[Influence of modeling of gravitational unloading on the postsynaptic acetylcholine receptor organization and acetylcholinesterase activity in neuromuscular synapses of rat fast and slow muscles].

Using immunofluorescent techniques, we have revealed that, after 35 days of rats hindlimb unloading, neuromuscular synapses of fast and slow muscles show enhanced fluorescence intensity and decreased area of fluorescent staining of acetylcholine receptors; increased fluorescent intensity and area of fluorescent staining for acetylcholinesterase. The ratio of the number of postsynaptic acetylcholine receptors and the amount of acetylcholinesterase changed as well as their spatial position in relation to each other. These rearrangements correspond to electrophysiological data on the reduction of the amplitude of the miniature endplate currents in both muscles. Identified synapses restructuring accompanied by a decrease in the volume of muscle fibers. Hindlimb unloading (simulation of hypogravity) leads to an increase in functional activity of acetylcholinesterase on the background of reduced postsynaptic membrane area occupied by acetylcholine receptors. This leads to a decrease in the amplitude of excitatory postsynaptic potentials thereby reducing the nerve-muscle excitation transmission safety factor.

Rapid and modifiable neurotransmitter receptor dynamics at a neuronal synapse in vivo.

Synaptic plasticity underlies the adaptability of the mammalian brain, but has been difficult to study in living animals. Here we imaged the synapses between pre- and postganglionic neurons in the mouse submandibular ganglion in vivo, focusing on the mechanisms that maintain and regulate neurotransmitter receptor density at postsynaptic sites. Normally, synaptic receptor densities were maintained by rapid exchange of receptors with nonsynaptic regions (over minutes) and by continual turnover of cell surface receptors (over hours). However, after ganglion cell axons were crushed, synaptic receptors showed greater lateral mobility and there was a precipitous decline in insertion. These changes led to near-complete loss of synaptic receptors and synaptic depression. Disappearance of postsynaptic spines and presynaptic terminals followed this acute synaptic depression. Therefore, neurotransmitter receptor dynamism associated with rapid changes in synaptic efficacy precedes long-lasting structural changes in synaptic connectivity.

Structure and gating mechanism of the acetylcholine receptor pore.

The nicotinic acetylcholine receptor controls electrical signalling between nerve and muscle cells by opening and closing a gated, membrane-spanning pore. Here we present an atomic model of the closed pore, obtained by electron microscopy of crystalline postsynaptic membranes. The pore is shaped by an inner ring of 5 alpha-helices, which curve radially to create a tapering path for the ions, and an outer ring of 15 alpha-helices, which coil around each other and shield the inner ring from the lipids. The gate is a constricting hydrophobic girdle at the middle of the lipid bilayer, formed by weak interactions between neighbouring inner helices. When acetylcholine enters the ligand-binding domain, it triggers rotations of the protein chains on opposite sides of the entrance to the pore. These rotations are communicated through the inner helices, and open the pore by breaking the girdle apart.

The ultrastructure of patch-clamped membranes: a study using high voltage electron microscopy.

We have developed techniques for studying patch-clamped membranes inside glass pipettes using high voltage electron microscopy (HVEM). To preserve the patch structure with the least possible distortion, we rapidly froze and freeze dried the pipette tip. The pipette is transparent for more than 50 microns from the tip. HVEM images of patches confirm light microscopy observations that the patch is not a bare bilayer, but a membrane-covered bleb of cytoplasm that may include organelles and cytoskeleton. The membrane that spans the pipette is commonly tens of micrometers from the tip of the pipette and occasionally as far as 100 microns. The structure of patches taken from a single cell type is variable but there are consistent differences between patches made from different cell types. With suction applied to the pipette before seal formation, we have seen in the light microscope vesicles swept from the plasmalemma up the pipette. These vesicles are visible in electron micrographs, particularly those made from chick cardiac muscle. Colloidal gold labeling of the patch permitted identification of lectin-binding sites and acetylcholine receptors. In young cultures of Xenopus myocytes, the receptors were diffuse. In 1-wk-old cultures, the receptors formed densely packed arrays. The patch pipette can serve, not only as a recording device, but as a tool for sampling discrete regions of the cell surface. Because the pipette has a constant path length for axial rotation, it is a unique specimen holder for microtomography. We have made preliminary tomographic reconstructions of a patch from Xenopus oocyte.

