Rotational diffusion of acetylcholine receptors on cultured rat myotubes

The rotational mobility of acetylcholine receptors (AChR) in the plasma membrane of living rat myotubes in culture is measured in this study by polarized fluorescence recovery after photobleaching (PFRAP). These AChR are known to exist in two distinct classes, evident by labeling with rhodamine alpha-bungarotoxin; clustered AChR that are aggregated in a pattern of highly concentrated speckles and streaks, with each cluster occupying an area of approximately 1,000 microns 2; and nonclustered AChR that appear as diffuse labeling. PFRAP results reported here show that: (a) most clustered AChR (approximately 86%) are rotationally immobile within a time scale of at least several seconds; and (b) most nonclustered AChR (approximately 76%) are rotationally mobile with characteristic times ranging from less than 50 ms to 0.1 s. External cross-linking with the tetravalent lectin concanavalin A immobilizes many nonclustered AChR. PFRAP experiments in the presence of carbachol or cytochalasin D show that the restraints to rotational motion in clusters are remarkably immune to treatments that disperse clusters or disrupt cytoplasmic actin. The experiments also demonstrate the feasibility of using PFRAP to measure rotational diffusion on selected microscopic areas of living nondeoxygenated cells labeled with standard fluorescence probes over a very wide range of time scales, and they also indicate what technical improvements would make PFRAP even more practicable.     

Agonists cause endocytosis of nicotinic acetylcholine receptors on cultured myotubes

Regulated trafficking of neurotransmitter receptors in excitable cells may play an important role in synaptic plasticity. In addition, agonist‐induced endocytosis of nicotinic acetylcholine receptors (nAChRs) in particular might be involved in nicotine tolerance and addiction. The existing evidence concerning regulated internalization of cell‐surface nAChRs is indirect and equivocal, however. In the present study, radioligand binding and fluorescence microscopy were used to show that agonists cause substantial endocytosis of nAChRs on cultured myotubes. Exposure to carbachol or nicotine caused a decrease in the intensity of fluorescent labeling of clusters of cell‐surface nAChRs that was blocked by low temperature. Overall, myotubes exposed to carbachol or nicotine bound 50–70% less [¹²⁵I]‐α‐bungarotoxin on the cell surface than untreated cells. The effect of carbachol was significant within 5 min, increased progressively for at least 4 h, and had a sensitivity of 100 nM or less. Exposure to carbachol caused the appearance or dramatic expansion of an intracellular pool of nAChRs, which were localized to discrete, largely perinuclear structures. A pulse‐chase labeling protocol allowed the selective labeling and localization of nAChRs that had been internalized from the cell surface. In untreated cells, very little internalization of nAChRs occurred over a period of 3 h, indicating that constitutive endocytosis of receptors over this period was minimal. Exposure to carbachol, however, caused a dramatic increase in the endocytosis of nAChRs. These results provide direct evidence that agonists, including the tobacco alkaloid nicotine, can cause substantial endocytosis of cell‐surface nAChRs. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 212–223, 2001     

Ionic properties of the acetylcholine receptor in cultured rat myotubes

The acetylcholine reversal potential (Er) of cultured rat myotubes is -3mV. When activated, the receptor is permeable to K+ and Na+, but not to Cl- ions. Measurement of Er in Tris+-substituted, Na-free medium also indicated a permeability to Tris+ ions. Unlike adult frog muscle the magnitude of Er was insensitive to change in external Ca++ (up to 30 mM) or to changes in external pH (between 6.4 and 8.9). The equivalent circuit equation describing the electrical circuit composed of two parallel ionic batteries (EK and ENa) and their respective conductances (gK and gNa), which has been generally useful in describing the Er of adult rat and frog muscle, could also be applied to rat myotubes when Er was measured over a wide range of external Na+ concentrations. The equivalent circuit equation could not be applied to myotubes bathed in media of different external K+ concentrations. In this case, the Er was more closely described by the Goldman constant field equation. Under certain circumstances, it is known that the receptor in adult rat and frog muscle can be induced to reversibly shift from behavior described by the equivalent circuit equation to that described by the Goldman equation. Attempts to similarly manipulate the responses of cultured rat myotubes were unsussessful. These trials included a reduction in temperature (15 degress C), partial alpha-bungarotoxin blodkade, and activation of responses with the cholinergic agonist, decamethonium.     

Agonists cause endocytosis of nicotinic acetylcholine receptors on cultured myotubes.

