Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses.

To elucidate the nature of signals that control the level and spatial distribution of mRNAs encoding acetylcholine receptor (AChR), alpha-, beta-, gamma-, delta- and epsilon-subunits in muscle fibers chronic paralysis was induced in rat leg muscles either by surgical denervation or by different neurotoxins that cause disuse of the muscle or selectively block neuromuscular transmission pre- or postsynaptically and cause an increase of AChRs in muscle membrane. After paralysis, the levels and the spatial distributions of the different subunit-specific mRNAs change discoordinately and seem to follow one of three different patterns depending on the subunit mRNA examined. The level of epsilon-subunit mRNA and its accumulation at the end-plate are largely independent on the presence of the nerve or electrical muscle activity. In contrast, the gamma-subunit mRNA level is tightly coupled to innervation. It is undetectable or low in innervated normally active muscle and in innervated but disused muscle, whereas it is abundant along the whole fiber length in denervated muscle or in muscle in which the neuromuscular contact is intact but the release of transmitter is blocked. The alpha-, beta-, and delta-subunit mRNA levels show a different pattern. Highest amounts are always found at end-plate nuclei irrespective of whether the muscle is innervated, denervated, active, or inactive, whereas in extrasynaptic regions they are tightly controlled by innervation partially through electrical muscle activity. The changes in the levels and distribution of gamma- and epsilon-subunit-specific mRNAs in toxin-paralyzed muscle correlate well with the spatial appearance of functional fetal and adult AChR channel subtypes along the muscle fiber. The results suggest that the focal accumulation at the synaptic region of mRNAs encoding the alpha-, beta-, delta-, and epsilon-subunits, which constitute the adult type end-plate channel, is largely determined by at least two different neural factors that act on AChR subunit gene expression of subsynaptic nuclei.

MuSK induces in vivo acetylcholine receptor clusters in a ligand-independent manner.

Muscle-specific receptor tyrosine kinase (MuSK) is required for the formation of the neuromuscular junction. Using direct gene transfer into single fibers, MuSK was expressed extrasynaptically in innervated rat muscle in vivo to identify its contribution to synapse formation. Spontaneous MuSK kinase activity leads, in the absence of its putative ligand neural agrin, to the appearance of epsilon-subunit-specific transcripts, the formation of acetylcholine receptor clusters, and acetylcholinesterase aggregates. Expression of kinase-inactive MuSK did not result in the formation of acetylcholine receptor (AChR) clusters, whereas a mutant MuSK lacking the ectodomain did induce AChR clusters. The contribution of endogenous MuSK was excluded by using genetically altered mice, where the kinase domain of the MuSK gene was flanked by loxP sequences and could be deleted upon expression of Cre recombinase. This allowed the conditional inactivation of endogenous MuSK in single muscle fibers and prevented the induction of ectopic AChR clusters. Thus, the kinase activity of MuSK initiates signals that are sufficient to induce the formation of AChR clusters. This process does not require additional determinants located in the ectodomain.

Local neurotrophic repression of gene transcripts encoding fetal AChRs at rat neuromuscular synapses.

The spatio-temporal expression patterns of mRNA transcripts coding for acetylcholine receptor (AChR) subunits and myogenic factors were measured in denervated rat soleus muscle and in soleus muscle chronically paralyzed for up to 12 d by conduction block of the sciatic nerve by tetrodotoxin (TTX). In denervated muscle the AChR alpha-, beta-, gamma-, and delta-subunit mRNAs were elevated with highest expression levels in the former synaptic and the perisynaptic region and with lower levels in the extrasynaptic fiber segments. In muscle paralyzed by nerve conduction block the alpha-, beta-, gamma-, and delta-subunit mRNA levels increased only in extrasynaptic fiber segments. Surprisingly, in the synaptic region the gamma-subunit mRNA that specifies the fetal-type AChR, and alpha-, beta-, delta-subunit mRNAs were not elevated. The expression of the gene encoding the epsilon-subunit, which specifies the adult-type AChR, was always restricted to synaptic nuclei. The mRNA for the regulatory factor myogenin showed after denervation similar changes as the subunit transcripts of the fetal AChR. When the muscle was paralyzed by nerve conduction block the increase of myogenin transcripts was also less pronounced in synaptic regions compared to extrasynaptic fiber segments. The results suggest that in normal soleus muscle a neurotrophic signal from the nerve locally down-regulates the expression of fetal-type AChR channel in the synaptic and perisynaptic muscle membrane by inhibiting the expression of the gamma-subunit gene and that inhibition of the myogenin gene expression may contribute to this down-regulation.

