Calcitonin gene-related peptide regulates muscle acetylcholine receptor synthesis

Innervation of muscle by motoneurones induces the development of a characteristic, high density cluster of acetylcholine receptors (AChRs) at the neuromuscular junction. Studies in vitro show that the accumulation of AChRs at nerve-muscle contacts results from both increased insertion of new AChRs into the muscle plasma membrane beneath nerve terminals and redistribution of preexisting AChRs; these two modes of AChR accumulation may be separately controlled since factors have been identified that influence AChR redistribution but not synthesis. Although many aspects of muscle development are regulated by nerve-dependent muscle activity, junctional AChR clusters still develop when neuromuscular transmission is blocked by either curare or alpha-bungarotoxin, suggesting that their formation is mediated by nerve-derived trophic factors other than activity. A molecule immunologically related to calcitonin gene-related peptide (CGRP-I) has been found in motoneurones in a variety of mammals including man. Here we provide indirect evidence that CGRP-I may be a motoneurone-derived trophic factor that increases AChR synthesis at vertebrate neuromuscular junctions.     

Acetylcholine receptor-aggregating factor is similar to molecules concentrated at neuromuscular junctions

The basal lamina in the synaptic cleft of the vertebrate skeletal neuromuscular junction contains molecules that direct the formation of synaptic specializations in regenerating axons and muscle fibres. We have undertaken a series of experiments aimed at identifying and characterizing the molecules responsible for the formation of one of these specializations, the aggregates of acetylcholine receptors (AChRs) in the muscle fibre plasma membrane. We began by preparing an insoluble, basal lamina-containing fraction from Torpedo californica electric organ, a tissue which has a far higher concentration of cholinergic synapses than muscle, and showing that this fraction caused AChRs on cultured chick myotubes to aggregate. A critical step is learning whether or not the electric organ factor is similar to the receptor-aggregating molecule in the basal lamina at the neuromuscular junction. The importance of this problem is emphasized by reports that clearly non-physiological agents, such as positively charged latex beads, can cause AChR aggregation on cultured muscle cells. We have already shown that Torpedo muscle contains an AChR-aggregating factor similar to that of electric organ, although in much lower amounts. Here we demonstrate, using monoclonal antibodies, that the AChR-aggregating factor in our extracts of electric organ is, in fact, antigenically related to molecules concentrated in the synaptic cleft at the neuromuscular junction.     

Channel properties of an insect neuronal acetylcholine receptor protein reconstituted in planar lipid bilayers

A pentameric membrane protein composed of four types of polypeptide has been identified as the minimal structural unit responsible for the electrogenic action of acetylcholine on electrocytes and muscle cells. Because many populations of central and peripheral neurons also have nicotinic acetylcholine receptors (AChRs), considerable effort has recently gone into identifying the neuronal receptor. The central nervous tissue of insects contains very high concentrations of nicotinic AChRs, and we have recently purified an alpha-toxin binding protein, a putative AChR, from neuronal membranes of locusts. It is a component of high relative molecular mass, clearly composed of identical subunits, a structure predicted for an ancestral AChR protein. To verify that the purified polypeptides not only represent ligand binding sites but that they are indeed functional receptors, we have now reconstituted the isolated protein in a planar lipid bilayer. We show that in this system cholinergic agonists activate functional ion channels, that have properties comparable to those exhibited by the peripheral AChRs in vertebrates; thus, for the first time a functional acetylcholine receptor channel has been identified in nerve cells.     

Aggregates of acetylcholinesterase induced by acetylcholine receptor-aggregating factor

Basal lamina-rich extracts of Torpedo californica electric organ contain a factor that causes acetylcholine receptors (AChRs) on cultured myotubes to aggregate into patches. Our previous studies have indicated that the active component of these extracts is similar to the molecules in the basal lamina which direct the aggregation of AChRs in the muscle fibre plasma membrane at regenerating neuromuscular junctions in vivo. Because it can be obtained in large amounts and assayed in controlled conditions in cell culture, the AChR-aggregating factor from electric organ may be especially useful for examining in detail how the postsynaptic apparatus of regenerating muscle is assembled. Here we demonstrate that the electric organ factor causes not only the formation of AChR aggregates on cultured myotubes, but also the formation of patches of acetylcholinesterase (AChE). This finding, together with the observation that basal lamina directs the formation of both AChR and AChE aggregates at regenerating neuromuscular junctions in vivo, leads us to hypothesize that a single component of the synaptic basal lamina causes the formation of both these synaptic specializations on regenerating myofibres.     

