Linking the Acetylcholine Receptor-Channel Agonist-Binding Sites with the Gate

The gating isomerization of neuromuscular acetylcholine receptors links the rearrangements of atoms at two transmitter-binding sites with those at a distant gate region in the pore. To explore the mechanism of this reversible process, we estimated the gating rate and equilibrium constants for receptors with point mutations of α-subunit residues located between the binding sites and the membrane domain (N95, A96, Y127, and I49). The maximum energy change caused by a side-chain substitution at αA96 was huge (∼8.6 kcal/mol, the largest value measured so far for any α-subunit amino acid). A Φ-value analysis suggests that αA96 experiences its change in energy (structure) approximately synchronously with residues αY127 and αI49, but after the agonist molecule and other residues in loop A. Double mutant-cycle experiments show that the energy changes at αA96 are strongly coupled with those of αY127 and αI49. We identify a column of mutation-sensitive residues in the α-subunit that may be a pathway for energy transfer through the extracellular domain in the gating isomerization.     

The fractionation and partial characterization of membrane fragments derived from the electric organ of Electrophorus electricus

Two types of membrane particles, both binding α-[¹²⁵I]bungarotoxin, were obtained from electric tissue of Electrophorus electricus. They were both separated from acetyleholinesterase-containing particle by centrifugation in sucrose density gradients. The differing properties of the bungarotoxin-binding particles suggest that they may represent synaptic and extrasynaptic membrane structures containing acetylcholine receptors.     

Acetylcholine receptors and nerve terminal distribution at the neuromuscular junction of long-term regenerated muscle fibers

Mdx mice are deficient in dystrophin and show muscle fiber regeneration. Changes in the distribution of acetylcholine receptors have been reported at the neuromuscular junction of mdx mice and may be a consequence of muscle fiber regeneration. In this study, we examined whether the distribution of receptors was still altered in long-term, regenerated muscle fibers from C57Bl/10 mice. The left sternomastoid muscle of adult mice was injected with 60 μl of lidocaine hydrochloride to induce muscle degeneration-regeneration. In some mice, the sternomastoid muscle was denervated at the time of lidocaine injection. After 90 and 150 days, the nicotinic acetylcholine receptors were labeled with rhodamine-α-bungarotoxin for confocal microscopy. At both intervals studied, the receptors were distributed in spots. In denervated-regenerated fibers, the receptors were distributed as regular branches similar to denervated muscles without lidocaine treatment. These findings suggested that nerve-dependent mechanisms were involved in the changes in receptor distribution seen in regenerated muscle fibers after lidocaine treatment, and that a similar phenomenon could explain the changes in receptor distribution seen in dystrophic muscle fibers.     

Localization of surface and internal acetylcholine receptors in developing fast and slow muscles of the chick embryo

The localization of surface and internal acetylcholine (ACh) receptors was investigated in the developing anterior and posterior latissimus dorsi (ALD and PLD) muscles in the chick embryo (11, 15, and 19 days) by autoradiography using ¹²⁵I-α-bungarotoxin (BTX). At 11 days, ACh receptors were already preferentially at neuromuscular junctions. Internal ACh receptors, measured using muscles made permeable to BTX by saponin treatment, were scattered throughout the length of each muscle fiber with or without a slight increase in their number around neuromuscular junctions. Quantitative analysis of grains in montage electron micrographs of muscle fibers from 11-day embryos revealed that intracellular specific BTX binding sites were the Golgi complex and multivesicular bodies. The number of silver grains over the Golgi complex decreased greatly after puromycin treatment of organ-cultured muscles. These findings strongly suggest that the Golgi complex is one of the sites involved in the production of ACh receptors in the skeletal muscle cells in vivo. Multivesicular bodies are assumed to be involved in the degradation of ACh receptors.     

Common Molecular Mechanisms in Field- and Agrin-Induced Acetylcholine Receptor Clustering

1. The aggregation of acetylcholine receptors at the developing neuromuscular junction is critical to the development and function of this synapse. In vitro studies have shown that receptor aggregation can be induced by the finding of agrin to the muscle cell surface and by the electric field-induced concentration of a (nonreceptor) molecule at the cathodal cell pole.2. We report here on the interaction between agrin binding and electric fields with respect to the distribution of receptors and agrin binding sites.3. (a) Pretreatment of cells with agrin completely blocks the development of field-induced receptor clusters. (b) Field-induced aggregation of receptors precedes the field-induced aggregation of agrin binding sites by approximately 30min. (c) Electric fields prevent agrin-induced receptor clustering despite the presence of agrin binding sites and freely diffusing receptors.4. These results indicate that another membrane component—but not the agrin binding site and not the receptor—is required for agrin-induced receptor clustering. They also suggest that electric fields and agrin cause receptor clustering via common molecular mechanisms.     