Argiotoxin detects molecular differences in AMPA receptor channels.

Argiotoxin, a component of the spider venom from Argiope lobata, blocks AMPA receptor channels expressed in homomeric and heteromeric configuration in Xenopus oocytes. Argiotoxin acts as an open channel blocker in a voltage-dependent manner and discriminates between the functionally diverse AMPA receptors. Importantly, a transmembrane region 2 determinant for divalent cation permeability also determines argiotoxin sensitivity. Subunit-specific differences in the time courses of block and recovery demonstrate that heteromeric AMPA receptors can assemble in variable ratios. Thus, argiotoxin can be used as a tool in analyzing the subunit composition of AMPA receptors in native membranes.

The separation and characterization of two forms of Torpedo electric organ calelectrin.

Two methods for extracting calelectrin, a Ca2+-regulated membrane-binding protein from the electric organ of Torpedo marmorata, have been compared and the more promising one was modified to increase the yield to 7-8 mg.kg-1 wet weight of tissue, that is 4-5 times greater than the original method. The calelectrin so obtain could be resoloved into a minor component (designated L-calelectrin) eluted from an anion-exchange column at relatively low ionic strength (100 mM NaCl) and a major component (H-calelectrin) eluted at higher ionic strength (300 mM NaCl). The two forms were also separated by chromatography on a hydrophobic resin. Electrophoresis on cellulose acetate indicated that L-calelectrin had a lower mean isoelectric point that the H-form and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed that under reducing conditions (presence of 5% beta-mercaptoethanol) both forms migrated as single species, the L-form having a lower apparent relative molecular mass (Mr 32,000) that the H-form (34,000). Under non-reducing conditions, there was no change in the migration of L-calelectrin but the H-form was resolved into two components of Mr 34,000 and 32,000. The addition of 2 mM Ca2+ had no effect on the migration of either form. Both forms were equally recognized by an anti-calelectrin antiserum and were microheterogeneous with respect to their isoelectric points (pH 4.3-5.5) in two-dimensional gel electrophoresis. Physical measurements were carried out on the major H-form. The Stokes radius was estimated to be 3nm, corresponding to an apparent Mr of 44,000. It was unaffected by changes in ionic strength, pH or Ca2+ concentration. Analytical ultracentrifugation gave a sedimentation constant of 2.9 S and an apparent Mr of 36,000. Measurements of circular dichroism indicated that 78% of the molecule was in the alpha-helix configuration and 22% in random coil. Ca2+ had no significant effect on the conformation.

Ca2+- and phospholipid-binding properties of Torpedo electric organ calelectrin.

The Ca2+-regulated lipid-binding properties of the H- and L-forms of calelectrin present in the electric organ of Torpedo marmorata have been compared. Binding of H-calelectrin required Ca2+ in millimolar concentrations, whereas that of L-calelectrin occurred in the micromolar range. Dissociation of H-calelectrin previously bound to lipids in the presence of 2 mM Ca2+ took place only when the Ca2+ concentration was reduced to micromolar concentrations. Binding was most effective to acidic phospholipids such as phosphatidylserine. Both forms of calelectrin promoted the aggregation of membrane vesicles in the presence of Ca2+.Mg2+, Na+ and K+ inhibited the Ca2+-induced binding to phospholipid, decreasing in effectiveness in that order. Binding was also inhibited by high pH. The surface activity and hydrophobicity index showed that H-calelectrin is a hydrophilic molecule. It may represent a less active, more highly phosphorylated "down-regulated" form of L-calelectrin. The role of calcium in H-calelectrin binding to lipid appeared to be consistent with the formation of a ternary complex of the protein, an acidic lipid and Ca2+, rather than with a direct interaction of lipid with hydrophobic sequences in H-calelectrin whose accessibility is Ca2+-regulated.

