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Induction of ion-permeable channels by the venom of the fanged bloodworm Glycera dibranchiata
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INDUCTION OF ION-PERMEABLE CHANNELS BY THE VENOM OF THE FANGED BLOODWORM GLYCERA DIBRANCHIATA
HRUCB L. KAaAI~ t' HAxvmt B. PoLLAxD~ and ROHBRT B. HA1v1vA3 'Department d Phydobgy and Biophysics, Albert Einstein College of Medicine, New York, 10461 and Marine Biological Laboratory, woods Hole, Massachusetts 02543, 'Clinical Hematology Branch, National Institute dArthritis, Metabolism and Digestive Diseases National Institutes d Health, Betbesda, MD 20205 and Marine Biological Laboratory, Roods Hole, Massachusetts 02543 and 'College d Fa~estry sad Faviranmental Sciemoe State University d New York at Syracuse, Syracuse, New York and Marine Biological Laboratory, Woods Hok, Massachusetts 02543 (Acceptedfor puh&scion 27 March 1982) B. L. K~aex, H.B. Pa .uttuand R B. Hexx~. Induction of ion-permeable channel by thevenom of the fanged bloodworm Glyoera dibrancMata. Taxiton 20, 887-893,1982 Venom from the poism glaads d the polydtaebe annelid Glyoera consa6rta has bees reported to dramatically increase the 6roquemcy d miaiaàue ~m pokmtiab at the Frog and aayfieh nearamusailarjuoctims, without causing detxtable ultrastructural changes. we mport here that additim d venom from the related annelid Glycera rübranchlata to one side d a lipid bilayer results in the formation dion-permeable channels in the membrane. The channel forming activity was found in the void volume of a Sephadex ß-25 comma (mot wt. > 5000 The conductance d a single channel v shoat 330pmho in 0 .1 M NaCI and is ohmia The channel exhibit moderate (but not ideal) cation selectivity in NaCI or KCI gradients. Other selectivity measurements suggest that Cas+ and Mgs+ are also permeable. The flannel show a slight voltage sensitivity. The steady state conductance at - 70 mV (side opposite venom) is about 3 times the conductance at + 70 mV. we suggest that these channel in the venom may evoketransmitterrelease at nenromusailar junctions either by (1) depolaris~g the pre-synaptic terminal and thus opening voltage~ependent Cas+ channeL~, or (2) diroctly albwing Cas+ to eater the terminal Black widow spider venom l known to produce similar effects an neuromuscular junctions and lipid bilayets. The single channel ooaductaaoes and ionic selectivities of the channel found in the vraoms dGlycera and Latrodcctw are strikingly similar. Taken together, these results suggest that d~annel formation tan explain the electrophysiologic e8'ects of these two different venom'. INTRODUCTION
Glycera dibranchiata is a segmented worm found along the coast ofthe Eastern United States and Canada. It possesses four fangs on its proboscis which can be evened with great rapidity when the worm is aggravated. This "sting may result in an itchy inflammation for a cetele~ investigator and can be bthal to a avstaoeaa (HAISreADS 1965 ; KLAWE and Dlcl~ 1957). A venom extract of the related polychaete annelid Glycera coreooluta has previously been shown to dramatically increase the fiequency of miniature end-plate potentials (m.e.p.p.s) at crustacean and frog neuromuscular junctions, without inducing concomitant ultrastructural changes (MANARANCHE et a1.,1980). A similar increase in m.e.p.p. frequenc y is observed when black widow spider venom is applied to neuromuscular preparations " To whom reprint requests should be addressed. Present addren : Department d Psychiatry, UCLA-Neuropsychiatry Lastitate, Los Angeles, CA, U.SA 887
888
BRUCE L. KAGAN, HARVEY B. POLLARD and ROBERT B. HANNA