Regulated trafficking of neurotransmitter receptors in excitable cells may play an important role in synaptic plasticity. In addition, agonist-induced endocytosis of nicotinic acetylcholine receptors (nAChRs) in particular might be involved in nicotine tolerance and addiction. The existing evidence concerning regulated internalization of cell-surface nAChRs is indirect and equivocal, however. In the present study, radioligand binding and fluorescence microscopy were used to show that agonists cause substantial endocytosis of nAChRs on cultured myotubes. Exposure to carbachol or nicotine caused a decrease in the intensity of fluorescent labeling of clusters of cell-surface nAChRs that was blocked by low temperature. Overall, myotubes exposed to carbachol or nicotine bound 50-70% less [(125)I]-alpha-bungarotoxin on the cell surface than untreated cells. The effect of carbachol was significant within 5 min, increased progressively for at least 4 h, and had a sensitivity of 100 nM or less. Exposure to carbachol caused the appearance or dramatic expansion of an intracellular pool of nAChRs, which were localized to discrete, largely perinuclear structures. A pulse-chase labeling protocol allowed the selective labeling and localization of nAChRs that had been internalized from the cell surface. In untreated cells, very little internalization of nAChRs occurred over a period of 3 h, indicating that constitutive endocytosis of receptors over this period was minimal. Exposure to carbachol, however, caused a dramatic increase in the endocytosis of nAChRs. These results provide direct evidence that agonists, including the tobacco alkaloid nicotine, can cause substantial endocytosis of cell-surface nAChRs.     

Specificity of neuronal factors which aggregate acetylcholine receptors on cultured myotubes

Neuronal factors from conditioned medium of neuroblastoma X glioma hybrid cells or isolated from embryonic pig brain aggregate acetylcholine receptors (AChR) on cultured chicken and rat myotubes. A membrane surface protein labelled with a fluorescent monospecific antibody was not aggregated with the same treatment. Antibodies against AChR block the action of the aggregating factors but do not produce large aggregates themselves. These findings indicate that the factors specifically react with the AChR on developing myotubes.     

Metabolic properties of human acetylcholine receptors can be characterized on cultured human muscle

Experiments examining acetylcholine receptor (AChR) metabolism in tissue culture have hitherto been limited to animal systems. For many studies, the human AChR on human skeletal muscle provides a more physiologic target. However, previous studies suggested that the levels of AChR produced on cultured human muscle were inadequate for metabolic studies. We demonstrate here that the metabolism of human acetylcholine receptors can be analysed on pure human muscle fibers that develop in tissue culture. Degradation of AChR follows first-order kinetics and is inhibited 85% by leupeptin, demonstrating that proteolysis of human AChR occurs in the lysosome. New AChR continue to appear on the cell surface for 3 h in the presence of cycloheximide, indicating the existence of a pool of intracellular AChR destined for the cell membrane. This pool is equivalent to approximately one-third of the AChR present on the surface of the cell. At any given time, the rate of AChR accumulation on the cell surface can be quantitatively accounted for by the rates of synthesis and degradation. Our results demonstrate that studies on the effects of hormones, neurotoxins or antibodies from patients with autoimmune neuromuscular diseases are now possible with human AChR which develop on intact human muscle myotubes formed in tissue culture.     

Immunogold surface replica study on the distribution of acetylcholine receptors in cultured rat myotubes

We studied the distribution of acetylcholine receptors (AchRs) in rat skeletal muscles, Torpedo membrane fractions, and cultured rat skeletal myotubes. AchRs were first exposed to alpha-bungarotoxin (alpha-BTX) followed by anti-alpha-BTX antibodies. Bound antibodies were visualized with FITC-conjugated goat anti-rabbit antibodies for light microscopy or with peroxidase-conjugated goat anti-rabbit or gold-labeled protein A for electron microscopy. The protocol developed for the present studies detected AchRs with high specificity. In addition, we combined post-fixation immunogold cytochemical and surface replication techniques to study the distribution of AchRs on the dorsal surface of cultured myotubes in detail. Two distinct distribution patterns of AchRs on the cell surfaces of the myotubes were revealed; AchRs were either diffusely distributed or clustered. Dispersed AchRs usually surrounded clusters of AchRs. The AchRs in the clusters were characteristically arranged, and small aggregates of AchRs could also be observed within the clusters. The techniques used in this study are appropriate for studying the dynamics of AchR clustering.     

