Interaction of a synaptobrevin (VAMP)-syntaxin complex with presynaptic calcium channels.

Nerve terminal protein complexes implicated in exocytosis were examined by immuno-isolation from rat brain synaptosomes. Immunoprecipitation with anti-syntaxin or anti-VAMP antibodies revealed a syntaxin-SNAP25-VAMP-synaptotagmin complex. Anti-VAMP antibodies also trapped a distinct VAMP-synaptophysin complex. A similar fraction (about 70%) of N-type calcium channels ([125I]omega conotoxin GVIA receptors), was immunoprecipitated by either anti-syntaxin or anti-VAMP antibodies, but not by anti-synaptophysin antibodies (< 4%). The majority of N- but not L-type calcium channels ([3H]PN200-110 receptors), appear to be associated with a synaptic vesicle prefusion complex.

Expression of synaptotagmin and syntaxin associated with N-type calcium channels in small cell lung cancer.

The presence of synaptic proteins involved in excitation/secretion coupling was examined in ten small cell lung cancer lines. N-Type calcium channels (omega-conotoxin receptors), synaptotagmin (p65) and syntaxin (HPC-1) were detected in eight. Co-immunoprecipitation experiments indicated that syntaxin can form a complex with synaptotagmin and calcium channels. The expression of synaptotagmin in small cell lung cancer may elicit an autoimmune response that reduces transmitter release at the nerve terminal.

Binding of Ala-scanning analogs of omega-conotoxin MVIIC to N- and P/Q-type calcium channels.

omega-Conotoxin MVIIC binds to P/Q-type calcium channels with high affinity and N-type channels with low affinity. To reveal the residues essential for subtype selectivity, we synthesized Ala-scanning analogs of MVIIC. Binding assays using rat cerebellar P(2) membranes suggested that Thr(11), Tyr(13) and Lys(2) are essential for binding to both N- and P/Q-type channels, whereas Lys(4) and Arg(22) are important for binding to P/Q-type channels. These results suggest that MVIIC interacts with P/Q-type channels via a large surface, in good agreement with previous observations using chimeric analogs.

Role of Thr(11) in the binding of omega-conotoxin MVIIC to N-type Ca2+ channels.

As replacement of Thr(11) of omega-conotoxin MVIIC with Ala significantly reduced the affinity for both N- and P/Q-type calcium channels, we examined the effect of substitution at this position with other residues. Binding assays using rat cerebellar P2 membranes showed that the affinity is in the order of Leu>Val, aminobutyric acid, Thr>Asn&z.Gt;Ser, Ala, Asp, Phe, Tyr for N-type channels and Thr>Leu, Val, aminobutyric acid, Asn, Ser>Ala&z.Gt;Asp, Phe, Tyr for P/Q-type channels, suggesting that aliphatic amino acids with longer side chains are favorable for block of N-type channels. The effects of substitution were examined electrophysiologically in BHK cells expressing N-type Ca2+ channels. Inhibition of Ba2+ current by the analogs did not completely correlate with binding affinity, although binding to BHK cells was comparable to rat cerebellar membranes.

Interaction of synaptotagmin with voltage gated calcium channels: a role in Lambert-Eaton myasthenic syndrome?

Plasma from patients with Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disease of neuromuscular transmission, contains antibodies that bind to the synaptic vesicle protein synaptotagmin. Synaptotagmin associates with calcium channels and appears to regulate synaptic vesicle docking at the plasma membrane prior to rapid neurotransmitter release. Autoantibodies directed against a synaptotagmin-calcium channel complex may be involved in the etiology of LEMS. In the majority of patients LEMS is associated with small cell lung cancer (SCLC). We have detected the expression of proteins of the secretory pathway, including synaptotagmin, syntaxin and N-type calcium channels, in a panel of SCLC tumor lines. These observations are compatible with the hypothesis that the initial autoimmune response in LEMS is triggered by the tumor.

Synaptotagmin: a Lambert-Eaton myasthenic syndrome antigen that associates with presynaptic calcium channels.

Plasma from patients with Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disease of neuromuscular transmission, contains antibodies that immunoprecipitate 125I-omega-conotoxin GVIA labeled-calcium channels solubilized from rat brain. These antibodies label a 58-kDa protein in Western blots of partially purified 125I-omega-conotoxin receptor preparations. Monoclonal antibody 1D12, produced by immunizing mice with synaptic membranes, has similar properties as these LEMS IgG. 1D12 antigen was purified by immunoaffinity chromatography and shown to bind LEMS IgG. The antigen was identified by immunoscreening a rat brain cDNA library with mAb 1D12 and found to have strong homology to the synaptic vesicle protein synaptotagmin. These antibodies immunoprecipitate calcium channels by binding to synpatotagmin, an associated protein. We suggest that the interaction between synaptotagmin and omega-conotoxin sensitive calcium channels plays a role in docking synaptic vesicles at the plasma membrane prior to rapid neurotransmitter release. Autoantibody binding to a synaptotagmin-calcium channel complex may be involved in the etiology of LEMS.

