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Presynaptic polypeptide neurotoxins
TIPS -April
1982
167
Presynaptic polypeptide neurotoxins
BruccD.Howard lkpamnrn~ of /liu/o#ca/ Chcmisfry. SchdnJ
wxx4.
Mdwmr.
Unrwrun
OJ G?hh-mrcl. I.rn Anark
(.A
U.S.A
There exist several polypepride neurotoxins Ihat act on nerve termmalt IO ahrr the storage and release of neurotransmhten. Table I ILVLS the hcst characterixd of &SC* roxirrr. The references given in Table I are IO other rwent reviews that dtwrtbe the chemical and pharmacological properties ojthe toxins. The emphair this revwH will be on the nature of rhe sites to which rite toxins bind when they exert Ihew pharmacological effect%.-
of
sN&evenamtoxilw Several ptesynapticallyacting neumtok ins are found in snakevenoms. Each of tbe snake toxins listed in Table I has phos pholipase k activity. and a variety of studies indicate that the catalytic site of theseenzymes ftmctionsin their toxicity’. Paradoxically, mostknown phospholipascs An anz not neurotonic in spite of having a greaterspecificenzyme activity. PhospholipaseAZ neumtoxinsinhibit the evoked teeleaseof acetylcboline from the terminals of motor neurons and some but not all cholinergic neurons of the aut* nomic nervous system. In experiments in which the toxins are allowed to gain access
to neumns of the CNS they affect several types of terminals with no apparentpreference for cholinergic structutes. Phospholipase Ap toxin-induced neu~l~ muscular blockade occurs in three stages.
Within S-IO min after exposure to a toxin there occurs a reduction m the evoked release of acetylcholine This fust stage is followed by a 3040 min period of increasedevoked release. During ti third stage which lasts 30420 min. the evoked releasegradually decreasesto an undetecb able level. Only the first stage of blockade is observed under conditions in which the toxins’ phospholipaseactivity is inhibited; the decreasedtransmitterreleasein the first stagemay be a consequenceof toxin binding alone. There is evidence that the increasedtransmitterreleaseoccurringduring the secondstageis due to an increasein the level of free Cam’ in the cytosol of the nerve terminals. Studies in which a nerve-mu& pm paration was briefly exposed to & bungamtoxinand then washed have shown that within 5 to lOmin the toxin either binds ((1the preparationimversibly or produces effect. E+ irteversible so,me incubation of a nerve-muscle ptepatation
with a phospholipase& toxti.that had been inactivated by chemical moddicatlon caurd an incnzaw in tbc time required for blockade by subsequentlyapplied nanre toxin; thus. native toIm ma) bind to speclff nerve terminal sites. which can k competitively blocked by the modified toxin. However. the various neurutoxic phos pholipasesk may not bind to the samesite. An inactivated toxin protectedagainst the toxicity of native toxin only when it wa5 the same toxin’. The inactive toxin eilhcr had no effect or enhancedthe toxicity of other pbospholipase Aa to*ms. Furthermore. simultaneousincubatumwith two diffennt neurotoxic phospholipasescaused blockade much more rapidly tban did a double doseof any one toxin alone’. What is the nature of tlmzbmdmg sltet$) for the toxic phospbolipases? Since neurotoxic pbospholipa..s Aa appear to degladc the same types of common pho+ phoglycerides degradkd by nontoxic enzymes’. it is unlikely that the neuronal bindiig site for the ncurotoxicphospholip
ases is a common phosphoglycerideof the neumnal plasma membranr. Otherwlsc. essentiallyall phospholiws At shouldk neumtoxic and the neurotollc cnz:meb shou!dnot potentiateeachother in the mzm
There 1% reason tag Mu3r ihat rL molecular target 0i the ncurot011~pht~+ pbolip;nes tt 1s not d ltnkTure in~~dvcd spzcifwall) UI tramnutter rekar !5ome oi thl lMlrot0x1c phqhollp;ly, Aa are aho mvotoxr m that the) cause a degemation of s)sletal murk mdependent ot their effect* on neurons. Thenzfore. tbc mokcular target of the neurotoxlc phosphollpa5e3 & is Ilkelk to h unnc structure that 15 common to both nene terminal\ and muscles. Expenmenb on ljnaptosom~ trom mammahan bram mnduzate that ths cife fs ot