Concentration of acetylcholine receptor mRNA in synaptic regions of adult muscle fibres

Acetylcholine receptors (AChRs) are highly concentrated in the small fraction (approximately 0.1%) of the skeletal muscle fibre surface that comprises the postsynaptic membrane of the neuromuscular junction (Fig. 1a). In adult murine muscle, for example, AChRs are packed at a density of over 15,000 per micron2 in postsynaptic membrane, whereas their density is less than 30 per micron2 in extrasynaptic membrane. Because synaptic AChRs turn over they must be replaced, and it is interesting to consider where the new AChRs that maintain synaptic aggregates are synthesized. One possibility is that AChRs are synthesized uniformly along the length of the multinucleated muscle fibre; in this case, AChRs might be redistributed to or selectively stabilized at the synapse, as probably occurs during synapse formation. Alternatively, AChRs might be preferentially synthesized near synapses, a possibility that would suggest that innervation can influence not only where AChRs are inserted or accumulate but also where they are synthesized. In support of this second possibility, we report here that AChR messenger RNA is more abundant near to than far from synapses in adult muscle fibres.     

Imprinting of acetylcholine receptor messenger RNA accumulation in mammalian neuromuscular synapses

IN mammalian muscle, the subunit composition of the nicotinic acetylcholine receptor (AChR) and the distribution of AChRs along the fibre are developmentally regulated. In fetal muscle, AChRs are distributed over the entire fibre length whereas in adult fibres they are concentrated at the end-plate. We have used in situ hybridization techniques to measure the development of the synaptic localization of the messenger RNAs (mRNAs) encoding the alpha-subunit and the epsilon-subunit of the rat muscle AChR. The alpha-subunit is present in both fetal and adult muscle, whereas the epsilon-subunit appears postnatally and specifies the mature AChR subtype. The synaptic localization of alpha-subunit mRNA in adult fibres may arise from the selective down-regulation of constitutively expressed mRNA from extrasynaptic fibre segments. In contrast, epsilon-subunit mRNA appears locally at the site of neuromuscular contact and its accumulation at the end-plate is not dependent on the continued presence of the nerve terminal very early during synapse formation. This suggests that epsilon-subunit mRNA expression is induced locally via a signal which is restricted to the end-plate region and is dependent on the presence of the nerve only during a short period of early neuromuscular contact. Evidently, several mechanisms operate to confine AChR mRNAs to the adult end-plate region, and the levels of alpha-subunit and epsilon-subunit mRNAs depend on these mechanisms to differing degrees.     

SOL-1 is a CUB-domain protein required for GLR-1 glutamate receptor function in C. elegans

Ionotropic glutamate receptors (iGluRs) mediate most excitatory synaptic signalling between neurons. Binding of the neurotransmitter glutamate causes a conformational change in these receptors that gates open a transmembrane pore through which ions can pass. The gating of iGluRs is crucially dependent on a conserved amino acid that was first identified in the 'lurcher' ataxic mouse. Through a screen for modifiers of iGluR function in a transgenic strain of Caenorhabditis elegans expressing a GLR-1 subunit containing the lurcher mutation, we identify suppressor of lurcher (sol-1). This gene encodes a transmembrane protein that is predicted to contain four extracellular beta-barrel-forming domains known as CUB domains. SOL-1 and GLR-1 are colocalized at the cell surface and can be co-immunoprecipitated. By recording from neurons expressing GLR-1, we show that SOL-1 is an accessory protein that is selectively required for glutamate-gated currents. We propose that SOL-1 participates in the gating of non-NMDA (N-methyl-D-aspartate) iGluRs, thereby providing a previously unknown mechanism of regulation for this important class of neurotransmitter receptor.     