(Lor>c~nmc~it et al., 1970} Since black widow spider venom is known to contain an ionpermeable channel which may account for its electrophysiologic effect (FiNKELSTEIN et al., 1976), we decided to test Glycera dibranchiata venom for the ability to produce ionpermeable channels in phospholipid bilayer membranes. We report here that addition of Glycera venom to one side of a phospholipid bilayer results in the formation of ion-permeable channels strikingly similar to those produced by black widow spider venom, suggesting that channel formation is responsible for the electrophysiologic effects of both venoms. MATERIALS AND METHODS from Maine were immobilized on ice and provored into evening the proboscis which we rapidlyseverod five. Poison glands were dissectedaway from other tissue.Glands were then homogenized in 10 mM sodium phosphatebuRer (pH 7.2) and centrifuged at 48,000 pfor 30 min.The supernatant was passed over Sephadex G-25 and samplesfrom thevoid vohune (mol. wt. > 5000) were added directly to one aide of aplanar phoapholipid bilayermembrane. Membranes were formed at room temperature from theunion oftwomonolayera of lipideases a hole (0.1-0.2 mm diameter) in a Saranwrap partition separating twoaqueous phases of about S ml (Moxr~r.,1974} T~ hob was prewated with a solution of vaseline in petrobnm ether. In some experiments, 1 Kl of squabne was added to the monolayer to improve membrane stability, but this did not affect the properties of the d~aanela Monolayers wero sproad using 15 p1 of a 1% solution of phoapholipid. All ion sebdivity meaaurementa were performed on membranes formed from diphantaaoylphosphatidylcboline, which has no net surface char8e. Bilayer experiments wero performed under "Voltage lamp" conditions, as desalted elsewhere (Scru?nv et aL, 1976} Two calomel ebctrades, connected to thebath through saturatedKCljunctions, were used for both applying voltages and measuring the resulting current, which was recorded on ea oacillosoope and a chart recorder . The conductance (G) of the membrane is defined by G = I/V, where I is the measured currant and V is the applied voltage. The chamber to which venom was added is virtual ground Thus V is the sigo of the potential of the side opposite the venom (corresponding to the inside of a all} The ability ofvenom to robaae K* from unilamellar phospholipid veaicbs was studied in thefollowing manner. Single walled vesicbsof approximauly 1000 A diameter were made in theprawns of 100mM KG,10mM dimethyl glutaric acid, 0.2 mM EDTA,pH 5.5,by thefreeze-thaw technique(K~serut~+aad Htxttr~,1977} After sanitation of a 3% solution of uapurified aaobctin to clarity, the mixturo was subjectedto 1 or 2 cycles of rapid-fretting in dry ioe/ETOH, followed by thawing at room temperature. Twenty ~ of thin veaicb solution was added to 2 ml of a .1 mM KCI, pH 5.5. A K* solution containing 100mM LiCI, 10 mM dimethyl glutaric acid, 0.2 mM EDTA, 0 selective electrode (Orion Research) was connected to a pH meter and a chart recorder . Rebase of K* from inside the vesicba was manitorod as a change in [K*] of the solution, which the K* ebctrode signalled as a change in millivolta. Controlexperiments with gramicidin A indicated that the vesicbs hadan internal vohmre of about 10'/ of the volume of the original lipid solution. Glycera dibranchiata
RESULTS
Addition of a small amount of Glycera venom to one side of a lipid bilayer (to a final concentration of0.001-0.OS glands/ml) resultsin adramatic increase in the conductance of a bilayer (Fig. 1). Conductances of 10,000-SO,000pmho are commonly observed. In the absence of Glyceravenom, the conductance of a lipid bilayer is about 10 pmbo and is ohmic. At shorttimes aftervenom addition, discrete fluctuations ofthe current can be observed (Fig. 2) corresponding to an apparent single channel conductance of about 350 pmho in 100 mM NaCL Occasionally, conductance jumps of other sizes have been observed and, although these jumps are probably due to multiple conductance states of the channel, the possibility that more than one channel type is presentcannot yet be rigorously excluded. The magnitude of these conductance steps is much too large to be produced by a shuttling ion carrier (LAUGER, 1972 ; HLADKY and HAYDON, 1972) and must result from the formation of large aqueous channels in the membrane. The instantaneous conductance ofa single channel in the open state is ohmic (Fig. 3aß However, the steady-state I-V relation of a membrane containing many channels is not ohmic (Fig. 3bß The conductance at - 70 mV (a value close to the resting potential ofmost excitab~ cells) is about 6 timesgreater than the conductance at + 30 mV (a value corresponding to a highly depolarized all} Taken together, these two results imply that the probability ofa channel being open is a function ofthe transmembrane