foxic phospholipalesiL on nenc terminals are due to an altrntmn of ion fluxes across some neuronal mcmbtanc’ The mns involved have not been Identitied. but Ca“
133 good c:~nd&tr. Recall thin the =ond stage oi ncuromusular bl~ltadc could mult from mcreawd i&c Cs’- in the c)tosoI ot ncne termmats. Ftirrnorc. tbc toxK phosphohpzus.t inhibit the Ned uptake 10 Ca“ into isolated mnochondria from rat brain and inio ?;an-oplasmic reticulum vebtcles ptqxue~! from mam mahan skeletal mi&e. Of cm nontoxic phosphohpases also tnhibn Ca” uptake intothesr organelles; however. there is eve dence that the ~pholiluuc: toxins’ mtubitIon oi the Cal- uptake(a) differs ~mewhat m mechanism from that operating for no& IUUK enqmes. and tb) 13a rek~anr come-
168 late of their neumtoxicty and myotoxicit~. It has been proposedthat the toxic pbos ~~~~j~~~~e~~~~ cytoplasmand exerting their effect on some intemai sttuctme. I this WeR indeed the case, the C!aw tmnspozl systems nf w2we terminal mitochondria and muscle mw piasmic reticulum might be the actual targets, in viva, of ~~~~ A% neumtoxins and myotoxins, n!Spectively. The pathologicalef?&ts producedby these toxins ia P&O aie: consistent with this mecttanism.However, there is noevidence that the toxins enter ceils and they could @uce the same ~~~~~ e%cts by actingat and altering Ca’- transportacross the piiasmamembranes of nerve terminals and muscle. We have suggestedthat the toxic piaX+ phoiipaws lit! act by degrading lipids rusocriated w&b an ion chaouelor pamp and that they may be directed to these lipids by preferentiaifybinding to the ion channel or pump. Sian the toxinz;are krkowltto aiter Ca*+ transport, the binding site could be a Ca2’ channel or pump. However, the effects oa C8”* ,ranspon co&d be ~ecorr daty to an alterationof the channelfor some otherion, e.g. K’ , tlratfunc~iuns todrive or faciwe the transpwt of Cap’. Anather example of a pkosphoiipascAa toxin’saitering an ion channel is the desek s&ation of dte ~~yk~~~ receptor caused by cmtoxin when it is incubated with receptor-rich membranes from Tnrpado ekctiic argat?. Crotoxin isaeomplex of two noncovaientiy linked proteins: a phnspholi~ k and 3 small pmtein Med crota@n. Cmtapotin by its&f iaeks neurotoxicity and pbospholipaseactivity. bu! it potenfiatesthe neu:otoxicity of the #to@lof~ At componeot nf the cmtorin complex. It is si;nif63nt that the intwt ciotoxin complex is more potentthan ~~~~~~~t~~ desensitidng the acetyichniine receptor; flmkrmorr, the intact complex exhibits saturabiebinding to the receptor-ri& rnem bmnes witiie the phosphoiipaseAi corn ponentalone does noP. Most ei~~ys~o~~~~ stud%es of the neummuscuiar blockade caused by the phosphoiipase& toxins have failed to SRow~ypost-synapiiceaecoorthetoxins during the time required to produce%iockade; themfore, the desensitizationof tbg Torpedo acetyichoiiae mptor by cratoxin may he due to Ifouin’sbinding to and hydroiysis of @ids of contaminating presyna~&cmembranes. I is known &at the acetylchoiine receptor can be desensi&xi by smaii amounts of fatty acids and ~~~f~ds. Ott the other hand. crotoxin may indeed be capabte of specitlcalfy altering a post
TIPS -April
1982
synaptic ion channel in Torpedo but not K’, ti’, Ca”, Mg” and the organic ions post-synapticion channels in mammaiian methytatnine and choiine, but not ~u~~~ junctions. There is known glucosamine4. Various studiesindicatethatcr-iattutoxin to be a pronouncedspeciesdependencyto the ~~~~~~1 actions of thE phos. does nM exert its effects at netmnnusctdar @a&&se k toxins. ~ex~~~~ chltaxin ~~~~by~~~~~~~ transiently increases spontaneousquantai eithctNa’MCaa+,OT~h,ortheeMuxof reiease &om nerve endings of mouse dia. K’ from nerve tetminais. It has also betn arguedthat attitargh toxin forms ion ~~~~~~~~~ channelsin artiilcial lipid bitayers,it camtut the converSe is true for taipoxin. Also, taipoxin is three to five times more potent act phatmacologicaiiy suieiy by furming, titan ~~g~toxin or crotoxin at cattsing ion channelsin membmues~i~iy; neutomuscuiar blockade of the mouse otherwise it should affect all netnuns. but dihtagm. but it is 3&IOO times less invertebrate neurons am unaffected. Evi potent than ~~~~~~n or CFoloxiaat ~~~~a~~~~yof blocking the chick biventercervicis muscle binding’@.A study wm madeof the binding peparation. of In31labeled n-latmtoxin to synaptosomai ThLre have been only a few reports of ~rn~s~~~~~.