43K rapsyn is a peripheral protein specifically associated with the nicotinic acetylcholine receptor (nAChR) present in the postsynaptic membrane of the neuromuscular junction and of the electrocyte, and is essential for its clustering. Here, we demonstrate a novel specific phosphorylation of 43K rapsyn by endogenous protein kinase(s) present in Torpedo electrocyte nAChR-rich membranes and identify thiamine triphosphate (TTP) as the phosphate donor. In the presence of Mg(2+) and [gamma-(32)P]-TTP, 43K rapsyn is specifically phosphorylated with a (32)P-half-maximal incorporation at approximately 5-25 microM TTP. The presence of TTP in the cytosol and of 43K rapsyn at the cytoplasmic face of the postsynaptic membrane, together with TTP-dependent phosphorylation of 43K rapsyn without added exokinases, suggests that TTP-dependent-43K-rapsyn phosphorylation may occur in vivo. In addition, phosphoamino acid and chemical stability analysis suggests that the residues phosphorylated are predominantly histidines. Inhibition of phosphorylation by Zn(2+) suggests a possible control of 43K rapsyn phosphorylation state by its zinc finger domain. Endogenous kinase(s) present in rodent brain membranes can also use [gamma-(32)P]-TTP as a phosphodonor. The use of a phosphodonor (TTP) belonging to the thiamine family but not to the classical (ATP, GTP) purine triphosphate family represents a novel phosphorylation pathway possibly important for synaptic proteins.

Abstract. The subcellular distribution of the 43,000-D protein (43 kD or u~) and of some major cytoskeletal proteins was investigated in Torpedo marmorata electrocytes by immunocytochemical methods (immunofluorescence and immunogold at the electron microscope level) on frozen-fixed sections and homogenates of electric tissue. A monoclonal antibody directed against the 43-kD protein (Nghirm, H. O., J. Cartaud, C. Dubreuil, C. Kordeli, G. Buttin, and J. P. Changeux, 1983, Proc. Natl. Acad. Sci. USA, 80:6403-6407), selectively labeled the postsynaptic membrane on its cytoplasmic face. Staining by anti-actin and anti-desmin antibodies appeared evenly distributed within the cytoplasm: anti-desmin antibodies being associated with the network of intermediate-sized filaments that spans the electrocyte, and anti-actin antibodies making scattered clusters throughout the cytoplasm without preferential labeling of the postsynaptic membrane. On the other hand, a dense coating by anti-actin antibodies became apparent on the postsynaptic membrane in homogenates of electric tissue pointing to the possible artifactual redistribution of a soluble cytoplasmic actin pool.Anti-fodrin and anti-ankyrin antibodies selectively labeled the non-innervated membrane of the cell. F actin was also detected in this membrane. Filamin and vinculin, two actin-binding proteins recently localized at the rat neuromuscular junction (Bloch, R.J., and Z. W. Hall, 1983, J. CellBiol., 97:217-223), were detected in the electrocyte by the immunoblot technique but not by immunocytochemistry.The data are interpreted in terms of the functional polarity of the electrocyte and of the selective interaction of the cytoskeleton with the innervated and noninnervated domains of the plasma membrane.T HE postsynaptic membrane of the neuromuscular junction and of the electromotor synapse corresponds to a local differentiation of the plasma membrane characterized by an accumulation of the nicotinic acetylcholine receptor (Ach-R).t This membrane specialization persists for a long period after denervation (reviewed in references 10 and 18), indicating that physical constraints maintain the Ach-R molecules in place and, in particular, prevent against their lateral diffusion. Interactions of the Ach-R with extrinsic components from the extracellular matrix (13, 55) and/or with the cytoskeleton (17, 48, 67) have been postulated to contribute to this differentiation process. cell--the non-innervated face--is specialized in the regeneration of the electrochemical gradient (40). The Ach-R is present exclusively on the innervated membrane, and Na ÷ K ÷ ATPase accumulates on the non-innervated one. These membranes thus constitute fully differentiated and stable domains of the plasma membrane. From this standpoint the diskshaped electrocyte shows a striking functional and structural polarity ( 18, 21).Postsynaptic membrane fractions purified from Torpedo comprise essentially the intrinsic Ach-R polypeptides plus few extrinsic components of apparent molecular mass 43 kD (63, 64) na...

Subsynaptic membrane fragments prepared from Torpedo marmorata electric organ contain, in addition to the acetylcholine receptor polypeptides, a major protein band of apparent molecular mass 43,000 daltons. On two-dimensional gels, this band yields three spots referred to as v1, v2, and V3. Monoclonal antibodies against the 43,000-dalton proteins were developed in CBA mice. One of them reacted exclusively with the vP polypeptide but not with p2 and v3. Staining by the "immunogold" reaction followed by observation by electron microscopy showed that this antibody exclusively labeled the innervated membrane of T. marmorata electroplaque on its cytoplasmic face. Electroblots of onedimensional gels of membrane preparations from 80-mm embryo electric organ were prepared. After reaction with the anti-vP monoclonal antibody, a strongly stained 43,000-dalton band was revealed.The acetylcholine receptor (AcChoR) protein (1) makes densely packed and stable aggregates in the subsynaptic membrane of the adult neuromuscular junction and of the electromotor synapse. This supramolecular organization develops during embryogenesis from a diffuse and labile distribution of AcChoR molecules present in the membrane of the noninnervated myotube or electrocyte. The mechanisms postulated to account for this important morphogenetic step (for refs., see ref.