Channel Formation by Glyoera Venom
889
500 pAI
60er V(mV)
-70 0~ +70
FIG. 1 . CURRENT RESPONSE OF GIyCera VENOM-TREAT® MEMBRANE TO VOLTAGE When a step of - 70mV is applied to the membrane, the current (and therdore conductance) rises rapidly to a steady-state level of about 1800pA (26,000pmho~ When the voltage is reversed to + 70 mV the current instantaneously has the same magnitude but opposite sign) andthen decays to a steady-state value of about 300pA (4300 pmho)or about 1/6 thevalueof thecurrent at - 70 mV . The membrane was formed from DPPC (diphantanoylphoaphatidylcholine} The solutions were symmetric 2(10 mM NaCI, 5 n>IVi CaCl s, 5 mM Hepea (pH 7.Oj, 0.2 mM F.DTA. Venom was added 15 min prior to the recording to a final concentration of 0.05 glands/mL
electrical field. Experiments with membranes containing a single channel confirm the expectation that a channel is more likely to be open at - 70 than at + 30 mV. Venom-treated membranes show moderate, but not ideal, cation selectivity in gradients of KCl or NaCL For NaCI a diffusion potential ofabout 33 mV/10-fold gradient and for KCl a diffusion potential of about 42 mV/10-fold gradient is observed (ideal selectivity = 58 mV/10-fold gradient). Thus, the channel admits Cl - in addition to Na+ and K +. IfCaC12 or MgCl 2 is added to one side of a venom-treated membrane separating symmetricNaCI or KCl solutions, the result is complicated Immediately after addition ofthe divalent cation, the reversal potential [E, determined from I = G(V-E)] of the membrane takes on a value indicative of anion selectivity (divalent side positive). However, this potential decays and changes sign over the course of several min. A steady-state value i8 reached which indicates
v
IOpA I 500 porno
12 s~e FIG. 2. SINGIE CHANNEL CURRENT FLUCTUATIONS INDUCED BY Glycera vENOM. The membrane voäa8e was hdd cmetaat at -20mV. The membrane was termed from diphsataaoylphosphatidylcholiae and separated symmetrical solutions containing 100mM NaCI, 5 mM CaC13, 5 mM Hepea, 0 .2 mM i3DTA all adjusted to pH 7.0 Glyaera venom was added to ace side to a concentration of about 0.005 glandsy/mL The rgaosd was obtained 5 min after Glyoera sddiäeu, when many channels were catering the film. Note that channel closures (arrow) as well as opeainga occur, eves at this voltage where channels are usually open . Note also that the channel ftuduatiooa are all approximately (but not precisely) the same amplitude.
BRUCE L. KAGAN, HARVEY B . 1?OLLARD and ROBERT B. HANNA
890
I(PA)
V(mV)
38. INSTANTANEOUS CURRENT-VOLTAGE P1.0'r OF A SINGLE CHANNEL PRODUCED HY Glycaa VENOM. The open c~lannel is essentially ohmic in this range. The asolectin (soybean phospholipids ; KAGAWA and RACICER,1971) membrane separated symmetrical solutions of 100 mM KCI, S mM Hepes, S mM CaCl z, 0 .2mM EDTA, all adjusted to pH 7.0 . The slope of the line gives a conductance of about 350 pmho. FIG.
I(PA)
V(mV) -IÔO
-g0
-20
t20
+60
t100
-40 -80 -120 -160 -200 FLG. 3b. STEADY-STATE CURRENT-VOLTAGE PLOT FOR A MEMHRANB CONTAINING MANY CHANNELS The membrane lipid was diphentanoylphaphatidylchaJine . The aqueous phases were both 200mM NaCI, 5 mM CaCl 3 , S mM Hepes (pH 7.0) 0.2 mM EDTA. While the instantaneous I-V plot for this membrane was ohmic (Fig. 3aß the steady-stau plot shows significant recti5cation . Note that conductance dxreases with increasing "depolariTStion" of the membrane tslative to the typical excitable sell resting potential. This is due to a reduced probability of the channel being in the open state (data not shown) .