~ direct measurement of phosphoiipaseAn iodinated protein was able to cause binding to neumnaior muscle membranes. nearomuscuiar blockade but the loss (if This is becatiseof the diificulty encoun. any) of potency due to ~~~ was not tered in radioactivity labeling the loxins preciselydetermined. Nonspecificbinding. without inactivatingthem, which was estimatedby co-incubationwith an excess(r Swfold~ of mdabeledtoxin, u_LacWtaxln accountedfor less than 109h of the total The black widow spider Lactmdecm binding of the iaheied a-latrotoxin. The mrtcMns ~~~~~g~~~ and the biown speeif@binding wry fit) sammble;@I BOB widow spider L. geotnemkus pmduce cooperative; (c) high affiity with an toxinsthat uiterthe stnictunrand haactionof appatent KD of i no; aad (d) complete& ~&o&?rgic, noradrunergk and amillergik: inhibited by p&r treatment of the mem. nerve tetminals in vertcbmtesand invelt6 branes with trypsin. suggestingchat the bmtesl’ Almost aii of the pharmamkrgical receptoris a pmtein. The labeled toxin did Sudii vyitb these toxins have em@yed not exhibit speei%cbinding to iiver memunfiitianated venom or mote commoniy branes. an extract of venom glands from the spiders. rtre effects of the crude gkmd exBacrerbrltoxiins; tract on cholinergic neuromu.uiar juncTetanus toxin is pm&ced by the KioKts of vertebratesNIX now known to he anaerobic bacterium, Closttidiwn due to a singf+toxin, o+Krotoxin. F%rif& te&mP. fn mammais the toxin producesa tr-latrotoxin also induces an efflux of spasticparaiysis which has been attributed wetykhoiine, norepinephrine and gamma to a tetanustoxi&duced blockade of the ~~~$~~ acid from marmn&iw brain &ease of transmittersthat mediate inbibislices bu?*isinactive on invertebratenerv- tory tleitmnaipathwaysin theCNS. Support ous tissue. for thishypophesis c-es fmm theability of A few minutes after appfiiation of retanustoxin to b&k&e releaseof glycine, cr.iatmtoxin tv a venebrate nerve-muscle gamma aminobutyric acid and other puta. preparation there occurs a burst of span. rive amino acid transmitters, fmm nerve taneous quantat r&ease producing minia terminals of mammalian brain and spinal tute endpiate potentials of normal amp cold. litude but markedly increased frequency. The toxin appears to reach the CNS via The m r&easecontinuesat maxi. m-grade axon& tmnsport through mai levels for 5 min or so and then neuronsthat have axonai processesextendgndIJaiiy de&es over the nexK 3tl-50 ing from the CNS. There is evidence that minces. The decfine is accompanied by tetanustoxin can aiso be transportedtrans complete inhibition of evoked tr~smiffer syna~ic~ly. release. The nerve terminals treated with Tetanustoxin can act peripbentllyas well u-fatmloxin become depicted of synaptic and inhibit the evoked release of acetyl. vesicles. choiine from motor nerve aims. Little The mechanismof action of a-iatmtoxin is known about its mechanismof action. ~s~n.~-~~ox~~~~~~ cat. Severai studiiesindiiate that the receptor ion conductanceof ~nifiiial lipid biiayer for tetamtstoxin is seiectiveiy iocaIized to membraues appartmtiy by inserting itself neumns and contains gangliosidesof the into ibe bilayer and forming ion cba~tnets Glb series or a carbohydrate component rather Kttanacting as an ion car&f. it similar to that found in these~~g~~~~. makes the membranespermeable to Na’, Sialicacklisthwghttok:oneoftheciucial
TIPS -April
I982
