314 and QX-222 did not protect alphaL258C and alphaE262C against reaction with MTSEA in the presence of ACh implies that protection of the other residues was due to occlusion of the channel and not to the promotion of a less reactive state from a remote site. Given the 12-A overall length of the blockers and the alpha-helical conformation of M2 in the open state, the binding site for both blockers extends from alphaVal255 down to alphaSer248.

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.

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.

Interactions of general anesthetics within the pore of an ion channel

(1) We review the electrophysiological evidence that the ion channel pore is the site at which general anesthetics bind to inhibit muscle-type acetylcholine receptor channels. (2) The amphipathic character of a pore certainly offers a suitable environment for the binding of amphipathic anesthetics. (3) The absence of direct information about the binding sites of these rather non-specific drugs, forces us to rely on indirect information provided by kinetic experiments. (4) We also discuss the implications of these findings for the interaction of general anesthetics with other ion channels.

Nicotinic receptors are regulated by protein kinase C activated via a nicotinic receptors-mediated signaling pathway

The present study was conducted to examine the effect of protein kinase C (PKC) on nicotinic acetylcholine (ACh) receptors expressed in Xenopus oocytes by monitoring single-channel currents. In an outside-out patch-clamp configuration, ACh (1 μM) elicited single channel currents with a slope conductance of 31 pS (control) in normal Torpedo ACh receptors. Activation of PKC via an endogenous phosphatidylinositol signaling pathway elevated the slope conductance to 41 pS, which effect was blocked by the selective PKC inhibitor, staurosporine. Mutant ACh receptor channels, which mimic PKC phosphorylation of the receptors, exhibited a slope conductance of 41 pS. Notably, pretreatment with a higher concentration of ACh (100 μM) caused an increase in the slope conductance of the channels for 1 μM ACh (43 pS), which was the same level as obtained with either PKC activation or mutant ACh receptors, and this effect was also inhibited by staurosporine. In addition, the control slope conductance was reduced by PKC inhibitor peptide (24 pS), which corresponded to that obtained with another mutant ACh receptors lacking PKC phosphorylation sites (18 pS). Mouse muscle ACh receptors were also regulated by the same mechanism. The results of the present study suggest that ACh activates PKC via nicotinic ACh receptors, which alternatively, modulates the properties of the receptor channels.

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.

A Distinct Contribution of the δ Subunit to Acetylcholine Receptor Channel Activation Revealed by Mutations of the M2 Segment

Acetylcholine receptor (AChR) channels with proline (P) mutations in the putative pore-forming domain (at the 12′ position of the M2 segment) were examined at the single-channel level. For all subunits ( α, β, ϵ, and δ), a 12′P mutation increased the open channel lifetime >5-fold. To facilitate the estimation of binding and gating rate constants, subunits with 12′P mutations were co-expressed with α subunits having a binding site mutation that slows channel opening ( αD200N). In these AChRs, a 12′P mutation in ϵ or β slowed the closing rate constant ∼6-fold but had no effect on either the channel opening rate constant or the equilibrium dissociation constant for ACh ( K d). In contrast, a 12′P mutation in δ slowed the channel closing rate constant only ∼2-fold and significantly increased both the channel opening rate constant and the K d. Pairwise expression of 12′P subunits indicates that mutations in ϵ and β act nearly independently, but one in δ reduces the effect of a homologous mutation in ϵ or β. The results suggest that a 12′P mutation in ϵ and β has mainly local effects, whereas one in δ has both local and distributed effects that influence both agonist binding and channel gating.

Mutations at lipid-exposed residues of the acetylcholine receptor affect its gating kinetics.

