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Toxins that block neuronal potassium channels and affect transmitter release
152
8th European Symposium-Abstracts
Toxins that block neuronal potassium channels and affect transmitter release. AuN L. HARVEY (Department of Physiology and Pharmacology, Strathclyde University, Glasgow G1 1XW, U.K .) . SEVERAL, toxins are known to affect the release of acetylcholine at the neuromuscularjunction. Snake toxins with phospholipase activity can block release, although they initially cause a stimulation. Facilitation of release in mammalian nerve-muscle preparations by e.g. ß-bungarotoxin is associated with block of some nerve terminal K channels (RowAN and HARVEY, 1988). Presumably this slows repolarüation after action potentials, and thus allows Ca ions longer to enter the terminal and trigger release . Another facilitatory toxin is dendrotoxin from mamba snakes . It also blocks K channels, although a different subtype to those affected by phospholipase toxins. Dendrotoxin causes repetitive firing of nerve terminals and increases acetylcholine release (ANnERSON and HARVEY, 1988). High affinity binding sites for ß-bungarotoxin and dendrotoxin exist on synaptosomal membranes (e.g. BREEZE aIId DOLLY, 1989). Another toxin blocking neuronal K channels is noxiustoxin from the scorpion Centruroides noxius (PossANl et al ., 1982). It also facilitates release of acetylcholine, although its selectivity for K channel subtypes has not been established. Noxiustoxin competes with radiolabelled dendrotoxin for binding to rat synaptosomal membranes (HARVEY et al., 1989a). Similar scorpion toxins are present in Pandinus imperator venom (MARSHALL and HARVEY, 1989). Other K channels are activated by internal Ca ions. Charybdotoxin, from the scorpion Leiurus quinquestriatus (MILLER et al., 1985), blocks Ca-activated K currents at motor nerve terminals, but this does not normally affect acetylcholine release unless there is simultaneous block of voltage-dependent K currents (ANDERSON et al., 1988). Despite this, charybdotoxin shows high affinity for dendrotoxin binding sites on synaptosomal membranes (HARVEY et al ., 1989b). REFERENCES ANDERSON, A. J. and HARVEY, A. L. (1988) Br. J. Pharmac. 93, 215. ANDERSON, A. J., HARVEY, A. L., RoweN, E. G. and STRONG, P. N. (1988) Br . J. Pharmac. 9S, 1329. BREEEZE, A. L. and DOLLY, J. O. (1989) Eur. J. Biochem. 178, 771. HARVEY, A. L., MARSHALL, D. L. and PossANl, L. D. (1989a) Toxicon, in press. HARVEY, A. L., MARSHALL, D. L., DE-ALL~, F. A. and STRONG, P. N. (1989b) Biochem. Biophys. Res. Commun . 163, 394. MARSHAU., D. L. and HARVEY, A. L. (1989) Toxicon 27, 493. MILLER, C., Moc2YDLOwsx1, E., LATORRE, R. and PHaurs, M. (1985) Nature 313, 316. PossANl, L. D., MARTIN, B. M. and SvexDSEN, T. (1982) Carlsberg Res. Commun. 47, 285. ROWAN, E. G. and HARVEY, A. L. (1988). Br . J. Pharmac. 94, 839. Noxiustoxin blocks newonalpotassium channels andfacilitates the evoked release of acetylcholine. A. L. HARVEY, D. L. MARSHAU. and L. D. POSSANI' (Department of Physiology and Pharmacology, Strathclyde University, Glasgow G1 1XW, U.K . and' Departmento de Bioquimica, Centro de Investigation sobre Ingenieria Genetics y Biotecnologia-UNAM, Cuernavaca Morelos 62290, Mexico). NOXIUSTOXIN is a 39 residue polypeptide isolated from the venom of the scorpion Centruroides noxius (POSSANI et
al ., 1982) that is known to block some neuronal potassium currents (CARaoNE Pt al., 1982, 1987). We have tested noziustoxin for effects on acetylcholine release using isolated nerve-muscle preparations and examined the ability of noxiustoxin to interfere with the binding of another potassium channel toxin, dendrotoxin to rat brain synaptosomal membranes. On chick biventer cervicis preparations, noxiustoxin (0 .1-0 .2 pM) produced a slow, progressive increase in responses to indirect stimulation, with little change in postjunctional sensitivity as monitored by responses to acetylcholine, carbachol and KCI. After 60 min exposure, responses to nerve stimulation had doubled. Responses to direct muscle stimulation were only increased by about 20% after 90 min exposure to noxiustoxin. Toxin I from the black mamba Dendroaspis polylepis was labelled with ' 25I and specific binding to synaptosomal membranes from rat brain was determined (HARVEY et al ., 1989). Cold toxin I displaced the labelled toxin with a K, of I .1 ±0.5 x 10 - ' ° M. Noxiustoxin also displaced the specific binding of the labelled dendrotoxin with a K, almost identical to that of toxin I. REFERENCES CARBONE, E ., WANKE, E ., PAESTIPINO, G ., POSSANI, L. D. and MAEUCKE, A. (1982) . Nature 296, 90 . CARBONE, E., PRESTIPINO, G., SPADAVECCFDA, L ., FRANCIOLINI, F. and PossANl, L. D. (1987) . Pflugers Arch. 408,
423.
HARVEY, A. L., MARSHALL, D . L., DE-ALLIE, F . A . and STRONG, P. N. (1989) . Biochem. Biophys. Res. Commun .
163, 394.
POSSANI, L. D., MARTIN, B. M. and SveNDSex, I. (1982) . Carlsberg Res. Commun. 47, 285.
Acknowledgement-this work was supported by a grant from the Science and Engineering Research Council.