moieties in the receptor. Tetanus toxin binds IO neumns bum not to notuneumnal cells in cultures of rodent and chicken brain, ganglia and retina. plasma mcmbmnserrrichcd suWis of mam maliin brain synaproaomesbind tetanus toxin IO a much greater extent than do sub. frnctions enrkhed in synaptic vesicks or mitochondria. The binding of J”Clabekd teranustoxin to a crude synaptosomalfraction occumzdwith a dis~~ti~ constantof 1IM andwascompetitively inhibited by the sialic acid-containing gangliosider GTI b (KI = 6 nht) and GDlb (K, = 10 mu)“. Tetanus toxin spacifkally binds ro gang. IiosidesGDlb, GTlband GQlbandako, with kss afftnity, to otikr ganglksides’*. Tkt gangliosidebinding site appearsto be on the larger subunitof the toxin. Nothing kk~wn~t~~~~~of~ mokcuk that serves as the receptor for tetanustoxin. The neumnal receptor for tetanus toxin intriguingly appears to be similar to the thymid receptor for ihe peptide thymtropinJ5.onder the binding conditions used, tetanus toxin reversibly binds to thyroid membranes. and this binding is blocked or reversedby thymtropin but not by any of severa!other pep&e hormones. Conversely, thytotropin exhibits a reves ible bindingto rat brain membranesand this binding is blocked by tetanustoxin. However. the binding of tetanus toxin to the brain membranesis enhancedby thymtropin. It has heen suggestedthat some of ihe symptoms of tetanus toxicity may be causs by tetanus toxin-induced thyroid hyperfunction. Another similarity between tetanus toxin and thymtmpin is that each can affect the membrane potential of a target cell; tetanustoxin has recently been foundto alter the ion permeability of synap tosomesfrom guinea pig brain. ruling toxin is ptuduced by Cfasrridiwn ~~ufi~um’,~. There am at least eight immunologically distinct types of toxin, which ate similar in structure and pharmacological properties. Botulinurn toxin inhibits the evoked rekase of mans muter from cholineqk nerve terminals.
iw Very little : rufinum toxin is required to produce neunmuscular blockade; it has heen estimated that no mote than IO molecules of toxin are tequired to block a singk cbolinergic synapse. Nerve-muscle ~~~~s that alp @ated with ~ulinum toxin have a dtxwa#l spontaneous rekau of acetylcholine tdectua& frequency and amplitude of miniature end-pl_atepotentials) while the pnst-synapticresponseto acetylcholine is unaltered. Eventually the toxin causescompkte inhihitinnof evoked release of acetyicholine. Although these effects have heen characterized in gmat detail’-“. httk has heen learned about the molecular larger nf the toxin. ‘“ElaMcd bntulinum toxin exhibits utumbk binding to synaptosomcs fmm rat brain” and unlabeledtoxin that was boundIO ~ynapb sames was found by ekctron microscopyto be localizedon tbe~~~1~0~1 andextrajumtional areas of presynapticmembranesbut not on post-synapticmembranesJS. Tetanus toxin and botulinum toxin are similar with respect to bacterial genus of origin. structute. and effectson neummus cular transmission.Tetanustoxin of course affects a broader mnp of nerve terminals. It is tempting to speculatethat tetanu toxin and ~ulinum toxin have a rommon molecular mechanism of action (e.g. enzymic activity) but \I ith different target cells that have different n ceptm molecules.
cWhile the pharmacologicaleffect* of the polypeptide toxins mat inhihit u-drummer releasehave been well characterized.little is known about the actionsof the toxins at the molecular kvei. Ahnost all the evidence indicatesthat there are \pccitic binding sites for these toxins on the plasma membmne of nerve terminals. Pmgres\ should soon be made in un~~tand~ne the mokcular structureof thesebinding &es. However, a word of caution seemsappm piate in this regtud. It is p&bk that tluse toxinsdo not inhibit transmitterteleaseby a direct alteration of the tekase apparatus it\ei:. For exrmpk. changesin membrane
~~abiiil~ Ict km\ ha\e hrm tmphcated tn Ihe pnmary acoon 0i rt-iatioroxm. Mamu Inxin. attd the phusphohpasc& toxins l uggc*ting that tlku cftect~ on trausmittcr\lorage and mka+e may be twig secondaryto the ion stability change\. Thus. knowhdge TPrhe molecular me&m. irms of action of these lntinr wdl nut ttecessardykad to an unde’nanding of Ihc biochemistq of the release iqpG&b. Nevedekss. all of the roxim &ould bc urful for faheling and characteriring pro syttaptr componentslb1 arc tmpv-timt for uBmeneuronai’funcuan.
