Catch-and-Hold Activation of Muscle Acetylcholine Receptors Having Transmitter Binding Site Mutations

Agonists turn on receptors because their target sites have a higher affinity in the active versus resting conformation of the protein. We used single-channel electrophysiology to measure the lower-affinity (LA) and higher-affinity (HA) equilibrium dissociation constants for acetylcholine in adult-type muscle mouse nicotinic receptors (AChRs) having mutations of agonist binding site amino acids. For a series of agonists and for all mutations of αY93, αG147, αW149, αY190, αY198, εW55, and δW57, the change in LA binding energy was approximately half that in HA binding energy. The results were analyzed as a linear free energy relationship between LA and HA agonist binding, the slope of which (κ) gives the fraction of the overall binding chemical potential where the LA complex is established. The linear correlation between LA and HA binding energies suggests that the overall binding process is by an integrated mechanism (catch-and-hold). For the agonist and the above mutations, κ ∼ 0.5, but side-chain substitutions of two residues had a slope that was significantly higher (0.90; αG153) or lower (0.25; εP121). The results suggest that backbone rearrangements in loop B, loop C, and the non-α surface participate in both LA binding and the LA ↔ HA affinity switch. It appears that all of the intermediate steps in AChR activation comprise a single, energetically coupled process.     

A Novel Agonist Binding Site on Nicotinic Acetylcholine Receptors

Abstract

This report provides evidence that physostigmine (Phy) and benzoquinonium (BZQ) are able to activate nicotinic acetylcholine receptors (nAChRs) through binding site(s) distinct from those of the natural transmitter, ACh. Such findings are in agreement with a second pathway of activation of nAChRs. Receptor activation may be modulated through the novel site, and, consequently, physiological processes involving nicotinic synapses could be controlled. Using patch clamp techniques, single channel currents activated by ACh and anatoxin were recorded from frog interosseal muscle fibers under cell-attached condition and outside-out patches excised from cultured rat hippocampal neurons. Whole cell nicotinic currents were also studied in the cultured neurons. In most of the neurons, nicotinic responses were blocked by the nicotinic antagonists methyllycaconitine (MLA) and α-bungarotoxin (α-BGT). Evaluation of the effects of Phy and BZQ on the muscle and on the α-BGT- and MLA-sensitive neuronal nAChRs demonstrated that both compounds were open channel blockers at these receptors. Furthermore, at low micromolar concentrations, Phy and BZQ activated the nAChRs of all preparations tested, such an effect being unexpectedly resistant to α-BGT or MLA. Thus, the nAChRs could be activated via two distinct binding sites: one for ACh and the other for Phy and BZQ. These findings and previous biochemical results led us to suggest that a putative endogenous ligand could bind to the new site and thereby regulate the activation of nAChRs in nicotinic synapses.     

Acute Activation,Desensitization and Smoldering Activation of Human Acetylcholine Receptors

The behavioral effects of nicotine and other nicotinic agonists are mediated by AChRs in the brain. The relative contribution of acute activation versus chronic desensitization of AChRs is unknown. Sustained “smoldering activation” occurs over a range of agonist concentrations at which activated and desensitized AChRs are present in equilibrium. We used a fluorescent dye sensitive to changes in membrane potential to examine the effects of acute activation and chronic desensitization by nicotinic AChR agonists on cell lines expressing human α4β2, α3β4 and α7 AChRs. We examined the effects of acute and prolonged application of nicotine and the partial agonists varenicline, cytisine and sazetidine-A on these AChRs. The range of concentrations over which nicotine causes smoldering activation of α4β2 AChRs was centered at 0.13 µM, a level found in smokers. However, nicotine produced smoldering activation of α3β4 and α7 AChRs at concentrations well above levels found in smokers. The α4β2 expressing cell line contains a mixture of two stoichiometries, namely (α4β2)₂β2 and (α4β2)₂α4. The (α4β2)₂β2 stoichiometry is more sensitive to activation by nicotine. Sazetidine-A activates and desensitizes only this stoichiometry. Varenicline, cytisine and sazetidine-A were partial agonists on this mixture of α4β2 AChRs, but full agonists on α3β4 and α7 AChRs. It has been reported that cytisine and varenicline are most efficacious on the (α4β2)₂α4 stoichiometry. In this study, we distinguish the dual effects of activation and desensitization of AChRs by these nicotinic agonists and define the range of concentrations over which smoldering activation can be sustained.     