Three-dimensional structure of the acetylcholine receptor by cryoelectron microscopy and helical image reconstruction.

Long tubular vesicles have been grown from isolated Torpedo postsynaptic membranes, in which the receptors are arranged helically on the vesicle surface. The structures of these tubes have been analyzed by cryoelectron microscopy of specimens embedded in thin films of ice, combined with helical image reconstruction. Complete data sets from tubes belonging to several helical families have been obtained to a resolution of 17 A in all directions. Confirming a preliminary study (Toyoshima, C., and N. Unwin. 1988. Nature (Lond.). 336:247-250), the central ion channel has an almost constant diameter throughout the molecule except for the portion extending through the hydrophobic part of the lipid bilayer, where the pore is too small to be resolved. However, the density on the pseudo fivefold axis running through the pore is consistently highest in the cytoplasmic half of the bilayer, suggesting the gate is located in that region. The path followed by each subunit has been identified throughout the length of the receptor. The two alpha subunits follow equivalent paths. All subunits have similar features which change in character at the same level relative to the membrane.

Acetylcholine receptor in a C2 muscle cell variant is retained in the endoplasmic reticulum.

We have examined the properties and intracellular localization of acetylcholine receptors in the C2 muscle cell line and in a variant (T-) that accumulates AChR intracellularly. On immunoblots, the subunit structures of the AChR from wild-type and T- cells were similar except that the gamma and delta subunits of the variant AChR had altered mobilities. Digestion with endoglycosidases H and F demonstrated that this difference results from a failure of high-mannose N-linked oligosaccharides on AChR subunits to be processed to complex forms in the variant. N-linked glycosylation of other proteins in the variant was normal. When examined by immunocytochemistry, the distribution of internal AChR in wild-type cells was consistent with a location both in the endoplasmic reticulum and in the Golgi. Variant cells, however, showed no evidence of Golgi staining. Subcellular fractionation experiments also demonstrated AChR in the Golgi fractions of wild-type cells, but not in those derived from T- cells. We conclude that in T- myotubes most of the AChR fails to be transported out of the endoplasmic reticulum.

Tyrosine-phosphorylated and nonphosphorylated isoforms of alpha-dystrobrevin: roles in skeletal muscle and its neuromuscular and myotendinous junctions.

alpha-Dystrobrevin (DB), a cytoplasmic component of the dystrophin-glycoprotein complex, is found throughout the sarcolemma of muscle cells. Mice lacking alphaDB exhibit muscular dystrophy, defects in maturation of neuromuscular junctions (NMJs) and, as shown here, abnormal myotendinous junctions (MTJs). In normal muscle, alternative splicing produces two main alphaDB isoforms, alphaDB1 and alphaDB2, with common NH2-terminal but distinct COOH-terminal domains. alphaDB1, whose COOH-terminal extension can be tyrosine phosphorylated, is concentrated at the NMJs and MTJs. alphaDB2, which is not tyrosine phosphorylated, is the predominant isoform in extrajunctional regions, and is also present at NMJs and MTJs. Transgenic expression of either isoform in alphaDB-/- mice prevented muscle fiber degeneration; however, only alphaDB1 completely corrected defects at the NMJs (abnormal acetylcholine receptor patterning, rapid turnover, and low density) and MTJs (shortened junctional folds). Site-directed mutagenesis revealed that the effectiveness of alphaDB1 in stabilizing the NMJ depends in part on its ability to serve as a tyrosine kinase substrate. Thus, alphaDB1 phosphorylation may be a key regulatory point for synaptic remodeling. More generally, alphaDB may play multiple roles in muscle by means of differential distribution of isoforms with distinct signaling or structural properties.