Detection and photoaffinity labeling of the Ca2+-activated K+ channel-associated apamin receptor in cultured astrocytes from rat brain.

Apamin, an 18-amino acid bee venom peptide, is a specific blocker of a class of Ca2+ activated K+ channels. Mono 125I-iodoapamin was used to detect the K+ channel-associated receptor site in cultured astrocytes from rat brain. Specific high-affinity binding to intact glial cells with a Kd of about 90 pM at 1 degree C and pH 7.5 was demonstrated by equilibrium and kinetic methods. The average receptor capacity was 3 fmol/mg cell protein which is 2 to 3-fold lower than in primary cultured neurons. Binding was stimulated by K+ ions, but to a lesser extent than with neuronal receptors. Photoaffinity labeling of receptor/ion channel components using an arylazide derivative of 125I-monoiodoapamin revealed the presence of the 86- and 33-kDa polypeptides, previously detected in neurones. However a 59-kDa peptide which is present in synaptic membrane preparations from adult rat brain, but not in cultured neurons, was also clearly labeled in intact astrocytes. This indicates that the 59-kDa polypeptide is not a proteolytic fragment of the 86-kDa chain but an associated subunit which is only accessible to photolabeling in certain apamin receptor preparations. Apamin-sensitive Ca2+-activated K+ channels in astrocytes may be one of the pathways by which glial cells redistribute K+ in the central nervous system (CNS).

Mouse motor nerve terminal immunoreactivity to synaptotagmin II during sustained quantal transmitter release.

An antibody directed against the lumenal NH2-terminus of synaptotagmin II was used to examine the distribution of this vesicular protein either after spontaneous acetylcholine release or after sustained release induced by La3+ or alpha-latrotoxin, in conditions that prevent endocytosis. The detection of the epitope was examined in the presence or absence of Triton X-100. We show that, in resting conditions of transmitter release, permeabilization of nerve terminal membranes is required for obvious detection of synaptotagmin Ii immunoreactivity whereas during sustained rates of quantal release, permeabilization is not necessary. These data indicate that, in the latter conditions, synaptotagmin II is incorporated into the terminal axolemma and its intravesicular domain exposed at the extracellular nerve terminal surface.

Omega-conotoxin sensitive calcium channels in cerebellar granule cells are not coupled to [3H]glutamate release.

We have studied the biochemical and functional aspects of omega-conotoxin GVIA (omega-CgTx)-sensitive calcium channels in cerebellar granule cells in vitro. 125I-omega-Conotoxin GVIA (125I-omega-CgTx) binding sites were detected in intact cultured cerebellar granule cells and binding parameters were measured (Bmax: 134 fmol/mg protein; kinetic association constant kappa: 3.10(6) M-1.s-1). [3H]Glutamate release was assessed under different release paradigms (namely release triggered by calcium, voltage, and sodium channel agonists) and different times (15 s and 2 min). However, in all cases, [3H]glutamate release was found to be completely insensitive to omega-CgTx. Conversely, voltage-dependent release was inhibited in a dose-dependent fashion by cadmium chloride, with total inhibition at 10(-4) M. These results indicate that N-type calcium channels are not involved in glutamate secretion from granule neurons.

Interactions of the neurotoxin apamin with a Ca2+-activated K+ channel in primary neuronal cultures.

Mono[125I]iodoapamin bound to specific sites on cultured rat embryonic neurons. The dissociation constant for the receptor-neurotoxin complex measured at equilibrium was 60-120 pM at pH 7.2 and 4 degrees C, with a maximal binding capacity of 3-8 fmol/mg of cell protein. Apamin inhibited calcium ionophore-induced 86Rb+ release from cell cultures. The dose effect curve for this pharmacological test corresponded closely to the displacement of 125I-apamin by native apamin in binding experiments. Formation of the 125I-apamin receptor complex requires exogenous K+. Reduced binding in the absence of K+ was due to diminished binding capacity rather than a lower affinity. The apamin receptor seems to be associated with a cell surface K+ site which shows 50% occupancy at 1.6 mM, and which could be involved in the regulation of channel activity. Apamin sites were present at the earliest developmental stage tested and their number did not evolve during 8 days in culture. In the same period, however, alpha-scorpion toxin binding increased by a factor of 10. The ontogenesis of Ca2+-activated K+ channels does not seem to occur in parallel with that of voltage-sensitive Na+ channels.