Insertion and internalization of acetylcholine receptors at clustered and diffuse domains on cultured myotubes

Two populations of acetylcholine receptors (AChRs) are present in cultured myotubes. One forms large aggregates or clusters and the other has a much lower density of AChRs, which are diffusely distributed. Both clustered and diffuse AChRs are inserted and removed (internalized) from the sarcolemma. To determine the insertion and removal rates of AChRs in these two plasma membrane domains, we used a double label technique to distinguish and quantitate newly inserted and "old" AChRs. Application of our method revealed that the rate of AChR internalization is the same at the clustered and diffuse regions of the plasma membrane, whereas the rate of insertion is threefold greater at the clusters than elsewhere in the plasma membrane. Thus, the increase in AChR number at the clusters is not due to an increase in their half-life, but to an increase in their rate of insertion.     

Mobility and detergent extractability of acetylcholine receptors on cultured rat myotubes: a correlation

On aneurally cultured rat primary myotubes, 10% of the acetylcholine receptors (AChR) are found aggregated and immobilized in endogenous clusters. The remaining receptors are diffusely distributed over the cell membrane and the majority of these are free to diffuse in the plane of the membrane. This study correlates the mobility of AChR (as measured with the fluorescence photobleaching recovery technique, FPR) with the detergent extractability of this receptor. Gentle detergent extraction of the cells removes the lipid membrane and the soluble cytoplasmic proteins but leaves an intact cytoskeletal framework on the substrate. Two studies indicate a correlation between mobility and extractability: (a) mobility of diffusely distributed AChR decreases as myotubes age in culture; previous work showed that extractability of AChR decreases as myotubes age in culture (Prives, J., C. Christian, S. Penman, and K. Olden, 1980, In Tissue Culture in Neurobiology, E. Giacobini, A. Vernadakis, and A. Shahar, editors, Raven Press, New York, 35-52); (b) mobility of clustered AChR increases when cells are treated with metabolic inhibitors such as sodium azide (NaN3); extractability of clustered AChR also increases with this treatment. From these results we suggest the involvement of a cytoskeletal framework in the immobilization of AChR on the cell surface.     

The pericellular boundary layer modulates acetylcholine receptor stability in cultured myotubes

Degradation of acetylcholine receptors in cultured chicken myotubes was measured by release into the medium of radioactivity from 125I-labeled alpha-bungarotoxin. Disturbance of the pericellular boundary layer by stirring of the culture medium shortened the half-life of receptor in the membrane from 24 to 12 h. The effect could not be explained by dissociation of toxin-receptor complexes or by conditioning of the bulk phase of the medium. The rates of synthesis and degradation of total cell protein and the degradation of lactoperoxidase-iodinated surface protein were not affected by medium stirring. The loss of glucosamine-labeled material from the cells was enhanced by stirring, however, and this resulted entirely from the increased shedding of high molecular weight glycosubstances from the cells. Cells in stirred cultures contained lower levels of surface coat material stainable with colloidal thorium. These results indicate that glycosubstances of the pericellular matrix protect ACh receptors from degradation.     

The relationship of the postsynaptic 43K protein to acetylcholine receptors in receptor clusters isolated from cultured rat myotubes

We have examined the relationship of acetylcholine receptors (AChR) to the Mr 43,000 receptor-associated protein (43K) in the AChR clusters of cultured rat myotubes. Indirect immunofluorescence revealed that the 43K protein was concentrated at the AChR domains of the receptor clusters in intact rat myotubes, in myotube fragments, and in clusters that had been purified approximately 100-fold by extraction with saponin. The association of the 43K protein with clustered AChR was not affected by buffers of high or low ionic strength, by alkaline pHs up to 10, or by chymotrypsin at 10 micrograms/ml. However, the 43K protein was removed from clusters with lithium diiodosalicylate or at alkaline pH (greater than 10). Upon extraction of 43K, several changes were observed in the AChR population. Receptors redistributed in the plane of the muscle membrane in alkali-extracted samples. The number of binding sites accessible to an anti-AChR monoclonal antibody directed against cytoplasmic epitopes (88B) doubled. Receptors became more susceptible to digestion by chymotrypsin, which destroyed the binding sites for the 88B antibody only after 43K was extracted. These results suggest that in isolated AChR clusters the 43K protein covers part of the cytoplasmic domain of AChR and may contribute to the unique distribution of this membrane protein.     

Organization of acetylcholine receptor clusters in cultured rat myotubes is calcium dependent

The effect of extracellular Ca2+ concentration and myasthenic globulin on the distribution and appearance of acetylcholine receptor (AChR) clusters on rat myotubes was studied with tetramethyl-rhodamine-labeled alpha BTX. Low Ca2+ medium (2.5 X 10(-5) M) caused a time-dependent loss of AChR clusters, and a concomitant increase in small punctate areas of fluorescence. High Ca2+ concentrations (1.5 X 10(-2) M) increased the size of AChR clusters without altering AChR synthesis. These changes were not observed with other divalent ions. In the presence of myasthenic globulin, the rate of AChR turnover increases, and AChR clusters are rapidly dispersed. High Ca2+ concentration partially protects the AChR clusters from dispersal and decreases the rate of receptor turnover.     