Enhanced expression of the alpha 7 beta 1 integrin reduces muscular dystrophy and restores viability in dystrophic mice.

Muscle fibers attach to laminin in the basal lamina using two distinct mechanisms: the dystrophin glycoprotein complex and the alpha 7 beta 1 integrin. Defects in these linkage systems result in Duchenne muscular dystrophy (DMD), alpha 2 laminin congenital muscular dystrophy, sarcoglycan-related muscular dystrophy, and alpha 7 integrin congenital muscular dystrophy. Therefore, the molecular continuity between the extracellular matrix and cell cytoskeleton is essential for the structural and functional integrity of skeletal muscle. To test whether the alpha 7 beta 1 integrin can compensate for the absence of dystrophin, we expressed the rat alpha 7 chain in mdx/utr(-/-) mice that lack both dystrophin and utrophin. These mice develop a severe muscular dystrophy highly akin to that in DMD, and they also die prematurely. Using the muscle creatine kinase promoter, expression of the alpha 7BX2 integrin chain was increased 2.0-2.3-fold in mdx/utr(-/-) mice. Concomitant with the increase in the alpha 7 chain, its heterodimeric partner, beta 1D, was also increased in the transgenic animals. Transgenic expression of the alpha 7BX2 chain in the mdx/utr(-/-) mice extended their longevity by threefold, reduced kyphosis and the development of muscle disease, and maintained mobility and the structure of the neuromuscular junction. Thus, bolstering alpha 7 beta 1 integrin-mediated association of muscle cells with the extracellular matrix alleviates many of the symptoms of disease observed in mdx/utr(-/-) mice and compensates for the absence of the dystrophin- and utrophin-mediated linkage systems. This suggests that enhanced expression of the alpha 7 beta 1 integrin may provide a novel approach to treat DMD and other muscle diseases that arise due to defects in the dystrophin glycoprotein complex. A video that contrasts kyphosis, gait, joint contractures, and mobility in mdx/utr(-/-) and alpha 7BX2-mdx/utr(-/-) mice can be accessed at http://www.jcb.org/cgi/content/full/152/6/1207.

The brain ryanodine receptor: a caffeine-sensitive calcium release channel.

The release of stored Ca2+ from intracellular pools triggers a variety of important neuronal processes. Physiological and pharmacological evidence has indicated the presence of caffeine-sensitive intracellular pools that are distinct from the well-characterized inositol 1,4,5,-trisphosphate (IP3)-gated pools. Here we report that the brain ryanodine receptor functions as a caffeine- and ryanodine-sensitive Ca2+ release channel that is distinct from the brain IP3 receptor. The brain ryanodine receptor has been purified 6700-fold with no change in [3H]ryanodine binding affinity and shown to be a homotetramer composed of an approximately 500 kd protein subunit, which is identified by anti-peptide antibodies against the skeletal and cardiac muscle ryanodine receptors. Our results demonstrate that the brain ryanodine receptor functions as a caffeine-sensitive Ca2+ release channel and thus is the likely gating mechanism for intracellular caffeine-sensitive Ca2+ pools in neurons.

Mutation of the acetylcholine receptor alpha subunit causes a slow-channel myasthenic syndrome by enhancing agonist binding affinity.