Inhibitory effects of tunicamycin and 2-deoxyglucose on thyroglobulin synthesis.

The kinetic incorporation of labelled sugars and amino acids by rat thyroid hemilobes was measured in the presence of 2-deoxyglucose and tunicamycin, inhibitors of the glycosylation of glycoproteins. With either inhibitor the carbohydrate content of exocytosed thyroglobulin was only slightly decreased (less than 20% of control) whereas the rate of exocytosis was strongly inhibited (by 60-80%). As no intracellular accumulation or proteolysis of non-glycosylated molecules was detected, the reduced rate of thyroglobulin release seems essentially due to a decrease in protein synthesis. In a whole cell system (hemilobes), it is impossible to uncouple glycosylation and protein synthesis by incubation with tunicamycin; 50 micrograms/ml tunicamycin for 270 min inhibited total [3H]-glucosamine and 14C-labelled amino acid incorporation by 65% and 33% respectively. This can be contrasted with cell-free incubation of thyroid rough microsomes where glycosylation was blocked by the same tunicamycin concentration (90% inhibition of N-[3H]acetylglucosamine transfer from UDP-N-[3H]acetylglucosamine) whilst ongoing protein synthesis was not significantly modified (less than 4% inhibition). This clearly suggests that, in thyroid follicular cells, a regulatory link exists between the synthesis of the peptide moiety of a glycoprotein and its glycosylation.

Solubilization of the apamin receptor associated with a calcium-activated potassium channel from rat brain.

The apamin binding protein was solubilized from rat brain synaptic membranes using sodium cholate. Receptor yield and stability depended closely on the detergent/protein ratio. In optimum conditions the receptor retained high affinity for mono 125I-iodoapamin with Kd = 40 pM at pH 7.5 and 1 degree C and a binding capacity of 17 fmol/mg protein. 125I-apamin binding was stimulated by K+ ions with a K0.5 = 0.6 mM, demonstrating that the regulatory K+ site is also part of the soluble complex. Other ions could be substituted for K+ with an affinity sequence Tl+ = K+ = Rb+ greater than Cs+ greater than NH4+ greater than Li+ or Na+. Binding was inhibited by the neuromuscular blockers gallamine and tubocurarine and by the K+ channel blockers quinidine and tetraethylammonium chloride but not by 4-aminopyridine, in agreement with known pharmacological profile for inhibition of apamin-sensitive K+ permeability. Increasing the K+ concentration did not reverse inhibition by tetraethylammonium ions demonstrating that it does not bind competitively to the regulatory cationic site. Analysis of the covalently labeled apamin binding protein/sodium cholate complex by density gradient centrifugation indicated a high molecular weight with S20,w = 20 S.

Photoaffinity labeling of the K+-channel-associated apamin-binding molecule in smooth muscle, liver and heart membranes.

High-affinity binding sites for mono[125I]iodoapamin were detected in membranes (Kd = 59 pM, Bmax = 24 fmol/mg protein) and cultured cells (Kd = 69 pM, Bmax = 2.8 fmol/mg protein) from rat heart and in membranes from guinea-pig ileum (Kd = 67 pM, Bmax 42 fmol/mg protein) and liver (Kd = 15 pM, Bmax = 43 fmol/mg protein). Binding was stimulated by K+ ions (K0.5 = 0.3-0.5 mM). Covalent labeling with arylazide [125I]iodoapamin derivatives showed that smooth muscle, liver and heart binding molecules are associated with a 85-87-kDa polypeptide. A second strongly labeled 57-kDa component was identified in liver membranes only.

Photoaffinity labeling of components of the apamin-sensitive K+ channel in neuronal membranes.

An azidonitrophenylaminoacetyl mono[125I]iodoapamin derivative was prepared which showed specific binding to rat neuronal membranes. UV photolysis lead to the irreversible occupation of binding sites. Photo-labeling of intact primary cultured rat neurones followed by membrane solubilization, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography revealed the covalent incorporation of radioactivity into 3 main components with Mr = 86,000, 30,000, and 23,000. Labeling was completely prevented by a competing excess of native apamin. Similar studies on purified synaptic membranes from the rat brain showed another labeling pattern with major bands corresponding to Mr = 86,000 and 59,000. Although the reasons for the partial discrepancy between cultured embryonic neurons and an adult brain membrane fraction are not yet clear, we conclude that these proteins are intimately associated with the apamin binding site and are probably components of a type of Ca2+-activated K+ channel.