8th European Symposium-Abstracts
Toxins that block neuronal potassium channels and affect transmitter release. AuN L. HARVEY (Department of Physiology and Pharmacology, Strathclyde University, Glasgow G1 1XW, U.K .) . SEVERAL, toxins are known to affect the release of acetylcholine at the neuromuscularjunction. Snake toxins with phospholipase activity can block release, although they initially cause a stimulation. Facilitation of release in mammalian nerve-muscle preparations by e.g. ß-bungarotoxin is associated with block of some nerve terminal K channels (RowAN and HARVEY, 1988). Presumably this slows repolarüation after action potentials, and thus allows Ca ions longer to enter the terminal and trigger release . Another facilitatory toxin is dendrotoxin from mamba snakes . It also blocks K channels, although a different subtype to those affected by phospholipase toxins. Dendrotoxin causes repetitive firing of nerve terminals and increases acetylcholine release (ANnERSON and HARVEY, 1988). High affinity binding sites for ß-bungarotoxin and dendrotoxin exist on synaptosomal membranes (e.g. BREEZE aIId DOLLY, 1989). Another toxin blocking neuronal K channels is noxiustoxin from the scorpion Centruroides noxius (PossANl et al ., 1982). It also facilitates release of acetylcholine, although its selectivity for K channel subtypes has not been established. Noxiustoxin competes with radiolabelled dendrotoxin for binding to rat synaptosomal membranes (HARVEY et al., 1989a). Similar scorpion toxins are present in Pandinus imperator venom (MARSHALL and HARVEY, 1989). Other K channels are activated by internal Ca ions. Charybdotoxin, from the scorpion Leiurus quinquestriatus (MILLER et al., 1985), blocks Ca-activated K currents at motor nerve terminals, but this does not normally affect acetylcholine release unless there is simultaneous block of voltage-dependent K currents (ANDERSON et al., 1988). Despite this, charybdotoxin shows high affinity for dendrotoxin binding sites on synaptosomal membranes (HARVEY et al ., 1989b). REFERENCES ANDERSON, A. J. and HARVEY, A. L. (1988) Br. J. Pharmac. 93, 215. ANDERSON, A. J., HARVEY, A. L., RoweN, E. G. and STRONG, P. N. (1988) Br . J. Pharmac. 9S, 1329. BREEEZE, A. L. and DOLLY, J. O. (1989) Eur. J. Biochem. 178, 771. HARVEY, A. L., MARSHALL, D. L. and PossANl, L. D. (1989a) Toxicon, in press. HARVEY, A. L., MARSHALL, D. L., DE-ALL~, F. A. and STRONG, P. N. (1989b) Biochem. Biophys. Res. Commun . 163, 394. MARSHAU., D. L. and HARVEY, A. L. (1989) Toxicon 27, 493. MILLER, C., Moc2YDLOwsx1, E., LATORRE, R. and PHaurs, M. (1985) Nature 313, 316. PossANl, L. D., MARTIN, B. M. and SvexDSEN, T. (1982) Carlsberg Res. Commun. 47, 285. ROWAN, E. G. and HARVEY, A. L. (1988). Br . J. Pharmac. 94, 839. Noxiustoxin blocks newonalpotassium channels andfacilitates the evoked release of acetylcholine. A. L. HARVEY, D. L. MARSHAU. and L. D. POSSANI' (Department of Physiology and Pharmacology, Strathclyde University, Glasgow G1 1XW, U.K . and' Departmento de Bioquimica, Centro de Investigation sobre Ingenieria Genetics y Biotecnologia-UNAM, Cuernavaca Morelos 62290, Mexico). NOXIUSTOXIN is a 39 residue polypeptide isolated from the venom of the scorpion Centruroides noxius (POSSANI et
al ., 1982) that is known to block some neuronal potassium currents (CARaoNE Pt al., 1982, 1987). We have tested noziustoxin for effects on acetylcholine release using isolated nerve-muscle preparations and examined the ability of noxiustoxin to interfere with the binding of another potassium channel toxin, dendrotoxin to rat brain synaptosomal membranes. On chick biventer cervicis preparations, noxiustoxin (0 .1-0 .2 pM) produced a slow, progressive increase in responses to indirect stimulation, with little change in postjunctional sensitivity as monitored by responses to acetylcholine, carbachol and KCI. After 60 min exposure, responses to nerve stimulation had doubled. Responses to direct muscle stimulation were only increased by about 20% after 90 min exposure to noxiustoxin. Toxin I from the black mamba Dendroaspis polylepis was labelled with ' 25I and specific binding to synaptosomal membranes from rat brain was determined (HARVEY et al ., 1989). Cold toxin I displaced the labelled toxin with a K, of I .1 ±0.5 x 10 - ' ° M. Noxiustoxin also displaced the specific binding of the labelled dendrotoxin with a K, almost identical to that of toxin I. REFERENCES CARBONE, E ., WANKE, E ., PAESTIPINO, G ., POSSANI, L. D. and MAEUCKE, A. (1982) . Nature 296, 90 . CARBONE, E., PRESTIPINO, G., SPADAVECCFDA, L ., FRANCIOLINI, F. and PossANl, L. D. (1987) . Pflugers Arch. 408,
423.
HARVEY, A. L., MARSHALL, D . L., DE-ALLIE, F . A . and STRONG, P. N. (1989) . Biochem. Biophys. Res. Commun .
163, 394.
POSSANI, L. D., MARTIN, B. M. and SveNDSex, I. (1982) . Carlsberg Res. Commun. 47, 285.
Acknowledgement-this work was supported by a grant from the Science and Engineering Research Council.