Differential activation of myotube nuclei following exposure to an acetylcholine receptor-inducing factor

A glycoprotein purified from chick brain, of relative molecular mass 42,000, increases the rate of appearance of acetylcholine receptors (AChRs) on the surface of chick myotubes. RNase protection assays have shown that this AChR-inducing activity (ARIA) increases the amount of mRNA encoding the alpha-subunit of the AChR, with little or no effect on the amounts of gamma- and delta-mRNAs2. Here, we report that the mRNAs encoding the alpha- and gamma-subunits of the receptor detected by in situ hybridization are concentrated around nuclei in cultured myotubes. Consistent with previous results, ARIA selectively increased the amount of alpha-subunit mRNA, but we now find that all nuclei were not activated to the same extent, with a substantial number not responding at all. Assuming that ARIA is released by motor nerve terminals, our results indicate that only a subset of muscle nuclei are capable of contributing to the accumulation of AChRs at developing neuromuscular junctions.     

Distinct roles of nerve and muscle in postsynaptic differentiation of the neuromuscular synapse

The development of chemical synapses is regulated by interactions between pre- and postsynaptic cells. At the vertebrate skeletal neuromuscular junction, the organization of an acetylcholine receptor (AChR)-rich postsynaptic apparatus has been well studied. Much evidence suggests that the nerve-derived protein agrin activates muscle-specific kinase (MuSK) to cluster AChRs through the synapse-specific cytoplasmic protein rapsyn. But how postsynaptic differentiation is initiated, or why most synapses are restricted to an 'end-plate band' in the middle of the muscle remains unknown. Here we have used genetic methods to address these issues. We report that the initial steps in postsynaptic differentiation and formation of an end-plate band require MuSK and rapsyn, but are not dependent on agrin or the presence of motor axons. In contrast, the subsequent stages of synaptic growth and maintenance require nerve-derived agrin, and a second nerve-derived signal that disperses ectopic postsynaptic apparatus.     

Location of a delta-subunit region determining ion transport through the acetylcholine receptor channel

The combination of complementary DNA expression and single-channel current analysis provides a powerful tool for studying the structure-function relationship of the nicotinic acetylcholine receptor (AChR) (refs 1-5). We have previously shown that AChR channels consisting of subunits from different species, expressed in the surface membrane of Xenopus oocytes, can be used to relate functional properties to individual subunits. Here we report that, in extracellular solution of low divalent cation concentration, the bovine AChR channel has a smaller conductance than the Torpedo AChR channel. Replacement of the delta-subunit of the Torpedo AChR by the bovine delta-subunit makes the channel conductance similar to that of the bovine AChR channel. To locate the region in the delta-subunit responsible for this difference, we have constructed chimaeric delta-subunit cDNAs with different combinations of the Torpedo and bovine counterparts. The conductances of AChR channels containing chimaeric delta-subunits suggest that a region comprising the putative transmembrane segment M2 and the adjacent bend portion between segments M2 and M3 is involved in determining the rate of ion transport through the open channel.     

Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance

The structure-function relationship of the nicotinic acetylcholine receptor (AChR) has been effectively studied by the combination of complementary DNA manipulation and single-channel current analysis. Previous work with chimaeras between the Torpedo californica and bovine AChR delta-subunits has shown that the region comprising the hydrophobic segment M2 and its vicinity contains an important determinant of the rate of ion transport through the AChR channel. It has also been suggested that this region is responsible for the reduction in channel conductance caused by divalent cations and that segment M2 contributes to the binding site of noncompetitive antagonists. To identify those amino acid residues that interact with permeating ions, we have introduced various point mutations into the Torpedo AChR subunit cDNAs to alter the net charge of the charged or glutamine residues around the proposed transmembrane segments. The single-channel conductance properties of these AChR mutants expressed in Xenopus laevis oocytes indicate that three clusters of negatively charged and glutamine residues neighbouring segment M2 of the alpha-, beta-, gamma- and delta-subunits, probably forming three anionic rings, are major determinants of the rate of ion transport.     