Channel Formation by Glyoaa Venom
89 1
cation selectivity (divalent side negative} Although further investigation of this effect is warranted, the result suggests that (a) divalent canons interactwith thechannel, perhapswith negative fixed charges, and (b) divalent canons are permeable through the channel Addition ofsmall amounts (final concentrations 0.005-0.010 glands/ml) of Glycera venom to a solution ofK+-loaded asolectin vesicles causes release ofmore than 70'/ of the internal K+ within 1 min. The remaining K+ can be released by gramicidin A and is probably contained in small (250 A diameter)vesicles ofvery high curvature. This failure ofa channel to release K + from a population of smallervesicles hasvoen observed with aaother largeprotein channel, the B,s fragment of diphtheria toxin (B. L. Kagan and A. Finkelstein, unpublished observations This K+ release assay is simple and rapid enough to use to follow the purification of the channel forming molecule from Glycera venom. DISC[JSSION
Thevenom of Glycera dibranchiata contains an ion-permeable channel quite similar to the black widow spider venom channel, which causes an increased frequency of m.e.p.p.s. at the frog neuromuscularjunction. Since thevenom ofthe closely related spocies Glycera convolute also causes an increase in the frequency of m.e.p.p.s. at the frog neuromuscularjunction, it is not unreasonable to suspect that a similar diannel in Glyoera convolute v~om may be responsible for the observed ei;octs at the neuromusailar junction. ReoeatlY, the validity of the comparison between Glyoera dibranchiata aad Glyoera convoluta has bcen called into question by Israel, IVIanaranche and Thieffry (pa~sonal oommunication~ who report that the venom of Glycera dibranchiata does not cause an increase in the frequency of m.e.p.p.s. at the frog or crayfish neuromuscular junctions, although it does appear to depolarize the axonal membrane. There are several possible reasons for this discrepaacy. (1) The crayfish axonal membrane may be the biological target of Glycera dibranchiata venom, while thefunctional membrane may be the target for the Glycera convolute venom. (2) Glycera convolute and Glycera dibranchiata toxins may have different~a.~nities for phospholipids and thus different abilities to insert into bilayer membranes. Since we used lipids which are quite different from those found in the frog and crayfish neuromuscularjunction, it is quite conceivable that the toxin may form channels in our bilayers while not being able to form channels in frog or crayfish membranes. Since these preparations are not necessarily the natural biologic targets of the Glycera venom, the channel may be effective on the membranes of the true biologic targets of the bloodworm. There is in fact a precedent for this type of behavior. We have previously found (Sct~Net a1.,1978) that bilayer membranesformed from asolectin are more sensitive to a channel forming toxin than bilayer membranes formed from the lipids of the target cell. (3) Thevoltage-dependent behavior ofthe channel formed in Glycera dibranchiata venom may be different in neuromuscular junction membranes and asolectin membranes. Thus the channel might be closed more often in the neuromuscularjunction membrane and the "lealvness" caused by the channel could be limited (see abave~ and thus more difficult to detect . In a true biologic target membrane, the channel might exhibit voltage~ependent behavior more in keeping with its biologic action. (4) Thechannelforming activity on bilayer membranes might be unrelated to the action of Giycera convolute venom on neuromuscular junctions. Although this is possible, we feel that the effects of Glycera dibranchiata venom and black widow spider venom on bilayer membranes are so similar qualitatively and quantitatively that these two venoms probably work in a similar fashion. (5) Finally we should note the case ofblack widow spider venom, a channel forming toxin which is effective on the frog and crayfish neuromuscular junctions, but is ineffective on the crayfish stretch
892
BRUCE L. KAGAN, HARVEY B. POLLARD and ROBERT B. HANNA
receptor. As FIx1cEISTaIx et al., (1976) noted, the effectiveness of the channel in depolarizing the cell membrane depends on the amount of electrical shunt the channel introduces relative to the resistance of the entire membrane. Both the size of the single channel conductance and the number of channels actually inserted in the membrane are important in this regard. The results described here for Glycera venom are remarkably reminiscent of the results described by FiNKELSTEIx et al. (1976) for the BS fraction of black widow spider venom (alatrotoxin). Both venoms contain ion-permeable channels which are water-soluble and yet are capable of inserting spontaneously into lipid bilayers. Even more remarkable is the similarity of the sizes of the single