Azidophenantridinium compounds as photoaffinity labels of cholinergic proteins

The synthesis of diazidopropidium and diazidoethidium is described. The applicability of these compounds as photoaffinity labels for cholinergic proteins has been investigated: diazidopropidium inhibits neuromuscular transmission. This inhibition is reversible if the compound is applied in the dark but becomes irreversible after irradiation with white light. Inhibition is accompanied by a disappearance of miniature endplate potentials. Electrophysiological analysis of this effect indicates that diazidopropidium acts postsynaptically by blocking the acetylcholine receptors. At the molecular level the action of diazidopropidium and diazidoethidium on acetylcholinesterase has been investigated: both compounds appear to bind to a peripheral acetylcholine binding site of this enzyme. Binding of ¹²⁵I-labeled α-neurotoxin from Naja naja siamensis to purified membranes from Torpedo californica electric tissue rich in acetylcholine receptors is diminished after incubation and irradiation with diazidopropidium. About half of the toxin binding sites appear to be blocked by the photoaffinity label.     

Formation of functional synaptic connections between cultured cortical neurons from agrin-deficient mice.

Numerous studies suggest that the extracellular matrix protein agrin directs the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). Strong support for this hypothesis comes from the observation that the high density of acetylcholine receptors (AChR) normally present at the neuromuscular junction fails to form in muscle of embryonic agrin mutant mice. Agrin is expressed by many populations of neurons in the central nervous system (CNS), suggesting that this molecule may also play a role in neuron-neuron synapse formation. To test this hypothesis, we examined synapse formation between cultured cortical neurons isolated from agrin-deficient mouse embryos. Our data show that glutamate receptors accumulate at synaptic sites on agrin-deficient neurons. Moreover, electrophysiological analysis demonstrates that functional glutamatergic and gamma-aminobutyric acid (GABA)ergic synapses form between mutant neurons. The frequency and amplitude of miniature postsynaptic glutamatergic and GABAergic currents are similar in mutant and age-matched wild-type neurons during the first 3 weeks in culture. These results demonstrate that neuron-specific agrin is not required for formation and early development of functional synaptic contacts between CNS neurons, and suggest that mechanisms of interneuronal synaptogenesis are distinct from those regulating synapse formation at the neuromuscular junction.     

Changes in synaptic potential properties during acetylcholine receptor accumulation and neurospecific interactions in Xenopus nerve-muscle cell culture

Acetylcholine receptors in the muscle cell membrane accumulate at the nerve contact area in Xenopus cell cultures. The correlation between spontaneous synaptic potential properties and extent of acetylcholine receptor accumulation was studied. Small and infrequent miniature endplate potentials were measured before acetylcholine receptor accumulation which was observed with fluorescence microscopy using tetramethylrhodamine-conjugated α-bungarotoxin. As acetylcholine receptors accumulate at the nerve contact area, these synaptic potentials become larger and their frequency increases dramatically. In nerve-contacted muscle cells where spontaneous synaptic activity could not be detected, extensive acetylcholine receptor accumulation was not found at sites of nerve contact. Furthermore, muscle cells which exhibited extensive acetylcholine receptor accumulation along the nerve always produced miniature endplate potentials. Thus acetylcholine receptor accumulation and the presence of miniature endplate potentials were strongly correlated. Noncholinergic neurons from dorsal root ganglia did not form functional synaptic contacts with muscle cells nor acetylcholine receptor accumulation along the path of contact. Furthermore, explants from tadpole spinal cord formed functional synaptic contacts with muscle cells but rarely caused AChR localization. These data are discussed in terms of developmental processes during neuromuscular junction formation.     

Role of Myosin Va in the Plasticity of the Vertebrate Neuromuscular Junction In Vivo

Background

Myosin Va is a motor protein involved in vesicular transport and its absence leads to movement disorders in humans (Griscelli and Elejalde syndromes) and rodents (e.g. dilute lethal phenotype in mice). We examined the role of myosin Va in the postsynaptic plasticity of the vertebrate neuromuscular junction (NMJ).