Two neurotoxic alkaloids, anatoxin-a and its homologue homoanatoxin-a, were purified from the filamentous cyanobacteria Oscillatoria sp. strain 193 (PCC 9240) and Oscillatoria formosa NIVA CYA-92 (PCC 10111), respectively, and characterized by mass spectrometry. Biological activity was determined by examining the capacity of the toxins to competitively inhibit the binding of radiolabelled bungarotoxin to acetylcholine receptors, using post-synaptic membrane fractions of Torpedo electric tissue. Inhibition was concentration dependent, with a K i of 5?4±1?1610 "8 M for anatoxin-a and 7?4±0?9610 "8 M for homoanatoxin-a. Their high affinities for the nicotinic cholinergic receptors were exploited to adapt the radioligand-binding assay for routine detection of this class of neurotoxins directly in low-molecular-mass cell extracts of cyanobacteria. Confirmation of the results and toxin identification were achieved by coupled gas chromatography-mass spectrometry (GC/MS). Seventy-six axenic strains, representative of 13 genera, were analysed. Five strains of the genus Oscillatoria, hitherto unknown for their toxicity, inhibited bungarotoxin binding. GC/MS revealed that Oscillatoria sp. strains PCC 6407, PCC 6412 and PCC 9107 synthesized exclusively anatoxin-a, whereas both anatoxin-a and homoanatoxin-a were produced by strain PCC 9029. Oscillatoria sp. strain PCC 6506, an isolate co-identic with strain PCC 9029, also produced both neurotoxins, but their respective presence depended upon growth conditions. The latter results suggest that regulatory differences in at least some of the cyanobacterial strains may account for the preferential synthesis of only one of the two neurotoxins or for their simultaneous occurrence. INTRODUCTIONWorldwide proliferation of cyanobacterial blooms constitutes a serious environmental and economic problem that menaces wildlife and human health. Bloom development, a phenomenon related to water eutrophication, is favoured by the ability of planktonic cyanobacteria to synthesize gas vesicles that allow them to float to the surface layers of the water column. The massive growth and accumulation of cyanobacteria greatly impair food-web dynamics and the physico-chemical factors inside a given aquatic ecosystem. Moreover, many cyanobacteria are able to produce potent hepatotoxins such as microcystin, cylindrospermopsin and nodularin, and/or neurotoxins such as anatoxin-a, homoanatoxin-a, anatoxin-a(s) and saxitoxin (Carmichael, 1994;Sivonen & Jones, 1999).Anatoxin-a (ANTX) (2-acetyl-9-azabicyclo[4.2.1]non-2-ene) and its methylene homologue homoanatoxin-a (HANTX) (2-(propan-1-oxo-1-yl)-9-azabicyclo[4.2.1]non-2-ene) (Devlin et al., 1977;Skulberg et al., 1992), are lowmolecular-mass bicyclic secondary amines synthesized by some planktonic and benthic strains of the genera Anabaena, Oscillatoria, Aphanizomenon and Cylindrospermum (Sivonen et al., 1989(Sivonen et al., , 1990. At nanomolar levels, ANTX is a specific cholinergic agonist whose potency relies upon its high affinity for the nicotinic acet...

We have analyzed the potential role of myogenin in the regulation by electrical activity of the expression of the acetylcholine receptor (AChR) alpha-subunit gene in cultured chick embryonic myotubes. The state of phosphorylation of myogenin was followed by 32P-labeling and immunoprecipitation with an anti-myogenin antibody. In electrically active myotubes myogenin is phosphorylated, while it is dephosphorylated in electrically silent myotubes following tetrodotoxin (TTX) treatment. Accordingly, nuclear protein kinase C (PKC) activity decreases in TTX-treated myotubes. Myogenin dephosphorylation is also observed upon incubation of myotubes with GF109203X, a pharmacological agent which specifically inhibits PKC activity. Both treatments cause similar increases in the expression of the AChR protein. The effects are not additive. Thus TTX and GF109203X most probably affect a common process. Recombinant chick myogenin binds to myogenic sites (E boxes) present in the AChR alpha-subunit promoter but loses this binding capacity after phosphorylation. As a working hypothesis we propose that repression of AChR biosynthesis by electrical activity results, at least partly, from phosphorylation of myogenin via the PKC pathway.

In Torpedo marmorata electroplaque, an extrinsic membrane protein of apparent mass 43,000 daltons colocalizes with the cytoplasmic face of the nicotinic acetylcholine receptor (AChR) in approximately 1:1 stoichiometry. We show that this 43K protein can be phosphorylated in vitro by endogenous protein kinases present in AChR-rich membranes. The extent of 43K protein phosphorylation exceeds that of the subunits of the AChR, well-established substrates for enzymatic phosphorylation. We demonstrate that significant 43K phosphoprotein exists in vivo. The kinetics of phosphate incorporation mediated by endogenous kinases differed significantly from those of the AChR subunits, suggesting that different phosphorylation cascades are involved. Use of specific inhibitors of a variety of protein kinases indicated that endogenous cAMP-dependent protein kinase catalyzes phosphorylation of the 43K protein in vitro. All of the phosphate incorporated into 43K protein was accounted for by phosphoserine (0.65 mol/mol of 43K protein). Potential structural and functional consequences of 43K protein phosphorylation are discussed.

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Abstract. The assembly of the nicotinic acetylcholine receptor (AchR) and the 43-kD protein 0'0, the two major components of the post synaptic membrane of the electromotor synapse, was followed in Torpedo marmorata electrocyte during embryonic development by immunocytochemical methods. At the first developmental stage investigated (45-mm embryos), accumulation of AchR at the ventral pole of the newly formed electrocyte was observed within columns before innervation could be detected. No concomitant accumulation of 43-kD immunoreactivity in AchR-rich membrane domains was observed at this stage, but a transient asymmetric distribution of the extracellular protein, laminin, which paralleled that of the AchR, was noticed. At the subsequent stage studied (80-mm embryos), codistribution of the two proteins was noticed on the ventral face of the cell.