Spreading Activation of End-plate Receptors by Single Transmitter Quanta

WHEN the contents of a synaptic vesicle are emptied into the synaptic space, the transmitter molecules not only diffuse radially, reaching the postsynaptic membrane, but tangentially as well. Although some of the quantitative aspects of tangential diffusion have been discussed¹, the physiological significance of this process has not been sufficiently, emphasized. Indeed, we can assume that those molecules diffusing through the cleft interact with more receptors before reaching the extracellular space. In this manner, the surface area of activated postsynaptic membrane as well as the intensity and duration of the changes elicited by the transmitter-receptor interactions may be increased.     

Messenger RNA Levels and Binding Sites of Muscarinic Acetylcholine Receptors in Gastrointestinal Muscle Layers from Healthy Dairy Cows

Acetylcholine interacts with muscarinic receptors (M) to mediate gastrointestinal (GI) smooth muscle contractions. We have compared mRNA levels and binding sites of M₁to M₅ in muscle tissues from fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows. The mRNA levels were measured by quantitative RT-PCR. The inhibition of [³H]-QNB (1-quinuclidinyl-[phenyl-4-³H]-benzilate) binding by M antagonists [atropine (M_{1 ? 5}), pirenzepine (M₁), methoctramine (M₂), 4-DAMP (M₃), and tropicamide (M₄)] was used to identify receptors at the functional level. Maximal binding (B_max) was determined through saturation binding with atropine as a competitor. The mRNA levels of M₁, M₂, M₃, and M₅ represented 0.2, 48, 50, and 1.8%, respectively, of the total M population, whereas mRNA of M₄ was undetectable. The mRNA levels of M₂ and of M₃ in the ileum were lower (P < 0.05) than in other GI locations, which were similar among each other. Atropine, pirenzepine, methoctramine, and 4-DAMP inhibited [³H]-QNB binding according to an either low- or high-affinity receptor pattern, whereas tropicamide had no effect on [³H]-QNB binding. The [³H]-QNB binding was dose-dependent and saturable. B_max in fundus, pylorus, and PLAC was lower (P < 0.05) than in the ELSC, and in the pylorus lower (P < 0.05) than in the ileum. B_max and mRNA levels were negatively correlated (r = -0.3; P < 0.05). In conclusion, densities of M are different among GI locations, suggesting variable importance of M for digestive functions along the GI tract.     

Evidence for Structural Dissimilarity in the Neurotransmitter Binding Region of Purified Acetylcholine Receptors from Human Muscle and Torpedo Electric Organ

Acetylcholine receptor (AChR) purified from human skeletal muscle affinity-alkylated with bromoacetyl[methyl-3H]choline bromide ([3H]BAC) in mildly reducing conditions to yield a specifically radiolabeled polypeptide, Mr 44,000, the alpha-subunit. The binding of [125I]alpha-bungarotoxin to AChR was completely inhibited by affinity-alkylation, indicating that the human AChR's binding site for alpha-bungarotoxin is closely associated with the alpha-subunit's acetylcholine binding site. Structures in the vicinity of the alpha-bungarotoxin binding sites of AChRs from human muscle and Torpedo electric organ were compared by varying the conditions of alkylation. Under optimal conditions of reduction and alkylation, both human and Torpedo AChR incorporated BAC in equivalence to the number of alpha-bungarotoxin binding sites. However, with limited conditions of reduction but sufficient BAC to alkylate 100% of the alpha-bungarotoxin binding sites of human AChR, only 71% of the Torpedo AChR's binding sites were alkylated. In optimal conditions of reduction but with the minimal concentration of BAC that permitted 100% alkylation of the human AChR's alpha-bungarotoxin sites, only 74% of the Torpedo AChR's binding sites were alkylated. These data suggest that the neurotransmitter binding region of human muscle AChR is structurally dissimilar from that of Torpedo electric organ, having a higher binding affinity for BAC and an adjacent disulfide bond that is more readily accessible to reducing agents.     