Methodology/Principal Findings

Dilute lethal mice showed a good correlation between the propensity for seizures, and fragmentation and size reduction of NMJs. In an aneural C2C12 myoblast cell culture, expression of a dominant-negative fragment of myosin Va led to the accumulation of punctate structures containing the NMJ marker protein, rapsyn-GFP, in perinuclear clusters. In mouse hindlimb muscle, endogenous myosin Va co-precipitated with surface-exposed or internalised acetylcholine receptors and was markedly enriched in close proximity to the NMJ upon immunofluorescence. In vivo microscopy of exogenous full length myosin Va as well as a cargo-binding fragment of myosin Va showed localisation to the NMJ in wildtype mouse muscles. Furthermore, local interference with myosin Va function in live wildtype mouse muscles led to fragmentation and size reduction of NMJs, exclusion of rapsyn-GFP from NMJs, reduced persistence of acetylcholine receptors in NMJs and an increased amount of punctate structures bearing internalised NMJ proteins.

Conclusions/Significance

In summary, our data show a crucial role of myosin Va for the plasticity of live vertebrate neuromuscular junctions and suggest its involvement in the recycling of internalised acetylcholine receptors back to the postsynaptic membrane.     

Assembly and trafficking of nicotinic acetylcholine receptors (Review)

Nicotinic acetylcholine receptors (nAChRs) are members of an extensive super-family of neurotransmitter-gated ion channels. In humans, nAChRs are expressed within the nervous system and at the neuromuscular junction and are important targets for pharmaceutical drug discovery. They are also the site of action for neuroactive pesticides in insects and other invertebrates. Nicotinic receptors are complex pentameric transmembrane proteins which are assembled from a large family of subunits; seventeen nAChR subunits (α1-α10, β1-β4, γ, δ and ε) have been identified in vertebrate species. This review will discuss nAChR subunit diversity and factors influencing receptor assembly and trafficking.     

Functional properties of acetylcholine receptors coexpressed with the 43K protein in heterologous cell systems.

The nicotinic acetylcholine (ACh) receptor is an integral membrane protein which mediates synaptic transmission at the skeletal neuromuscular junction. A key event in the development of the neuromuscular junction is the formation of high density aggregates of ACh receptors in the postsynaptic membrane. Receptor clustering has been attributed, in part, to their association with a peripheral membrane protein of Mr 43,000 (43K protein). We have addressed whether the association of the 43K protein can alter the single channel properties of the ACh receptor, and thus influence neuromuscular transmission at developing synapses, by expressing ACh receptors with and without the 43K protein in heterologous expression systems. We found that coexpression of the 43K protein with the receptor did not significantly alter either its single channel conductance or its mean channel open time. This was true in oocytes and also in COS cells where it was possible to localize 43K-induced clusters by fluorescence microscopy and to record from those clustered receptors. These data are in agreement with previous single channel studies which have shown that the properties of diffusely distributed and clustered receptors in native muscle cells from both mice and Xenopus do not differ.     

Cytoplasmic actin in postsynaptic structures at the neuromuscular junction

We used an antibody prepared against Aplysia (mollusc) body-wall actin that specifically reacts with certain forms of cytoplasmic actin in mammalian cells to probe for the presence of actin at the neuromuscular junction. Immunocytochemical studies showed that actin or an actinlike molecule is concentrated at neuromuscular junctions of normal and denervated adult rat muscle fibers. Actin is present at the neuromuscular junctions of fibers of developing diaphragm muscles as early as embryonic day 18, well before postsynaptic folds are formed. These results suggest that cytoplasmic actin may play a role in the clustering or stabilization of acetylcholine receptors at the neuromuscular junction.     