Microtubules, microfilaments and the transport of acetylcholine receptors in embryonic myotubes

Both microtubules and microfilaments have been implicated in the exocytotic and endocytotic transport of coated and smooth surfaced membrane vesicles. We have reexamined this question by using specific pharmacological agents to disrupt these filaments and assess the effect on the movement of acetylcholine receptor (AChR) containing membrane vesicles in embryonic chick myotubes. Myotube cultures treated with nocodazole (0.6 microgram/ml) or colcemid (0.5 microgram/ml) (to disrupt microtubules) show only a 20-25% decrease in the number of cell surface AChRs after 48 h. Addition of chick brain extract (CBE) to cultured myotubes causes a significant increase in the total number of cell surface AChRs (measured by [125I]alpha-bungarotoxin (alpha-BGT) binding), thus providing us with a way to manipulate receptor and transport vesicle populations. Cultures treated with CBE plus nocodazole or colcemid show a 1.7-fold increase in AChR number over drug treatment alone, the same increase seen in cultures treated with CBE alone, although the total number remains about 20-25% less than that seen in control cultures. In cultures treated with cytochalasin D (0.2 microgram/ml) or dihydrocytochalasin B (5.0 micrograms/ml) (to disrupt microfilaments), 35 and 65% decreases in cell surface AChR number were seen after 48 h. However, in cultures treated with CBE and cytochalasin D, the same total number of AChRs was found as in cultures treated with CBE alone. No significant effects were seen with any of these drugs on the receptor incorporation rate (the appearance of new alpha-BGT-binding sites) after 6 h. The half-life for AChRs in control cultures was 23.0 h. In cytochalasin D and dihydrocytochalasin B it was 21.9 and 19.0 h, respectively; with colcemid and nocodazole, it increased to 37.1 and 28.1 h. These results suggest that non-myofibrillar microfilament bundles are not involved in the movement of AChR-containing membrane vesicles; further, the small effects seen with microtubule inhibitors tend to rule out a major role for microtubules in this transport.     

Agents that activate protein kinase C reduce acetylcholine sensitivity in cultured myotubes

We have examined acetylcholine (ACh)-elicited potentials or currents in current- or voltage-clamped cultured myotubes exposed to 12-O- tetradecanoyl-phorbol-13-acetate (TPA), a potent tumor promoter that activates protein kinase C. Although this agent had little action on either membrane resting potential or electrical resistance, a reversible decrease in ACh sensitivity was induced on 3-4-d-old chick myotubes. Depression of transmitter action by TPA was extended to 7-8-d mouse myotubes only when they were treated with phosphatidylserine. Glyceryl dioleate had effects on myotubes similar to those of TPA but with a reduced efficacy. We conclude that the activation of protein kinase C might be involved with the capacity of ACh receptors to respond to transmitter stimulation.     

Direct observation of the rapid aggregation of acetylcholine receptors on identified cultured myotubes after exposure to embryonic brain extract

We have directly observed the redistribution of acetylcholine receptors (AChR) on the surface of cultured myobutbes, induced by a soluble brain extract. The AChR were fluorescently labeled with rhodamine-conjugated α-bungarotoxin and viewed under low incident illumination with a video image intensification system. The results of our sequential observations indicate that AChR aggregates can be assembled rapidly (30–120 min) from mobile, diffuse AChR. This assembly was characterized by the initial formation of microaggregates (<1 μm diameter) that increased in number and coalesced or grew to form larger aggregates. The redistribution of fluorescently labeled AChR was completely inhibited by illumination of cells at levels used for conventional fluorescence micrography and could be observed only by using low light levels.     

Acetylcholine stimulates phosphatidylinositol turnover at nicotinic receptors of cultured myotubes

Acetylcholine treatment of [3H]inositol pre-labelled cultured chick embryo myotubes results in the stimulation of phosphatidylinositol breakdown, as shown by the measurement of inositol-1-phosphate accumulating in the presence of lithium. The described effect is dependent on agonist concentration and incubation time, and is inhibited by tubocurarine and alpha-bungarotoxin. The activation of phosphatidylinositol breakdown by acetylcholine at extrajunctional nicotinic receptors is likely to be involved in the modulation of the functional activity of the receptor.     