1982
167
Presynaptic polypeptide neurotoxins
BruccD.Howard lkpamnrn~ of /liu/o#ca/ Chcmisfry. SchdnJ
wxx4.
Mdwmr.
Unrwrun
OJ G?hh-mrcl. I.rn Anark
(.A
U.S.A
There exist several polypepride neurotoxins Ihat act on nerve termmalt IO ahrr the storage and release of neurotransmhten. Table I ILVLS the hcst characterixd of &SC* roxirrr. The references given in Table I are IO other rwent reviews that dtwrtbe the chemical and pharmacological properties ojthe toxins. The emphair this revwH will be on the nature of rhe sites to which rite toxins bind when they exert Ihew pharmacological effect%.-
of
sN&evenamtoxilw Several ptesynapticallyacting neumtok ins are found in snakevenoms. Each of tbe snake toxins listed in Table I has phos pholipase k activity. and a variety of studies indicate that the catalytic site of theseenzymes ftmctionsin their toxicity’. Paradoxically, mostknown phospholipascs An anz not neurotonic in spite of having a greaterspecificenzyme activity. PhospholipaseAZ neumtoxinsinhibit the evoked teeleaseof acetylcboline from the terminals of motor neurons and some but not all cholinergic neurons of the aut* nomic nervous system. In experiments in which the toxins are allowed to gain access
to neumns of the CNS they affect several types of terminals with no apparentpreference for cholinergic structutes. Phospholipase Ap toxin-induced neu~l~ muscular blockade occurs in three stages.
Within S-IO min after exposure to a toxin there occurs a reduction m the evoked release of acetylcholine This fust stage is followed by a 3040 min period of increasedevoked release. During ti third stage which lasts 30420 min. the evoked releasegradually decreasesto an undetecb able level. Only the first stage of blockade is observed under conditions in which the toxins’ phospholipaseactivity is inhibited; the decreasedtransmitterreleasein the first stagemay be a consequenceof toxin binding alone. There is evidence that the increasedtransmitterreleaseoccurringduring the secondstageis due to an increasein the level of free Cam’ in the cytosol of the nerve terminals. Studies in which a nerve-mu& pm paration was briefly exposed to & bungamtoxinand then washed have shown that within 5 to lOmin the toxin either binds ((1the preparationimversibly or produces effect. E+ irteversible so,me incubation of a nerve-muscle ptepatation
with a phospholipase& toxti.that had been inactivated by chemical moddicatlon caurd an incnzaw in tbc time required for blockade by subsequentlyapplied nanre toxin; thus. native toIm ma) bind to speclff nerve terminal sites. which can k competitively blocked by the modified toxin. However. the various neurutoxic phos pholipasesk may not bind to the samesite. An inactivated toxin protectedagainst the toxicity of native toxin only when it wa5 the same toxin’. The inactive toxin eilhcr had no effect or enhancedthe toxicity of other pbospholipase Aa to*ms. Furthermore. simultaneousincubatumwith two diffennt neurotoxic phospholipasescaused blockade much more rapidly tban did a double doseof any one toxin alone’. What is the nature of tlmzbmdmg sltet$) for the toxic phospbolipases? Since neurotoxic pbospholipa..s Aa appear to degladc the same types of common pho+ phoglycerides degradkd by nontoxic enzymes’. it is unlikely that the neuronal bindiig site for the ncurotoxicphospholip
ases is a common phosphoglycerideof the neumnal plasma membranr. Otherwlsc. essentiallyall phospholiws At shouldk neumtoxic and the neurotollc cnz:meb shou!dnot potentiateeachother in the mzm
There 1% reason tag Mu3r ihat rL molecular target 0i the ncurot011~pht~+ pbolip;nes tt 1s not d ltnkTure in~~dvcd spzcifwall) UI tramnutter rekar !5ome oi thl lMlrot0x1c phqhollp;ly, Aa are aho mvotoxr m that the) cause a degemation of s)sletal murk mdependent ot their effect* on neurons. Thenzfore. tbc mokcular target of the neurotoxlc phosphollpa5e3 & is Ilkelk to h unnc structure that 15 common to both nene terminal\ and muscles. Expenmenb on ljnaptosom~ trom mammahan bram mnduzate that ths cife fs ot