Cytoskeletal proteins at the cholinergic synapse: distribution of desmin, actin, fodrin, neurofilaments, and tubulin in Torpedo electric organ.

Treatment of the electric organ of Torpedo marmorata with Triton X-100 in the presence of 2 mM MgCl2 generated a cytoskeletal fraction in which a 54 kDa polypeptide is a major constituent. This 54 kDa polypeptide accounted for about 8% of the cellular protein when total electric organ tissue was analyzed by two-dimensional gel electrophoresis. Immunoblotting experiments showed that this protein reacts with monoclonal antibodies to desmin, the major intermediate filament protein of avian and mammalian muscle tissue. Negative stain analysis revealed that filaments of about 10 nm diameter are the major structural elements of the electric organ cytoskeleton. In the presence of Ca2+ there was a rapid degradation of the desmin-like protein and intermediate filaments due to a Ca2+-activated protease. Some of the resulting fragments retained antigenic activity against the desmin antibodies. Immunoblotting of membrane fractions enriched in acetylcholine receptor revealed desmin in addition to some actin. A further cytoskeletal component was identified from biochemical and immunological properties as a homologue of the mammalian neurofilament L-polypeptide. Thus Torpedo expresses proteins homologous to the mammalian desmin and neurofilament L-protein which can be detected using immunological approaches. Immunofluorescence microscopy was used to map the location of various cytoskeletal proteins of the cholinergic synapse on paraffin sections and on en face preparations of membranes. Desmin staining was restricted to electrocytes and in en face preparations was seen associated with both the ventral receptor-containing membrane and with the non-innervated dorsal membrane. Antibodies to neurofilament L-protein stained only the axons and not the electrocytes. Staining for fodrin, a non-erythrocyte spectrin, resulted in submembraneous decoration of both the axons and the electrocytes. Axonal staining for neurofilaments and microtubules did not extend into the ends of the nerve terminal arborizations.

Differential regulation of MyoD and myogenin mRNA levels by nerve induced muscle activity.

The levels of mRNAs coding for the myogenic factors MyoD and myogenin were measured during synapse formation in developing muscle and in adult muscle, after denervation and reinnervation and after muscle paralysis induced by blocking of neuromuscular transmission by neurotoxins known to alter the density and localization of synaptic proteins such as the acetylcholine receptor (AChR). The mRNA levels of both factors depend on usage of the neuromuscular synapses, but they change to different extents. Myogenin mRNA levels decrease drastically with innervation and increase strongly following blocking of transmission whereas the level of MyoD mRNA showed only a small decrease in response to innervation, denervation or muscle paralysis by neurotoxins. Neither mRNA showed a synapse-related cellular distribution. The results suggest that nerve-induced electrical muscle activity determines the cellular ratio of MyoD and myogenin mRNAs in adult muscle.

PVP-containing solutions for analysis of divalent cation-dependent NMDA responses in Xenopus oocytes.

The electrophysiological analysis of Ca(2+)-conducting ion channels in Xenopus oocytes is difficult due to secondary intracellular effects induced by Ca2+. In the presence of polyvinylpyrrolidone (PVP) membrane currents can be recorded in nominally divalent cation-free solutions. The Ca(2+)-permeable recombinant NMDA receptors of the NR1/NR2A subtype were used as assay system and the results show that PVP has no effect on NMDA receptor-induced currents. Ca2+ and Ba2+ depress NMDA-induced currents at submillimolar concentrations probably by interfering with the Na+/K+ flux. This block is fully reversible as also observed for Mg2+ but shows in contrast no pronounced voltage dependence. PVP-containing solutions may be useful for the analysis of divalent cation-dependent ion channels.

Internodal cells of the giant green alga Chara as an expression system for ion channels.