In five members of a family and another unrelated person affected by a slow-channel congenital myasthenic syndrome (SCCMS), molecular genetic analysis of acetylcholine receptor (AChR) subunit genes revealed a heterozygous G to A mutation at nucleotide 457 of the alpha subunit, converting codon 153 from glycine to serine (alpha G153S). Electrophysiologic analysis of SCCMS end plates revealed prolonged decay of miniature end plate currents and prolonged activation episodes of single AChR channels. Engineered mutant AChR expressed in HEK fibroblasts exhibited prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Single-channel kinetic analysis of engineered alpha G153S AChR revealed a markedly decreased rate of ACh dissociation, which causes the mutant AChR to open repeatedly during ACh occupancy. In addition, ACh binding measurements combined with the kinetic analysis indicated increased desensitization of the mutant AChR. Thus, ACh binding affinity can dictate the time course of the synaptic response, and alpha G153 contributes to the low binding affinity for ACh needed to speed the decay of the synaptic response.

Molecular dissection of subunit interfaces in the acetylcholine receptor: identification of determinants of alpha-conotoxin M1 selectivity.

The acetylcholine receptor from vertebrate skeletal muscle is a pentamer of homologous subunits with composition alpha 2 beta gamma delta. Its two ligand binding sites, formed at alpha-gamma and alpha-delta interfaces, differ in their affinities for agonists and competitive antagonists, owing to different contributions of the gamma and delta subunits. To identify portions of the gamma and delta subunits that contribute to the binding sites, the experiments described here use gamma-delta subunit chimeras and site-specific mutants to determine the basis of the 10,000-fold selectivity of conotoxin M1 for the sites. Three distinct regions of the extracellular domain were found to contribute to conotoxin M1 selectivity, each containing a single residue responsible for the contribution of that region. Residues K34, S111, and F172 of the gamma subunit confer low affinity to the alpha-gamma binding site, whereas the corresponding residues of the delta subunit, S36, Y113, and I178, confer high affinity to the alpha-delta site. Identification of three separate determinants of ligand selectivity suggests a limited model of the folding pattern of the extracellular domain of the subunits.

Involvement of nitric oxide in depolarization-induced suppression of inhibition in hippocampal pyramidal cells during activation of cholinergic receptors.

Several types of neurons are able to regulate their synaptic inputs via releasing retrograde signal molecules, such as endocannabinoids or nitric oxide (NO). Here we show that, during activation of cholinergic receptors, retrograde signaling by NO controls CB1 cannabinoid receptor (CB1R)-dependent depolarization-induced suppression of inhibition (DSI). Spontaneously occurring IPSCs were recorded in CA1 pyramidal neurons in the presence of carbachol, and DSI was induced by a 1-s-long depolarization step. We found that, in addition to the inhibition of CB1Rs, blocking the NO signaling pathway at various points also disrupted DSI. Inhibitors of NO synthase (NOS) or NO-sensitive guanylyl cyclase (NO-sGC) diminished DSI, whereas a cGMP analog or an NO donor inhibited IPSCs and partially occluded DSI in a CB1R-dependent manner. Furthermore, an NO scavenger applied extracellularly or postsynaptically also decreased DSI, whereas L-arginine, the precursor for NO, prolonged it. DSI of electrically evoked IPSCs was also blocked by an inhibitor of NOS in the presence, but not in the absence, of carbachol. In line with our electrophysiological data, double immunohistochemical staining revealed an NO-donor-induced cGMP accumulation in CB1R-positive axon terminals. Using electron microscopy, we demonstrated the postsynaptic localization of neuronal NOS at symmetrical synapses formed by CB1R-positive axon terminals on pyramidal cell bodies, whereas NO-sGC was found in the presynaptic terminals. These electrophysiological and anatomical results in the hippocampus suggest that NO is involved in depolarization-induced CB1R-mediated suppression of IPSCs as a retrograde signal molecule and that operation of this cascade is conditional on cholinergic receptor activation.

Super-Resolution Microscopy Reveals a Nanoscale Organization of Acetylcholine Receptors for Trans-Synaptic Alignment at Neuromuscular Synapses.