foxic phospholipalesiL on nenc terminals are due to an altrntmn of ion fluxes across some neuronal mcmbtanc’ The mns involved have not been Identitied. but Ca“
133 good c:~nd&tr. Recall thin the =ond stage oi ncuromusular bl~ltadc could mult from mcreawd i&c Cs’- in the c)tosoI ot ncne termmats. Ftirrnorc. tbc toxK phosphohpzus.t inhibit the Ned uptake 10 Ca“ into isolated mnochondria from rat brain and inio ?;an-oplasmic reticulum vebtcles ptqxue~! from mam mahan skeletal mi&e. Of cm nontoxic phosphohpases also tnhibn Ca” uptake intothesr organelles; however. there is eve dence that the ~pholiluuc: toxins’ mtubitIon oi the Cal- uptake(a) differs ~mewhat m mechanism from that operating for no& IUUK enqmes. and tb) 13a rek~anr come-
168 late of their neumtoxicty and myotoxicit~. It has been proposedthat the toxic pbos ~~~~j~~~~e~~~~ cytoplasmand exerting their effect on some intemai sttuctme. I this WeR indeed the case, the C!aw tmnspozl systems nf w2we terminal mitochondria and muscle mw piasmic reticulum might be the actual targets, in viva, of ~~~~ A% neumtoxins and myotoxins, n!Spectively. The pathologicalef?&ts producedby these toxins ia P&O aie: consistent with this mecttanism.However, there is noevidence that the toxins enter ceils and they could @uce the same ~~~~~ e%cts by actingat and altering Ca’- transportacross the piiasmamembranes of nerve terminals and muscle. We have suggestedthat the toxic piaX+ phoiipaws lit! act by degrading lipids rusocriated w&b an ion chaouelor pamp and that they may be directed to these lipids by preferentiaifybinding to the ion channel or pump. Sian the toxinz;are krkowltto aiter Ca*+ transport, the binding site could be a Ca2’ channel or pump. However, the effects oa C8”* ,ranspon co&d be ~ecorr daty to an alterationof the channelfor some otherion, e.g. K’ , tlratfunc~iuns todrive or faciwe the transpwt of Cap’. Anather example of a pkosphoiipascAa toxin’saitering an ion channel is the desek s&ation of dte ~~yk~~~ receptor caused by cmtoxin when it is incubated with receptor-rich membranes from Tnrpado ekctiic argat?. Crotoxin isaeomplex of two noncovaientiy linked proteins: a phnspholi~ k and 3 small pmtein Med crota@n. Cmtapotin by its&f iaeks neurotoxicity and pbospholipaseactivity. bu! it potenfiatesthe neu:otoxicity of the #to@lof~ At componeot nf the cmtorin complex. It is si;nif63nt that the intwt ciotoxin complex is more potentthan ~~~~~~~t~~ desensitidng the acetyichniine receptor; flmkrmorr, the intact complex exhibits saturabiebinding to the receptor-ri& rnem bmnes witiie the phosphoiipaseAi corn ponentalone does noP. Most ei~~ys~o~~~~ stud%es of the neummuscuiar blockade caused by the phosphoiipase& toxins have failed to SRow~ypost-synapiiceaecoorthetoxins during the time required to produce%iockade; themfore, the desensitizationof tbg Torpedo acetyichoiiae mptor by cratoxin may he due to Ifouin’sbinding to and hydroiysis of @ids of contaminating presyna~&cmembranes. I is known &at the acetylchoiine receptor can be desensi&xi by smaii amounts of fatty acids and ~~~f~ds. Ott the other hand. crotoxin may indeed be capabte of specitlcalfy altering a post
TIPS -April
1982
synaptic ion channel in Torpedo but not K’, ti’, Ca”, Mg” and the organic ions post-synapticion channels in mammaiian methytatnine and choiine, but not ~u~~~ junctions. There is known glucosamine4. Various studiesindicatethatcr-iattutoxin to be a pronouncedspeciesdependencyto the ~~~~~~1 actions of thE phos. does nM exert its effects at netmnnusctdar @a&&se k toxins. ~ex~~~~ chltaxin ~~~~by~~~~~~~ transiently increases spontaneousquantai eithctNa’MCaa+,OT~h,ortheeMuxof reiease &om nerve endings of mouse dia. K’ from nerve tetminais. It has also betn arguedthat attitargh toxin forms ion ~~~~~~~~~ channelsin artiilcial lipid bitayers,it camtut the converSe is true for taipoxin. Also, taipoxin is three to five times more potent act phatmacologicaiiy suieiy by furming, titan ~~g~toxin or crotoxin at cattsing ion channelsin membmues~i~iy; neutomuscuiar blockade of the mouse otherwise it should affect all netnuns. but dihtagm. but it is 3&IOO times less invertebrate neurons am unaffected. Evi potent than ~~~~~~n or CFoloxiaat ~~~~a~~~~yof blocking the chick biventercervicis muscle binding’@.A study wm madeof the binding peparation. of In31labeled n-latmtoxin to synaptosomai ThLre have been only a few reports of ~rn~s~~~~~.