The Stability of Solubilized Mammalian Muscle Acetylcholine Receptors During Purification by Monoclonal Immunoadsorption

The stability of nicotinic acetylcholine receptors (AChR) solubilized from mammalian skeletal muscle in nonionic detergent was investigated under various conditions of pH, chaotropic ions, and unfolding reagents in order to allow its purification in high yield by immunoadsorption to monoclonal antibodies. Preservation of the antigenicity and/or binding sites for alpha-bungarotoxin was used as an indicator of the receptor protein's integrity. Both were preserved in the pH range 6.5-8.0, but when exposed for 1 h at 4 degrees C to a pH outside this range, greater than 50% activity was lost. Of the chaotropic ions studied (NaSCN, NaI, NaNO3, NaCl), only NaCl was tolerated. Most of the AChR's toxin-binding activity was preserved after exposure to 2 M NaCl, which was suitable for dissociating AChR when a monoclonal antibody with relatively low binding affinity was selected as the immunoadsorbent. Yields of purified AChR were optimal (30%) when a low amount of monoclonal antibody was coupled to cyanogen bromide-activated agarose (1 mg protein/ml gel).     

Determinants of Phencyclidine Potency on the Nicotinic Acetylcholine Receptors from Muscle and Electric Organ

1.Phencyclidine (PCP) is an inhibitor of the nicotinic acetylcholine receptor (AChR) with characteristics of an open-channel blocker. The location of PCP binding site on the AChR molecule is unknown.2.PCP inhibits the AChR from electric organ with a higher potency than muscle AChR. To find the molecular basis of this difference, we expressed the two native and six hybrid receptors, and two receptors containing mutated mouse subunits in Xenopus laevis oocytes. The inhibition of ACh-induced current in these receptors by PCP was studied using whole-cell voltage-clamp. All hybrid receptors generated robust ACh-induced currents, while incomplete receptors (-less or -less) did not.3.PCP potency was higher on hybrids containing Torpedo and subunits regardless of the and subunit origin. A mouse subunit containing the asparagine 6 to the serine mutation in the M2 segment conferred a high sensitivity to PCP.4.These results support the conclusion that the amino acid residues at the position 6 of the M2 segments contribute to the PCP potency difference between Torpedo and mouse receptors.5.Another noncompetitive inhibitor of the AChR, the cembranoid eupalmerin acetate (EUAC), also inhibited the electric organ receptor with a somewhat higher potency than muscle AChR. However, the IC₅₀ values for EUAC inhibition of hybrid receptors did not follow the pattern observed for PCP. Therefore, these two inhibitors interact differently with the AChR molecule.     

Binding of Agonists and Antagonists to Muscarinic Acetylcholine Receptors on Intact Cultured Heart Cells

The binding of agonists and antagonists to muscarinic acetylcholine receptors on intact cultured cardiac cells has been compared with the binding observed in homogenized membrane preparations. The antagonists [3H]quinuclidinyl benzilate and [3H]N-methylscopolamine bind to a single class of receptor sites on intact cells with affinities similar to those seen in membrane preparations. In contrast with the heterogeneity of agonist binding sites observed in membrane preparations, the agonist carbachol binds to a homogeneous class of low-affinity sites on intact cells with an affinity identical to that found for the low-affinity agonist site in membrane preparations in the presence of guanyl nucleotides. Kinetic studies of antagonist binding to receptors in the absence and presence of agonist did not provide evidence for the existence of a transient (greater than 30 s) high-affinity agonist site that was subsequently converted to a site of lower affinity. Nathanson N. M. Binding of agonists and antagonists to muscarinic acetylcholine receptors on intact cultured heart cells.     