The firmest candidate among the transmembrane portions of the nicotinic acetylcholine receptor (AChR) to be in contact with the lipid bilayer is the fourth segment, M4. To explore the contribution of alphaM4 amino acid residues of mouse AChR to channel gating, we combined site-directed mutagenesis with single-channel recordings. Two residues in alphaM4, Cys418 and Thr422, were found to significantly affect gating kinetics when replaced by alanine. AChRs containing alphaC418A and alphaT422A subunits form channels characterized by a 3- and 5-fold reduction in the mean open time, respectively, suggesting an increase in the closing rate due to the mutations. The calculated changes in the energy barrier for the channel closing process show unequal and coupled contributions of both positions to channel gating. Single-channel recordings of hybrid wild-type alpha/alphaT422A AChR show that the closing rate depends on the number of alpha subunits mutated. Each substitution of threonine to alanine changes the energy barrier of the closing process by approximately 0.5 kcal/mol. Recordings of channels activated by high agonist concentration suggest that these mutations also impair channel opening. Both Cys418 and Thr422 have been postulated to be in contact with the lipid milieu and are highly conserved among species and subunits. Our results support the involvement of lipid-exposed residues in alphaM4 in AChR channel gating mechanism.

The Location of the Gate in the Acetylcholine Receptor Channel

The cation-conducting channel of the nicotinic acetylcholine (ACh) receptor is lined by the first (M1) and second (M2) membrane-spanning segments of each of its five subunits. Six consecutive residues, αS239 to αT244, in the α subunit M1–M2 loop and at the intracellular end of M2 were mutated to cysteine. The accessibility of the substituted cysteines were probed with small, cationic, sulfhydryl-specific reagents added extracellularly and intracellularly. In the closed state of the channel, there is a barrier to these reagents added from either side between αG240 and αT244. ACh induces the removal of this barrier, which acts as an activation gate. The residues αG240, αE241, αK242, and αT244 line a narrow part of the channel, in which this gate is located.

State-dependent accessibility and electrostatic potential in the channel of the acetylcholine receptor. Inferences from rates of reaction of thiosulfonates with substituted cysteines in the M2 segment of the alpha subunit.

Ion channel function depends on the chemical and physical properties and spatial arrangement of the residues that line the channel lumen and on the electrostatic potential within the lumen. We have used small, sulfhydryl-specific thiosulfonate reagents, both positively charged and neutral, to probe the environment within the acetylcholine (ACh) receptor channel. Rate constants were determined for their reactions with cysteines substituted for nine exposed residues in the second membrane-spanning segment (M2) of the alpha subunit. The largest rate constants, both in the presence and absence of ACh, were for the reactions with the cysteine substituted for alpha Thr244, near the intracellular end of the channel. In the open state of the channel, but not in the closed state, the rate constants for the reactions of the charged reagents with several substituted cysteines depended on the transmembrane electrostatic potential, and the electrical distance of these cysteines increased from the extracellular to the intracellular end of M2. Even at zero transmembrane potential, the ratios of the rate constants for the reactions of three positively charged reagents with alpha T244C, alpha L251C, and alpha L258C to the rate constant for the reaction of an uncharged reagent were much greater in the open than in the closed state. This dependence of the rate constants on reagent charge is consistent with an intrinsic electrostatic potential in the channel that is considerably more negative in the open state than in the closed state. The effects of ACh on the rate constants for the reactions of substituted Cys along the length of alpha M2, on the dependence of the rate constants on the transmembrane potential, and on the intrinsic potential support a location of a gate more intracellular than alpha Thr244.

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.

A role for alpha(187-199) in the conversion of agonist binding energy to the opening of the acetylcholine receptor ion channel.

A role for alpha(187-199) in the conversion of agonist binding energy to the opening of the acetylcholine receptor ion 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.

A murine specific splicing variant of the acetylcholine receptor ε-subunit gene transcript

The acetylcholine receptor (AChR) is an ion channel involved in signal transmission from motorneuron to skeletal muscle. We describe here a murine-specific splicing variant of the ε subunit of the AChR ( ε s), which lacks exon 5. ε s has a truncated extracellular domain which may affect the physiological characteristics of the resulting AChR channel.

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.

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.