Antibodies to nicotinic acetylcholine receptors used to probe the structural and functional relationships between brain α-bungarotoxin binding sites and nicotinic receptors

Antibodies against peripheral nicotinic acetylcholine receptors (nAChR) were used to determine the proportion of brain α-bungarotoxin binding sites that are immunologically related to the peripheral nAChR. The α-bungarotoxin binding component partially purified from rat brain was labelled with [¹²⁵I]α-bungarotoxin and reacted with increasing concentrations of rabbit anti(nAChR) antisera. At least 75% of the brain protein could be immunoprecipitated by rabbit anti(rat muscle junctional nAChR) antiserum (M) whereas an antiserum against Torpedo nAChR (J) was without effect and clearly failed to cross-react with the brain component. Both antisera precipitated 100% of [¹²⁵I]α-bungarotoxin-labelled nAChR from Torpedo marmorata. The lower precipitation of the brain protein was not a consequence of [¹²⁵I]α-bungarotoxin dissociating during the precipitation. We conclude that the majority of α-bungarotoxin binding sites in brain are clearly recognised by the crossreacting antiserum.Release of [³H]dopamine from striatal synaptosomes could be elicited by nicotine in a dose-dependent manner and the response was prevented by the ganglionic blocker mecamylamine, although antagonism by α-bungarotoxin was less clearcut. Preincubation of the synaptosomes with antiserum M resulted in a statistically significant decrease in the [³H]dopamine response to nicotine at all agonist concentrations tested. Antiserum J, however, had no consistent effect on the response. Thus the actions of the antisera parallel their ability to recognise the brain α-bungarotoxin binding component. We conclude that the cholinergic regulation of dopamine release is in part mediated through a nAChR that is immunologically related to the nAChR of the neuromuscular junction and to the α-bungarotoxin binding component that can be isolated from rat brain.     

Formation of functional synaptic connections between cultured cortical neurons from agrin‐deficient mice

Numerous studies suggest that the extracellular matrix protein agrin directs the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). Strong support for this hypothesis comes from the observation that the high density of acetylcholine receptors (AChR) normally present at the neuromuscular junction fails to form in muscle of embryonic agrin mutant mice. Agrin is expressed by many populations of neurons in the central nervous system (CNS), suggesting that this molecule may also play a role in neuron–neuron synapse formation. To test this hypothesis, we examined synapse formation between cultured cortical neurons isolated from agrin‐deficient mouse embryos. Our data show that glutamate receptors accumulate at synaptic sites on agrin‐deficient neurons. Moreover, electrophysiological analysis demonstrates that functional glutamatergic and gamma‐aminobutyric acid (GABA)ergic synapses form between mutant neurons. The frequency and amplitude of miniature postsynaptic glutamatergic and GABAergic currents are similar in mutant and age‐matched wild‐type neurons during the first 3 weeks in culture. These results demonstrate that neuron‐specific agrin is not required for formation and early development of functional synaptic contacts between CNS neurons, and suggest that mechanisms of interneuronal synaptogenesis are distinct from those regulating synapse formation at the neuromuscular junction. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 547–557, 1999     

Binding-gating coupling in a nondesensitizing α7 nicotinic receptor: A single channel pharmacological study

The highly conserved αLys145 has been suggested to play an important role in the early steps of activation of the nicotinic acetylcholine receptor (nAChR) by acetylcholine. Both macroscopic and single channel currents were recorded in the slowly desensitizing mutants L248T- and K145A-L248T-α7 receptors expressed in Xenopus oocytes. On ACh-evoked currents, substitution of Lys145 by alanine showed the same effects that in wild type receptors: moderately decreased gating function and a more-than-expected loss of ACh potency, thus validating the experimental model. Single channel analysis quantitatively agreed with macroscopic data and revealed that impaired gating function in the double mutant α7K145A/L248T is the consequence of a slower opening rate, β. Several nicotinic agonists were also studied, showing important features. Particularly, dimethylphenylpiperazinium (DMPP), acting as an antagonist in α7K145A, became a full agonist in α7K145A/L248T. Single channel analysis of DMPP-evoked currents showed effects of Lys145 removal similar to those observed with ACh. Data suggest that α7Lys145 facilitates the early steps of channel activation. Moreover, the slowly desensitizing mutant α7L248T could be an interesting tool for the study of channel activation in α7 receptors. Nevertheless, its extensively altered pharmacology precludes the simple extrapolation of pharmacological data obtained in singly mutated α7 receptors.     

Sorbs1 and -2 Interact with CrkL and Are Required for Acetylcholine Receptor Cluster Formation

Crk and CrkL are noncatalytic adaptor proteins necessary for the formation of neuromuscular synapses which function downstream of muscle-specific kinase (MuSK), a receptor tyrosine kinase expressed in skeletal muscle, and the MuSK binding protein Dok-7. How Crk/CrkL regulate neuromuscular endplate formation is not known. To better understand the roles of Crk/CrkL, we identified CrkL binding proteins using mass spectrometry and have identified Sorbs1 and Sorbs2 as two functionally redundant proteins that associate with the initiating MuSK/Dok-7/Crk/CrkL complex, regulate acetylcholine receptor (AChR) clustering in vitro, and are localized at synapses in vivo.     