~ direct measurement of phosphoiipaseAn iodinated protein was able to cause binding to neumnaior muscle membranes. nearomuscuiar blockade but the loss (if This is becatiseof the diificulty encoun. any) of potency due to ~~~ was not tered in radioactivity labeling the loxins preciselydetermined. Nonspecificbinding. without inactivatingthem, which was estimatedby co-incubationwith an excess(r Swfold~ of mdabeledtoxin, u_LacWtaxln accountedfor less than 109h of the total The black widow spider Lactmdecm binding of the iaheied a-latrotoxin. The mrtcMns ~~~~~g~~~ and the biown speeif@binding wry fit) sammble;@I BOB widow spider L. geotnemkus pmduce cooperative; (c) high affiity with an toxinsthat uiterthe stnictunrand haactionof appatent KD of i no; aad (d) complete& ~&o&?rgic, noradrunergk and amillergik: inhibited by p&r treatment of the mem. nerve tetminals in vertcbmtesand invelt6 branes with trypsin. suggestingchat the bmtesl’ Almost aii of the pharmamkrgical receptoris a pmtein. The labeled toxin did Sudii vyitb these toxins have em@yed not exhibit speei%cbinding to iiver memunfiitianated venom or mote commoniy branes. an extract of venom glands from the spiders. rtre effects of the crude gkmd exBacrerbrltoxiins; tract on cholinergic neuromu.uiar juncTetanus toxin is pm&ced by the KioKts of vertebratesNIX now known to he anaerobic bacterium, Closttidiwn due to a singf+toxin, o+Krotoxin. F%rif& te&mP. fn mammais the toxin producesa tr-latrotoxin also induces an efflux of spasticparaiysis which has been attributed wetykhoiine, norepinephrine and gamma to a tetanustoxi&duced blockade of the ~~~$~~ acid from marmn&iw brain &ease of transmittersthat mediate inbibislices bu?*isinactive on invertebratenerv- tory tleitmnaipathwaysin theCNS. Support ous tissue. for thishypophesis c-es fmm theability of A few minutes after appfiiation of retanustoxin to b&k&e releaseof glycine, cr.iatmtoxin tv a venebrate nerve-muscle gamma aminobutyric acid and other puta. preparation there occurs a burst of span. rive amino acid transmitters, fmm nerve taneous quantat r&ease producing minia terminals of mammalian brain and spinal tute endpiate potentials of normal amp cold. litude but markedly increased frequency. The toxin appears to reach the CNS via The m r&easecontinuesat maxi. m-grade axon& tmnsport through mai levels for 5 min or so and then neuronsthat have axonai processesextendgndIJaiiy de&es over the nexK 3tl-50 ing from the CNS. There is evidence that minces. The decfine is accompanied by tetanustoxin can aiso be transportedtrans complete inhibition of evoked tr~smiffer syna~ic~ly. release. The nerve terminals treated with Tetanustoxin can act peripbentllyas well u-fatmloxin become depicted of synaptic and inhibit the evoked release of acetyl. vesicles. choiine from motor nerve aims. Little The mechanismof action of a-iatmtoxin is known about its mechanismof action. ~s~n.~-~~ox~~~~~~ cat. Severai studiiesindiiate that the receptor ion conductanceof ~nifiiial lipid biiayer for tetamtstoxin is seiectiveiy iocaIized to membraues appartmtiy by inserting itself neumns and contains gangliosidesof the into ibe bilayer and forming ion cba~tnets Glb series or a carbohydrate component rather Kttanacting as an ion car&f. it similar to that found in these~~g~~~~. makes the membranespermeable to Na’, Sialicacklisthwghttok:oneoftheciucial
TIPS -April
I982