Fluorescently labelled Na+ channels are localized and immobilized to synapses of innervated muscle fibres

Segregation of voltage-dependent sodium channels to the hillock of motoneurones and nodes of Ranvier in myelinated axons is crucial for conduction of the nerve impulse. Much less is known, however, about the distribution of voltage-dependent Na+ channels on muscle fibres. Recently, Beam et al. have shown that Na+ channels are concentrated near the neuromuscular junction. To determine the topography and mechanisms governing the distribution of voltage-dependent Na+ channels on muscle, microfluorimetry and fluorescence photobleach recovery (FPR) have now been used to measure the density and lateral mobility of fluorescently labelled Na+ channels on uninnervated and innervated muscle fibres. On uninnervated myotubes, Na+ channels are diffusely distributed and freely mobile, whereas after innervation the channels concentrate at neuronal contact sites. These channels are immobile and co-localize with acetylcholine receptors (AChRs). At extrajunctional regions the Na+ channel density is lower and the channels more mobile. The results suggest that the nerve induces Na+ channels to redistribute, immobilize and co-localize with AChRs at sites of neuronal contact.     

Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH

Acid-sensing ion channels (ASICs) are voltage-independent, proton-activated receptors that belong to the epithelial sodium channel/degenerin family of ion channels and are implicated in perception of pain, ischaemic stroke, mechanosensation, learning and memory. Here we report the low-pH crystal structure of a chicken ASIC1 deletion mutant at 1.9 A resolution. Each subunit of the chalice-shaped homotrimer is composed of short amino and carboxy termini, two transmembrane helices, a bound chloride ion and a disulphide-rich, multidomain extracellular region enriched in acidic residues and carboxyl-carboxylate pairs within 3 A, suggesting that at least one carboxyl group bears a proton. Electrophysiological studies on aspartate-to-asparagine mutants confirm that these carboxyl-carboxylate pairs participate in proton sensing. Between the acidic residues and the transmembrane pore lies a disulphide-rich 'thumb' domain poised to couple the binding of protons to the opening of the ion channel, thus demonstrating that proton activation involves long-range conformational changes.     

An essential role for a CD36-related receptor in pheromone detection in Drosophila

The CD36 family of transmembrane receptors is present across metazoans and has been implicated biochemically in lipid binding and transport. Several CD36 proteins function in the immune system as scavenger receptors for bacterial pathogens and seem to act as cofactors for Toll-like receptors by facilitating recognition of bacterially derived lipids. Here we show that a Drosophila melanogaster CD36 homologue, Sensory neuron membrane protein (SNMP), is expressed in a population of olfactory sensory neurons (OSNs) implicated in pheromone detection. SNMP is essential for the electrophysiological responses of OSNs expressing the receptor OR67d to (Z)-11-octadecenyl acetate (cis-vaccenyl acetate, cVA), a volatile male-specific fatty-acid-derived pheromone that regulates sexual and social aggregation behaviours. SNMP is also required for the activation of the moth pheromone receptor HR13 by its lipid-derived pheromone ligand (Z)-11-hexadecenal, but is dispensable for the responses of the conventional odorant receptor OR22a to its short hydrocarbon fruit ester ligands. Finally, we show that SNMP is required for responses of OR67d to cVA when ectopically expressed in OSNs not normally activated by pheromones. Because mammalian CD36 binds fatty acids, we suggest that SNMP acts in concert with odorant receptors to capture pheromone molecules on the surface of olfactory dendrites. Our work identifies an unanticipated cofactor for odorant receptors that is likely to have a widespread role in insect pheromone detection. Moreover, these results define a unifying model for CD36 function, coupling recognition of lipid-based extracellular ligands to signalling receptors in both pheromonal communication and pathogen recognition through the innate immune system.     