Internodal cells of the giant green alga Chara corallina were utilized as an expression system for two nicotinic acetylcholine receptor subtypes (nAChR) derived from rat muscle. From Chara internodes that were pressure-injected with the respective cRNA, cytoplasmic droplets were formed, and functional expression of channel proteins in the membrane delineating the droplets was confirmed by patch-clamp techniques. The droplet membrane was recently identified as the original tonoplast, patch-clamp recordings on cytoplasmic droplets were easily performed and single channel activity could be measured with high resolution. The properties of the recombinant nAChR observed in this membrane were similar to those reported for the channel expressed in Xenopus oocytes, and for the native channel recorded in situ. The Chara expression system is, therefore, suitable for functional expression of animal messenger RNAs, without the risk of signal contamination by intrinsic ion channels. It offers a low-cost and practical alternative to the use of Xenopus oocytes for the investigation of heterologously expressed ion channels.

Acetylcholine receptor gene expression in experimental autoimmune myasthenia gravis.

Acetylcholine receptor (AChR) gene expression was analyzed in experimental autoimmune myasthenia gravis (EAMG) in rabbits, rats and mice. An increase in AChR transcripts was demonstrated to be exclusively associated with myasthenic symptoms and with a severe loss in membrane AChR. An increase of alpha-, beta-, epsilon-, and delta-subunit specific mRNAs (5.2-, 1.6-, 3.2- and 3.7-fold, respectively), which code for the adult type of AChR (alpha 2 beta epsilon delta) was observed in EAMG in rats. The gamma-subunit transcript was not detectable in myasthenic or healthy rats. It appears that the regulatory control of AChR gene expression in EAMG is different from that observed upon denervation.

Identification and characterization of a novel splice variant of MuSK.

MuSK is a receptor tyrosine kinase that initiates the formation of neuromuscular junctions in response to agrin. Little is known about the ligand-induced activation and kinase-dependent signalling that leads to the clustering of acetylcholine receptors. The ectodomain of these molecule is composed of four Ig-like domains. We describe here the isolation of a novel MuSK splice variant that lacks the third Ig-like domain in its ectodomain. The corresponding RNA is the result of alternative splicing which eliminates two exons. There is 10 times less mRNA for this shorter form than for the long form of MuSK and both forms are regulated coordinately. They decrease strongly after birth and are elevated in denervated muscle. Gene transfer by muscle injection of MuSK DNA into individual muscle fibers demonstrates that kinase-induced acetylcholine receptor clustering caused by overexpression of the two kinases does not depend on the presence of the third Ig-like domain.

Differential regulation of muscle acetylcholine receptor gamma- and epsilon-subunit mRNAs.

The contents of the mRNAs encoding the gamma- and epsilon-subunits of the nicotinic acetylcholine receptor as well as the single-channel properties of the receptor have been assessed in innervated, denervated and reinnervated rat muscle. The changes in abundance of the gamma- and epsilon-subunit mRNAs correlate with the changes in relative density of two classes of acetylcholine receptor channels. The results support the view that a switch in the relative abundance of the gamma- and epsilon-subunit mRNAs is a major mechanism in regulating the properties of acetylcholine receptor channels in muscle.

Developmental regulation of five subunit specific mRNAs encoding acetylcholine receptor subtypes in rat muscle.

The muscular content of the mRNAs encoding the five subunits of the nicotonic acetylcholine receptor was measured during postnatal development in the rat. Subunit specific mRNAs show differential regulation. The levels of the alpha-, gamma- and delta-subunit specific mRNAs decrease steadily after birth, while the beta- and epsilon-subunit mRNAs increase transiently and then decrease. The adult pattern of subunit specific mRNA levels is reached at 4-6 weeks postnatally. The content of gamma- and epsilon-subunit mRNA changes in a reciprocal fashion during the first 2 postnatal weeks, supporting the view that differential regulation of gamma- and epsilon-subunit mRNA during development is one mechanism mediating the appearance of the adult, epsilon-subunit containing, subtype of end-plate channel. Denervation of neonatal muscle increases the levels of all subunit-specific mRNAs during further development. It prevents the postnatal decrease in gamma-subunit mRNA and enhances the initial increase in epsilon-subunit mRNA. This makes it appear that the epsilon-subunit gene is less sensitive to regulation by the nerve in the postnatal period than the gamma-subunit gene.