Bispyridinium Compounds Inhibit Both Muscle and Neuronal Nicotinic Acetylcholine Receptors in Human Cell Lines

Standard treatment of poisoning by organophosphorus anticholinesterases uses atropine to reduce the muscarinic effects of acetylcholine accumulation and oximes to reactivate acetylcholinesterase (the effectiveness of which depends on the specific anticholinesterase), but does not directly address the nicotinic effects of poisoning. Bispyridinium molecules which act as noncompetitive antagonists at nicotinic acetylcholine receptors have been identified as promising compounds and one has been shown to improve survival following organophosphorus poisoning in guinea-pigs. Here, we have investigated the structural requirements for antagonism and compared inhibitory potency of these compounds at muscle and neuronal nicotinic receptors and acetylcholinesterase. A series of compounds was synthesised, in which the length of the polymethylene linker between the two pyridinium moieties was increased sequentially from one to ten carbon atoms. Their effects on nicotinic receptor-mediated calcium responses were tested in muscle-derived (CN21) and neuronal (SH-SY5Y) cells. Their ability to inhibit acetylcholinesterase activity was tested using human erythrocyte ghosts. In both cell lines, the nicotinic response was inhibited in a dose-dependent manner and the inhibitory potency of the compounds increased with greater linker length between the two pyridinium moieties, as did their inhibitory potency for human acetylcholinesterase activity in vitro. These results demonstrate that bispyridinium compounds inhibit both neuronal and muscle nicotinic receptors and that their potency depends on the length of the hydrocarbon chain linking the two pyridinium moieties. Knowledge of structure-activity relationships will aid the optimisation of molecular structures for therapeutic use against the nicotinic effects of organophosphorus poisoning.     

The First Recombinant Viper Three-Finger Toxins: Inhibition of Muscle and Neuronal Nicotinic Acetylcholine Receptors

Genes encoding two three-finger toxins TFT-AF and TFT-VN, nucleotide sequences of which were earlier determined by cloning cDNA from venom glands of vipers Azemiops feae and Vipera nikolskii, respectively, were expressed for the first time in E. coli cells. The biological activity of these toxins was studied by electrophysiological techniques, calcium imaging, and radioligand analysis. It was shown for the first time that viper three-finger toxins are antagonists of nicotinic acetylcholine receptors of neuronal and muscle type.     

Activation of β-Adrenergic Receptors Stimulates Release of an Inhibitory Transmitter from Astrocytes

Activation of beta-adrenergic receptors on astrocytes in primary cell culture results in the release of taurine, an inhibitory transmitter. Taurine release occurs via a cyclic AMP-mediated intracellular pathway, because (a) taurine release and intracellular cyclic AMP accumulation have similar pharmacologies and time courses of activation and (b) N6,O2'-dibutyryl cyclic AMP stimulates release with a time course similar to that observed with the beta-adrenergic agonist isoproterenol. These results describe a previously unrecognized physiological function of astrocytes in the CNS-receptor-mediated release of the neuroactive amino acid taurine. This observation indicates that astrocytes may function as local regulators of neuronal activity.     

Constitutive Activity of Muscarinic Acetylcholine Receptors

We review the literature describing constitutive activity of the five muscarinic acetylcholine receptors in native and recombinant systems and discuss the effect of constitutive activity on muscarinic pharmacology in the context of modern models of receptor activation. We include a summary of mutations found to cause constitutive activity and discuss the implications of these data for the structure, function, and activation mechanism of muscarinic receptors. Finally, we discuss the possible physiological significance of constitutive activity of muscarinic receptors, incorporating information provided by targeted deletion of each of the muscarinic subtypes.     