A novel labeling approach identifies three stability levels of acetylcholine receptors in the mouse neuromuscular junction in vivo

Background

The turnover of acetylcholine receptors at the neuromuscular junction is regulated in an activity-dependent manner. Upon denervation and under various other pathological conditions, receptor half-life is decreased.

Methodology/Principal Findings

We demonstrate a novel approach to follow the kinetics of acetylcholine receptor lifetimes upon pulse labeling of mouse muscles with ¹²⁵I-α-bungarotoxin in vivo. In contrast to previous assays where residual activity was measured ex vivo, in our setup the same animals are used throughout the whole measurement period, thereby permitting a dramatic reduction of animal numbers at increased data quality. We identified three stability levels of acetylcholine receptors depending on the presence or absence of innervation: one pool of receptors with a long half-life of ∼13 days, a second with an intermediate half-life of ∼8 days, and a third with a short half-life of ∼1 day. Data were highly reproducible from animal to animal and followed simple exponential terms. The principal outcomes of these measurements were reproduced by an optical pulse-labeling assay introduced recently.

Conclusions/Significance

A novel assay to determine kinetics of acetylcholine receptor turnover with small animal numbers is presented. Our data show that nerve activity acts on muscle acetylcholine receptor stability by at least two different means, one shifting receptor lifetime from short to intermediate and another, which further increases receptor stability to a long lifetime. We hypothesize on possible molecular mechanisms.     

Degradation of the acetylcholine receptor in cultured muscle cells: Selective inhibitors and the fate of undegraded receptors

To learn more about the pathway for degradation of an intrinsic membrane protein, we studied in cultured chick myotubes the effects of certain protease inhibitors and chloroquine (an inhibitor of lysosomal function) on degradation of the acetylcholine receptor measured with the specific ligand ¹²⁵I-α-bungarotoxin. Leupeptin, chymostatin, anti-pain and chloroquine decreased by 2–10 fold the rate of degradation of the acetylcholine receptor-¹²⁵I-α-bungarotoxin complex to ¹²⁵I-tyrosine (p < 0.01). After removing the inhibitors, the degradative rate returned to control levels. Leupeptin and chloroquine did not appear toxic to the cells; these agents did not alter the overall rate of protein synthesis, and leupeptin did not decrease the incorporation of receptors into the surface membrane. Therefore these inhibitors probably inhibit the degradative process selectively. A lysosomal site for receptor degradation appears probable, since chloroquine slows this process; leupeptin, chymostatin and antipain all inhibit cathepsin B; and chloroquine and to a lesser extent leupeptin altered the ultrastructural appearance of this organelle. Cultures labeled with ¹²⁵I-α-bungarotoxin and then incubated with leupeptin or chloroquine contained more radioactive protein than control cells. This material co-electrophoresed with bungarotoxin on sodium dodecylsulfate-urea-polyacrylamide gels. Thus myotubes exposed to these inhibitors seemed to accumulate undegraded bungarotoxin. They did not, however, contain more acetylcholine receptors on their surface. Instead, the inhibitor-treated cells accumulate toxin and receptors at some internal site. Thus treatment with such inhibitors does not appear to be a useful approach to the therapy of myasthenia gravis. The additional ¹²⁵I-toxin found in cells incubated with leupeptin or chloroquine was less accessible to exogenous protease than the toxin bound to control cells and was more resistant to extraction by Triton X-100. Since internalization of the receptor continued in the presence of these inhibitors, this process must not be coupled tightly to subsequent proteolysis. Measurement of receptors within cells not exposed to ¹²⁵I-α-bungarotoxin showed that incubation of myotubes with leupeptin or chloroquine for 48 hr increased the number of internal bungarotoxin-binding sites 2–11 fold (p < 0.001). Thus cells treated with these agents accumulate receptors intracellularly in a form that sediments at 35,000 × g. Electron microscopy showed that these treated myotubes contain 3–6 times more coated vesicles within their cytoplasm than control cells (p < 0.001). Thus chloroquine and leupeptin may retard receptor degradation in part by interfering with the fusion of coated vesicles with lysosomes.