An amino acid exchange in the second transmembrane segment of a neuronal nicotinic receptor causes partial epilepsy by altering its desensitization kinetics.

The alpha4 subunit of the neuronal nicotinic acetylcholine receptor is the first gene shown to be involved in a human idiopathic epileptic disease. A missense mutation, leading to the replacement of serine 248 by phenylalanine in the second transmembrane segment, had been detected in patients with autosomal dominant nocturnal frontal lobe epilepsy. The properties of the wild type receptor composed of alpha4 and beta2 subunits and the mutant receptor where alpha4 subunits carried the mutation at serine 248 were compared by means of cDNA manipulation and expression in Xenopus oocytes. The mutant receptor exhibited faster desensitization upon activation by acetylcholine and recovery from the desensitized state was much slower than in the wild type receptor. We conclude that the reported mutation causes seizures via a diminution of the activity of the alpha4beta2 neuronal nicotinic acetylcholine receptor.

Subunit-specific block of cloned NMDA receptors by argiotoxin636.

Cloned NMDA receptor channels of the NR1-NR2A, NR1-NR2B and NR1-NR2C type show differences in argiotoxin636 block. Mutations of an asparagine residue located at a homologous position in the TM2 region of all NMDA receptor subunits, which corresponds to the Q/R site of the AMPA receptors, alters the argiotoxin636-induced block. The results suggest that the toxin interacts at this amino acid position with the putative pore forming TM2 region of the NMDA receptor subunits. Sequence differences in the TM2 segment of NR2A and NR2C subunits are not responsible for the subtype-specific sensitivity to argiotoxin636 as revealed by site-directed mutagenesis.

Torpedo electromotor system development: biochemical differentiation of Torpedo electrocytes in vitro.

The accumulation of 2 postsynaptic proteins--the acetylcholine receptor and acetylcholinesterase, total protein and lactate dehydrogenase levels, and the evolution of the multiple molecular forms of acetylcholinesterase (exhibiting apparent sedimentation coefficients of 17, 13, 11 and 6S) have been examined in aneural cultures of embryonic Torpedo electric organ explanted before, during or after electrocyte differentiation and the onset of synaptogenesis. During electrocyte differentiation in vitro, with explants taken before the 38 mm stage, the relative proportions of the 17, 13 and 11S forms change in vitro as in vivo but the 6S form remains abnormally dominant. In tissue explants taken from 38 to 47 mm stage embryos, the 4 major molecular forms of acetylcholinesterase differentiate in a manner identical to that observed in vivo. In explants taken after the onset of synaptogenesis (55-80 mm stages), the proportions of the acetylcholinesterase forms change as in vivo only during the first week in vitro whilst accumulation is occurring at the normal in vivo rate. The switch to the high acetylcholine receptor and acetylcholinesterase accumulation rate that occurs when synaptogenesis begins in vivo is not observed after any time lag in vitro with tissue explanted before the stage (55 mm) at which synaptogenesis begins. The effects on acetylcholinesterase and acetylcholine receptor accumulation of supplementing the medium with a neural tissue extract are described. The experiments were designed to elucidate the factors and mechanisms that regulate the differentiation and formation of chemical synapses using the electric organ of Torpedo marmorata as a model system. The results demonstrate that the complex changes occurring in the multiple molecular forms of acetylcholinesterase during electrocyte differentiation are not under direct neural control but that the switch to an increased acetylcholinesterase and acetylcholine receptor accumulation rate may be triggered by an external, possible neural factor.

Asymmetry of the rat acetylcholine receptor subunits in the narrow region of the pore.