moieties in the receptor. Tetanus toxin binds IO neumns bum not to notuneumnal cells in cultures of rodent and chicken brain, ganglia and retina. plasma mcmbmnserrrichcd suWis of mam maliin brain synaproaomesbind tetanus toxin IO a much greater extent than do sub. frnctions enrkhed in synaptic vesicks or mitochondria. The binding of J”Clabekd teranustoxin to a crude synaptosomalfraction occumzdwith a dis~~ti~ constantof 1IM andwascompetitively inhibited by the sialic acid-containing gangliosider GTI b (KI = 6 nht) and GDlb (K, = 10 mu)“. Tetanus toxin spacifkally binds ro gang. IiosidesGDlb, GTlband GQlbandako, with kss afftnity, to otikr ganglksides’*. Tkt gangliosidebinding site appearsto be on the larger subunitof the toxin. Nothing kk~wn~t~~~~~of~ mokcuk that serves as the receptor for tetanustoxin. The neumnal receptor for tetanus toxin intriguingly appears to be similar to the thymid receptor for ihe peptide thymtropinJ5.onder the binding conditions used, tetanus toxin reversibly binds to thyroid membranes. and this binding is blocked or reversedby thymtropin but not by any of severa!other pep&e hormones. Conversely, thytotropin exhibits a reves ible bindingto rat brain membranesand this binding is blocked by tetanustoxin. However. the binding of tetanus toxin to the brain membranesis enhancedby thymtropin. It has heen suggestedthat some of ihe symptoms of tetanus toxicity may be causs by tetanus toxin-induced thyroid hyperfunction. Another similarity between tetanus toxin and thymtmpin is that each can affect the membrane potential of a target cell; tetanustoxin has recently been foundto alter the ion permeability of synap tosomesfrom guinea pig brain. ruling toxin is ptuduced by Cfasrridiwn ~~ufi~um’,~. There am at least eight immunologically distinct types of toxin, which ate similar in structure and pharmacological properties. Botulinurn toxin inhibits the evoked rekase of mans muter from cholineqk nerve terminals.
iw Very little : rufinum toxin is required to produce neunmuscular blockade; it has heen estimated that no mote than IO molecules of toxin are tequired to block a singk cbolinergic synapse. Nerve-muscle ~~~~s that alp @ated with ~ulinum toxin have a dtxwa#l spontaneous rekau of acetylcholine tdectua& frequency and amplitude of miniature end-pl_atepotentials) while the pnst-synapticresponseto acetylcholine is unaltered. Eventually the toxin causescompkte inhihitinnof evoked release of acetyicholine. Although these effects have heen characterized in gmat detail’-“. httk has heen learned about the molecular larger nf the toxin. ‘“ElaMcd bntulinum toxin exhibits utumbk binding to synaptosomcs fmm rat brain” and unlabeledtoxin that was boundIO ~ynapb sames was found by ekctron microscopyto be localizedon tbe~~~1~0~1 andextrajumtional areas of presynapticmembranesbut not on post-synapticmembranesJS. Tetanus toxin and botulinum toxin are similar with respect to bacterial genus of origin. structute. and effectson neummus cular transmission.Tetanustoxin of course affects a broader mnp of nerve terminals. It is tempting to speculatethat tetanu toxin and ~ulinum toxin have a rommon molecular mechanism of action (e.g. enzymic activity) but \I ith different target cells that have different n ceptm molecules.
cWhile the pharmacologicaleffect* of the polypeptide toxins mat inhihit u-drummer releasehave been well characterized.little is known about the actionsof the toxins at the molecular kvei. Ahnost all the evidence indicatesthat there are \pccitic binding sites for these toxins on the plasma membmne of nerve terminals. Pmgres\ should soon be made in un~~tand~ne the mokcular structureof thesebinding &es. However, a word of caution seemsappm piate in this regtud. It is p&bk that tluse toxinsdo not inhibit transmitterteleaseby a direct alteration of the tekase apparatus it\ei:. For exrmpk. changesin membrane
~~abiiil~ Ict km\ ha\e hrm tmphcated tn Ihe pnmary acoon 0i rt-iatioroxm. Mamu Inxin. attd the phusphohpasc& toxins l uggc*ting that tlku cftect~ on trausmittcr\lorage and mka+e may be twig secondaryto the ion stability change\. Thus. knowhdge TPrhe molecular me&m. irms of action of these lntinr wdl nut ttecessardykad to an unde’nanding of Ihc biochemistq of the release iqpG&b. Nevedekss. all of the roxim &ould bc urful for faheling and characteriring pro syttaptr componentslb1 arc tmpv-timt for uBmeneuronai’funcuan.