A Drosophila tissue polarity locus encodes a protein containing seven potential transmembrane domains

The function of the frizzled (fz) locus in Drosophilia melanogaster is required to coordinate the cytoskeletons of epidermal cells to produce a parallel array of cuticular hairs and bristles (for example on the wild-type wing all hairs point towards the distal tip). In fz mutants it is not the structure of individual hairs and bristles that is altered, but their orientation with respect to their neighbours and the organism as a whole. Mitotic clone analysis indicates that fz has two functions in the developing wing. It is required for the proximal-distal transmission of an intercellular polarity signal, a process that is expected to be at least partly extracellular. It is also required for cells to respond to the polarity signal, which is expected to be a cytoplasmic function. The fz locus could encode either one bifunctional or two single-function proteins. We report here that, in pupae, fz produces a messenger RNA that encodes a protein with seven putative transmembrane domains. Thus, the Fz protein should contain both extracellular and cytoplasmic domains, which could function in the transmission and interpretation of polarity information, respectively. This is the first reported sequence for the protein product of a tissue polarity gene.     

Possible role of acetylcholinesterase in regulation of postsynaptic receptor efficacy at a central inhibitory synapse of Aplysia

Most of the effects of acetylcholinesterase (AChE) on synaptic transmission are considered to be related to its acetylcholine (ACh) hydrolysing properties. This is clearly apparent from changes which occur in the characteristics of the miniature endplate potential and of the endplate potential at neuromuscular junctions when AChE is inhibited1-4 and during the development of enzymatic AChE activity at maturing synapses5. However, we report here that after inhibiting AChE in a cholinergic synapse in Aplysia, we found an increase not only in postsynaptic responses to presynaptic stimulation and to ionophoretic application of ACh on postsynaptic receptors, but also to ionophoretic application of carbachol. This could not be explained by the inhibition of the ACh hydrolysing function of the enzyme, as carbachol is not hydrolysed by AChE. A possible explanation of these observations is that inhibition of the enzyme affects a property of the ACh receptor (AChR) itself.     

Embryonic acetylcholine receptors guarantee spontaneous contractions in rat developing muscle

Many proteins are expressed in distinct embryonic and adult forms. However, in most cases we do not know why the embryonic form of proteins is required. This question can be readily addressed for the acetylcholine receptor (AChR) because developmentally specified modifications of this ligand-gated ion channel can be directly related to changes in membrane currents. In developing rat soleus muscle, spontaneous transmitter release causes miniature end-plate currents (m.e.p.cs) to flow into the muscle cell. We show here that these m.e.p.cs in neonatal soleus trigger spontaneous contractions. By injecting m.e.p.cs into young fibres, we showed that only embryonic m.e.p.cs can trigger such contractions; adult m.e.p.cs do not last long enough. Developing muscle fibres must be active for synapse and muscle differentiation. Our experiments indicate that the embryonic form of the AChR is essential for spontaneous contractile activity and may therefore be required for normal neuromuscular development.     

Cloning, sequencing and expression of cDNA for a novel subunit of acetylcholine receptor from calf muscle

The nicotinic acetylcholine receptor (AChR) from fish electric organ has a subunit structure of alpha 2 beta gamma delta, and this is thought to be also the case for the mammalian skeletal muscle AChR. By cloning and sequencing the complementary or genomic DNAs, we have previously elucidated the primary structures of all four subunits of the Torpedo californica electroplax and calf muscle AChR and of the alpha- and gamma-subunits of the human muscle AChR; the primary structures of the gamma-subunit of the T. californica AChR and the alpha-subunit of the Torpedo marmorata AChR have also been deduced elsewhere. We have now cloned DNA complementary to the calf muscle messenger RNA encoding a novel polypeptide (the epsilon-subunit) whose deduced amino-acid sequence has features characteristic of the AChR subunits and which shows higher sequence homology with the gamma-subunit than with the other subunits. cDNA expression studies indicate that the calf epsilon-subunit, as well as the calf gamma-subunit, can replace the Torpedo gamma-subunit to form the functional receptor in combination with the Torpedo alpha-, beta- and delta-subunits.     

G0 is a major growth cone protein subject to regulation by GAP-43

G0, a GTP-binding protein that transduces information from transmembrane receptors, has been found to be a major component of the neuronal growth cone membrane. GAP-43, an intracellular growth cone protein closely associated with neuronal growth, stimulates GTP-gamma-S binding to G0. It does so through an amino-terminal domain homologous to G-linked transmembrane receptors. Thus, G0 in the growth cone may be regulated by intracellular as well as extracellular signals.