Aggregating factor from Torpedo electric organ induces patches containing acetylcholine receptors, acetylcholinesterase, and butyrylcholinesterase on cultured myotubes

A factor in extracts of the electric organ of Torpedo californica causes the formation of clusters of acetylcholine receptors (AChRs) and aggregates of acetylcholinesterase (AChE) on myotubes in culture. In vivo, AChRs and AChE accumulate at the same locations on myofibers, as components of the postsynaptic apparatus at neuromuscular junctions. The aim of this study was to compare the distribution of AChRs, AChE, and butyrylcholinesterase (BuChE), a third component of the postsynaptic apparatus, on control and extract-treated myotubes. Electric organ extracts induced the formation of patches that contained high concentrations of all three molecules. The extract-induced aggregation of AChRs, AChE, and BuChE occurred in defined medium, and these components accumulated in patches simultaneously. Three lines of evidence indicate that a single factor in the extracts induced the aggregation of all three components: the dose dependence for the formation of patches of AChRs was the same as that for patches of AChE and BuChE; the AChE- and BuChE-aggregating activities co-purified with the AChR-aggregating activity; and all three aggregating activities were immunoprecipitated at the same titer by a monoclonal antibody against the AChR-aggregating factor. We have shown previously that this monoclonal antibody binds to molecules concentrated in the synaptic cleft at neuromuscular junctions. Taken together, these results suggest that during development and regeneration of myofibers in vivo, the accumulation at synaptic sites of at least three components of the postsynaptic apparatus, AChRs, AChE, and BuChE, are all triggered by the same molecule, a molecule similar if not identical to the electric organ aggregating factor.     

Physiochemical and immunological properties of acetylcholine receptors from human muscle

Summary The acetylcholine receptor protein from human muscle was extracted with the non-ionic detergent Triton X-100 and purified by affinity chromatography on -Naja toxin sepharose 4B. Further purification on Dicap-MP sepharose 4B, a choline analog compound, led to ACHR preparations with specific activities of 2–7 nmol/mg protein. The isolated receptor, labeled with ¹²⁵I--bungarotoxin was characterized by different methods and compared to ACHRs from Torpedo californica electroplax and rat-denervated skeletal muscle. Gel filtration on Ultrogel AcA 34 resulted in a stokes radius of 70 Å for the receptor monomer and 99 Å for the dimeric form. Sucrose density gradient centrifugation showed sedimentation coefficients of 9.1 S and 13.5 S. From these data the molecular weight of the ACHR monomer was estimated as 254 000 D and 540 000 D for the receptor dimer. The isoelectric point of the ¹²⁵I--bgt-ACHR complex was determined by thin-layer isoelectric focussing to be pH 5.Purified ACHRs were used for immunization of rats and mice which developed an EAMG as verified by clinical observation and electrophysical measurements. Sera from the immunized animals as well as from myasthenia gravis patients were subsequently used to compare the cross-reactivity of ACHR preparations from different sources. While antibodies of rats immunized with Torpedo ACHRs cross-reacted with ACHR preparations from rat and human skeletal muscle, antibodies from mice immunized with rat ACHR only reacted with preparations from rats and mice. Antibodies from mice immunized with ACHR of human origin exhibited a broad cross-reactivity, as did antibodies from MG patients.Abbreviations AB antibody - ACHR nicotinic acetylcholine receptor - BSA bovine serum albumin - Dicap-MP methyl-[N-(6-aminocaproyl-6aminocaproyl)-3-amino]pyridinbromide - EAMG experimental autoimmune myasthenia gravis - EDTA ethylenediaminetetraaceticacid - MG myasthenia gravis - PMSF phenylmethylsulfonylfluoride Recipient of a postdoctoral grant from Deutsche Forschungsgemeinschaft; present address: Neurologische Klinik, Medizinische Einrichtungen der Universität Düsseldorf.     

Isolation of acetylcholine receptor clusters in substrate-associated material from cultured rat myotubes using saponin

After exposure of rat myotube cultures to saponin, less than 1% of the cellular protein was found to remain associated with the tissue culture substrate. This substrate-associated material contained approximately 10% of the acetylcholine receptors (AChRs) and greater than 80% of the large, ventral AChR clusters present in the original culture. The domain structure evident in intact cells was maintained in AChR clusters after isolation using saponin. However, vinculin, present at the clusters of intact cells, was absent from isolated clusters. Dodecyl sulfate PAGE showed that substrate-associated material enriched in AChR clusters contained a distinctive set of polypeptides, the major ones electrophoresing with apparent molecular weights of 43,000 and 49,000. Saponin extraction of cultures of established cell lines also yielded substrate-associated material with characteristics particular to the cell type.