The neuromuscular junction (NMJ) is a chemical synapse formed between motoneurons and skeletal muscle fibers. The vertebrate NMJ uses acetylcholine (ACh) as the neurotransmitter and features numerous invaginations of the postsynaptic muscle membrane termed junctional folds. ACh receptors (AChRs) are believed to be concentrated on the crest of junctional folds but their spatial organization remains to be fully understood. In this study, we utilized super-resolution microscopy to examine the nanoscale organization of AChRs at NMJ. Using Structured Illumination Microscopy, we found that AChRs appear as stripes within the pretzel-shaped mouse NMJs, which however, do not correlate with the size of the crests of junctional folds. By comparing the localization of AChRs with several pre- and postsynaptic markers of distinct compartments of NMJs, we found that AChRs are not distributed evenly across the crest of junctional folds as previously thought. Instead, AChR stripes are more closely aligned with the openings of junctional folds as well as with the presynaptic active zone. Using Stochastic Optical Reconstruction Microscopy (STORM) for increased resolution, we found that each AChR stripe contains an AChR-poor slit at the center that is equivalent to the size of the opening of junctional folds. Together, these findings indicate that AChRs are largely localized to the edges of crests surrounding the opening of folds to align with the presynaptic active zones. Such a nanoscale organization of AChRs potentially enables trans-synaptic alignment for effective synaptic transmission of NMJs.

A possible model for the inner wall of the acetylcholine receptor channel.

A structural model of the inner wall of the acetylcholine receptor (AChR) channel is developed using assumptions derived from the results of the recent labelling experiments of the MII helices by noncompetitive blockers. The assumption of steric blocking of the channel by chlorpromazine (CPZ) in the neighbourhood of the labelled serines imposes the MII helices to be in contact at this level and allows the calculation of their minimal interaxial distance. The assumption that CPZ diffuses to this position through the upper crowded part of the channel imposes that the helices are more distant in this region and permits the determination of a tilt of about 7 degrees with respect to the central axis. Electrostatic potentials are used to demonstrate the effect of the charged residues at the exit of the pore. A discussion is given on the possible aptitude of MI to satisfy the contacts necessary with the MII/s at the different heights of the model.

Immunoelectron microscopy of acetylcholine receptors and 43 KD protein after rapid freezing, freeze-substitution, and low-temperature embedding in lowicryl K11M.

To label intracellular determinants of the acetylcholine receptor and associated cytoplasmic proteins while preserving optimal ultrastructure, we developed a post-embedment labeling technique that uses rapid-frozen specimens and freeze-substitution without chemical fixatives. This procedure has been made possible through the use of a low-temperature resin (Lowicryl K11M) that can be polymerized with UV light at -60 degrees C. Rapid-frozen muscle cells were used to evaluate the preservation of structure, and Torpedo electroplaque cells and purified postsynaptic membranes were used to quantitatively evaluate the labeling specificity, efficiency, and resolution of the technique. The labeling efficiency of seven different monoclonal antibodies (MAb) to the acetylcholine receptor varied from 3-13%; there was a correlation between the degree of efficiency and the number of epitopes with which the antibodies reacted. The resolution of the technique was not sufficient to determine whether the anti-acetylcholine receptor MAb were bound to the cytoplasmic or the extracellular surface, but was sufficient to correctly determine the location of the receptor-associated 43 KD protein on the cytoplasmic surface.

Ultrastructure of acetylcholine receptor aggregates parallels mechanisms of aggregation.