The acetylcholine receptor (AChR) channel is a pentameric protein in which every subunit contributes to the conducting parts of the pore. Recent studies of rat nicotinic AChR channels mutated in the alpha-subunit revealed that a threonine residue (alpha T264) in the transmembrane segment M2 forms part of the narrow region of the channel. We have mutated the residues at homologous positions in the beta-, gamma-, and delta-subunits and measured the resulting change in channel conductance. For all subunits the conductance is inversely related to the volume of the amino acid residue, suggesting that they form part of the channel narrow region. Exchanges of residues between subunits do not alter the conductance, suggesting a ring-like structure formed by homologous amino acids. To investigate the relative contribution of amino acid residues at these positions in determining the channel conductance, receptors carrying the same amino acid in each subunit in the narrow region were constructed. They form functional channels in which the conductance is inversely related to the volume of the amino acids in the narrow region. Channels in which the narrow region is formed by four serines and one valine have the same conductance if the valine is located in the alpha-, beta-, or gamma-subunits, but it is smaller if the valine is located in the delta-subunit. The results suggest a structural asymmetry of the AChR channel in its narrow region formed by the hydroxylated amino acids of alpha-, gamma- and delta-subunits, where the delta-subunit serine is a main determinant of the channel conductance.

Laminar expression of m1-, m3- and m4-muscarinic cholinergic receptor genes in the developing rat visual cortex using in situ hybridization histochemistry. Effect of monocular visual deprivation.

The postnatal development of laminar pattern of m1-, m3- and m4-mRNA-muscarinic acetylcholine receptor subtypes in the visual cortex of both normally raised and monocularly deprived rats (one eyelid sutured at the age of 11 days) was studied using in situ hybridization histochemistry and computer-assisted image analysis. From birth until day 15 the level of m1-receptor transcript in layer II/III increases markedly as compared to deeper layers. From day 15 up to day 18 a transient bimodal pattern develops with peaks in layers II/III and VI. Already on day 35 a more homogeneous distribution of m1-receptor mRNA level is detectable persisting until adulthood. In contrast, the m3-receptor mRNA shows already at birth a bimodal distribution with peaks in layers II/III and VI. Further development until adulthood results in transient changes in the ratio of the mRNA levels in these layers. In the adult visual cortex a similar laminar pattern as at birth is observed. From day 1 up to day 10 a relative increase in the mRNA level of the m4-receptor in layers II to IV is observed. From day 10 until day 15 a bimodal distribution of receptor mRNA develops with peaks in layers III and VI which is similar to the adult stage. However, between days 18 and 35 a shift in the laminar receptor mRNA distribution occurs resulting in peaks in layers IV and VI. The labeling of the m5-receptor transcript in rat visual cortex was very weak and did not show any alteration with age. Unilateral eyelid closure from postnatal day 11 resulted in transient changes in the laminar distribution of m3- and m4-receptor mRNA between postnatal days 18 and 25, whereas the development of the laminar pattern of the m1-receptor mRNA was not affected regardless of the length of visual deprivation. The distinct laminar developmental pattern of mRNA muscarinic receptor subtypes in rat visual cortex suggests specific roles of the muscarinic receptor subtypes during the first weeks of postnatal maturation of visual function.

Differential laminar expression of AMPA receptor genes in the developing rat visual cortex using in situ hybridization histochemistry. Effect of visual deprivation.

The postnatal development of laminar pattern of AMPA receptor mRNA subtypes GluR-A through to GluR-D (flip variants) in the visual cortex of both normally raised and monocularly deprived rats (one eyelid sutured at the age of 11 days) was studied using in situ hybridization histochemistry and computer-assisted image analysis. The AMPA receptors GluR-A through to GluR-D transcripts exhibit a differential laminar expression pattern in the developing rat visual cortex. At birth the levels of GluR-A transcripts are lower by about 50% in each visual cortical layer as compared to the adult values. In contrast, GluR-B to GluR-D mRNAs are expressed in all cases at higher levels at birth than in the adult brain. Unilateral eyelid closure at postnatal day 11 for several periods of time resulted in both transient and permanent changes in the laminar development of GluR-A through to GluR-C transcripts but hardly affected the GluR-D mRNA subtype. The distinct laminar developmental pattern of AMPA receptor mRNAs in rat visual cortex as well as the differential effects of visual deprivation suggest specific roles of AMPA receptor subtypes during the early postnatal maturation of visual function.