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 α subunit were mutated one at a time to cysteine and expressed together with wild-type β, γ, and δ 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 αVal255, one third of the distance in from the extracellular end of M2, to αGlu241, flanking the intracellular end of M2, but not with cysteine substituted for αLeu258 or αGlu262, at the extracellular end of M2. The reactions of MTSEA with cysteines substituted for αLeu258 and αGlu262 were considerably faster in the presence of ACh than in its absence. That QX-314 and QX-222 did not protect αL258C and αE262C 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-Å overall length of the blockers and the α-helical conformation of M2 in the open state, the binding site for both blockers extends from αVal255 down to αSer248.     

Multiple Molecular Forms of Acetylcholine Receptors in Cultured Skeletal Muscle Cells: Subcellular Localization and Characterization

Abstract: Skeletal muscle cells of newborn rats, cultured in the absence of neuronal influence, were found to contain two types of cell surface acetylcholine receptors as demonstrated by isoelectric focusing. The isoelectric points of the two types of receptors were indistinguishable from those of junctional and extrajunctional types of receptors in mature animals. The cultured cells had two classes of intracellular α-bungarotoxin (αBT) binding components; one had the same sedimentation coefficient as that of surface receptors (9S), and the other had much smaller apparent molecular weights. Only a single major component was detected by isoelectric focusing analysis of the 9s intracellular aBT binding component, with a PI value close to that of the extra junctional receptor. These results suggest that the junctional and extrajunctional types of receptors may be synthesized through a common precursor.     

Dependence of Miniature Endplate Current on Kinetic Parameters of Acetylcholine Receptors Activation: A Model Study

Mathematical modeling was applied to study the dependence of miniature endplate current (MEPC) amplitude and temporal parameters on the values of the rate constants of acetylcholine binding to receptors (k₊) when cholinesterase was either active or inactive. The simulation was performed under two different sets of parameters describing acetylcholine receptor activation–one with high and another with low probability (Po_high and Po_low) of receptor transition into the open state after double ligand binding. The dependence of model MEPC amplitudes, rise times, and decay times on k₊ differs for set Po_low and set Po_high. The main outcome is that for set Po_high, the rise time is significantly longer at low values of k₊ because of the prolongation of ACh diffusion time to the receptor. For the set Polow, the rise time is shorter at low values of k1, which can be explained by the small probability of AChR forward isomerization after ACh binding and faster MEPC's peak formation.     

ß-Adrenergic Receptor Activation Increases Acetylcholine Receptor Number in Cultured Skeletal Muscle Myotubes

Treatment of embryonic chick muscle myotubes with the beta-adrenergic agonist isoproterenol increased the number of surface membrane nicotinic cholinergic receptors. Receptor degradation was unaffected by isoproterenol, suggesting that receptor synthesis was increased. The effect of isoproterenol appears to be mediated by the beta-adrenergic receptor adenylate cyclase system for the following reasons: (a) The response to isoproterenol was dose-dependent and stereospecific. (b) The response to catecholamines followed the order isoproterenol greater than epinephrine greater than norepinephrine. (c) Alprenolol, a beta-adrenergic antagonist, but not phentolamine, an alpha-antagonist, abolished the effect. (d) The maximal effects of isoproterenol and cholera toxin, an activator of adenylate cyclase, were not additive. These results suggest that under certain physiological states catecholamines may play an important role in the regulation of cholinergic receptors.     

The Intracellular Site of Calcium Activation of Contraction in Frog Skeletal Muscle

Radioautography has been used to localize ⁴⁵Ca in isotopically labeled frog skeletal muscle fibers which had been quickly frozen during a maintained tetanus, a declining tetanus, or during the period immediately following a tetanus or a contracture. During a tetanus almost all of the myofibrillar ⁴⁵Ca is localized in the region of the sarcomere occupied by the thin filaments. The amount varies with the tension being developed by the muscle. The movement of calcium within the reticulum from the tubular portion to the terminal cisternae during the posttetanic period has a half-time of about 9 sec at room temperature and a Q₁₀ of about 1.7. Repolarization is not necessary for this movement. Evidence is given to support the notion that most calcium efflux from the cell occurs from the terminal cisternae into the transverse tubules.