BACKGROUND: Acetylcholine receptors become aggregated at the developing neuromuscular synapse shortly after contact by a motorneuron in one of the earliest manifestations of synaptic development. While a major physiological signal for receptor aggregation (agrin) is known, the mechanism(s) by which muscle cells respond to this and other stimuli have yet to be worked out in detail. The question of mechanism is addressed in the present study via a quantitative examination of ultrastructural receptor arrangement within aggregates. RESULTS: In receptor rich cell membranes resulting from stimulation by agrin or laminin, or in control membrane showing spontaneous receptor aggregation, receptors were found to be closer to neighboring receptors than would be expected at random. This indicates that aggregation proceeds heterogeneously: nanoaggregates, too small for detection in the light microscope, underlie developing microaggregates of receptors in all three cases. In contrast, the structural arrangement of receptors within nanoaggregates was found to depend on the aggregation stimulus. In laminin induced nanoaggregates receptors were found to be arranged in an unstructured manner, in contrast to the hexagonal array of about 10 nm spacing found for agrin induced nanoaggregates. Spontaneous aggregates displayed an intermediate amount of order, and this was found to be due to two distinct population of nanoaggregates. CONCLUSIONS: The observations support earlier studies indicating that mechanisms by which agrin and laminin-1 induced receptor aggregates form are distinct and, for the first time, relate mechanisms underlying spontaneous aggregate formation to aggregate structure.

Immunogenicity of human recombinant acetylcholine receptor alpha subunit: cytoplasmic epitopes dominate the antibody response in four mouse strains.

In mysathenia gravis (MG) autoantibodies directed against acetylcholine receptors (AChR), at the neuromuscular junction lead to muscle weakness. These antibodies are directed against extracellular determinants, predominantly on the AChR alpha subunits. Similar antibodies can be induced in animals by immunisation with purified AChR, but immunisation of mice with recombinant human alpha subunit or its extracellular domain has produced conflicting results. To study further the immunogenicity of the human alpha subunit we immunised four inbred stains (C57B1/6, SJL, BALB/c, SWR) with almost full-length recombinant alpha subunit, r37-429, and looked at B cell epitopes by mapping with smaller recombinant fragments and synthetic peptides. The majority of anti-r37-429 antibodies bound to sequences within a region thought to be cytoplasmic, alpha 325-368, and reacted with human AChR. In two C57B1/6 sera, only, most antibodies were directed against an extracellular region, alpha 138-167, but the r37-429 used for immunisation of these two mice appeared to have lost the integrity of its cytoplasmic domain during preparation. Our results suggest that the antigenicity of the cytoplasmic region of the recombinant alpha subunit dominates the immune response in each of the four strains, and may even suppress the formation of antibodies to the extracellular domain. Moreover, although C57B1/6 and SJL mice were able to produce antibodies to alpha 138-167, these antibodies did not react with intact AChR, and none of the mice became weak.

A congenital myasthenic disorder with paucity of secondary synaptic clefts: deficiency and altered distribution of acetylcholine receptors.

Congenital myasthenia (CM) constitutes a heterogeneous group of disorders with different underlying defects. The authors investigated a case of CM, presenting with congenital contractures. Endplate studies in the first year of life showed a developmental disorder of postsynaptic membranes. Clinical follow-up demonstrated a beneficial effect of pyridostigmine, resulting in normal motor development. Results of a second biopsy at age 4 are reported in this paper. Microelectrode study showed small Mepp amplitudes, which returned to nearly normal in the presence of neostigmine. In the electronmicroscope the postsynaptic membranes showed a paucity of infoldings, as in the first biopsy. These membranes showed only scanty, patchy enhancement with two different methods for localization of AChR. The extrajunctional membranes showed evidence of local presence of AChR. Our results show a developmental disorder of postsynaptic membranes with a deficiency and altered distribution of AChRs.

Circular harmonic averaging of noisy single-molecule images.

A new method for averaging of noisy single-molecule images has been developed. Averaging is done over the circular harmonic components of the images instead of over the images themselves. Those images which show different molecules or molecules in different projections can be separated into classes by comparing the power of their circular harmonic components, which is rotationally invariant. Orientational alignment, based on the rotational correlation of each image with all others, and averaging is performed separately for each class. Applying a quality measure to the circular harmonic averages allows separation of significant and nonsignificant components. Computer simulations indicate that the method is quite stable against noise. One of the main advantages of "circular harmonic averaging" over existing methods is that no reference images are needed in the averaging procedure.