channel conductance, a value which is a unique characteristic of a given channel. The similarity between the conductances of these two channels may reflect similarities in function and in structure. Both channels exhibit cation selectivity, although file black widow spider venom shows ideal selectivity while the Glycera channel is somewhat less selective. Both channels also exhibit behaviors suggestive of the presence of negative fixed charges. As FINKELSTEIN et al. (1976) noted, there are at least two possible mechanisms by which the channels could cause the observed increase in m.e.p.p . frequency. (1) The insertion of channels in the presynaptic membrane could lead to depolarization of the membrane, opening endogenous votage~ependent Caz+ channels, allowing Caz+ entry, and thus stimulating transmitter release. (2) The channels could directly allow Caz+ to pass into the presynaptic terminal . Mechanism (1) seems unlikely in view of the results of MISLER and HURLHUT (1979 showing that the action of black widow spider venom depends on the presence of divalent rations (Caz+, Mgz+ , Co z+~ ~z+ or Z~z + ) in the bathing solution. Since all these ions except Caz + are relatively impermeable to the endogenous voltage-dependent Ca z+ channels of the presynaptic terminal, it seems likely that the increased levels of intracellular divalents are due to direct passage of divalent rations through the black widow spider venom channel The same reasoning applies to Glycera venom and explains the Caz+ requirement for venom action observed by MANARANCHE et al. (1980 since their Ringer's solution contained no Mgz+ . The voltage dependence of the Glycera dibranchiata channel may also be relevant to the mode of action of Glycera convoluta venom at neuromuscular junctions. Since the channel tends to turn off as the cell becomesdepolarized, the amount of "leak" induced by the Glycera channel may be self-regulating. This may account for the observed "bursts" in m.e.p .p . frequency ob®emd by MAxARANCH>Eet al. The alternation of "bursts" and quiet periods could be due to a channel opening and closing in the membrane. In lipid bilayers, channels from Glyceravenom are commonly observed to open and close in this fashion. Black widow spider venom channels close only rarely (FINKELSTEIN et al., (1976), and the "bursting" phenomenon is not seen on neuromuscularjunctions treated with black widow spider venom (LONGLINECr x>:R et al., 1970 Acknowledprnrmts-We gratefully acknowledge the wit and wisdom of ALAN Fltv~ceis~t~rN and SrANr..eY M~st.ea. We thank M. Isw~t., R McNtiewxct~ and M . TrmrrrRY for sharing their unpublished data with us. (Supported by NIH grant ST32 GM 7288 to B.L.K . and SUNY Research Foundation 210-7142 to R.B.H .) REFERENCES Fttv~cetsrwrq A., RUH~N, L. L., and Tzexc, M.-C. (1976) Hlack widow spidsr venom: Eliect of puriûed toxin on lipid bilayer membranes. Scknce, Wash. 193, 1009. H~tsrnwq B. W . (1965) Poiaonow and Yenonaus Morine Animals of flee World, VoL 1 . Washington, D.C. : U .S. Government Printing O®oe .
Channel Formation by Glyoera Venom
89 3
Ht.wutcv, S. B. aad Hwvnotv, D. A. (1972) Ion transfer sacs lipid membranes in the presence of granicidin A. I. Studies of the unit conductance chsnneL Biochim. biophys. Acts 274, 294. Kwcwww, Y. and Rwcs:Ert, E (1971) Partialresolution of theenzymes catalyting oxidative phasphorylation : XXV Reconstruction of vesicles cataly2ing 3= P adenosine triphoephate exchange. J. biol. Chem. 246, 5477. Kwswxwxw, M. and Htxt~t~, P. C. (1977) Reconstitution and purification of the n-glucose transporter from human erythrocytes. J. btoL Chem. 252, 7384. Kt,wws,W. L. and DtcrcrE, L. H. (1957) Biology of the bloodworm Glycera dtbranchiata Ehkrsanditsrelation to the bloodworm üshery of the maritime provinces. Bu1L Fish. Res. Bd Can. 115, 1 . Lwuc~x, P. (1972) Carrier-mediated ion transport. Science, N.Y. 178, 24. Loxc.~~xea, H. E, Hutu.avr, W. P., Mwuxor A. andCt.wax, A. W. (1970) Effects of black widow spider venom on the frog neuromuswlar junction. Natroe, load 225ti 701. MANARANCHF, R, Ttm~tev, M. and Isw+Er, M. (1980) E&ct of the venomof Glycaa conoohua as the spontaneous quantal release of transmitters . J. Cell Biol. 85, 446. Mut es, S.and Huxi .aur, W. P. (1979) Action of black widow spider venomon quantized release of acetykholine at the frog neuromuscular junction : Dependence upon external Mg=+ . Proc . natn. Acad. Sci., U.S.A . 76, 991 . Moxrwt, M. (1974) Formation of bimolecular membranes from lipid monolayers Meth. Enzym. 328, 545. Sc~N, S J. Cow~xt, M. and Frxtcersrettr, A. (1976) Reconstitution in planar lipid bilayers of a vohagedependent anionaelective channel obtained from Paranrciam mitochondria. J . Membrane BtoL 30, 99. Sc~tx, S. J., Kwcwx, B. L. and Ftxrc~srax, A. (1978) Colicin K acts by forming voltage~peadent ~annels in phospholipid bilayer membranes Natm, f ond . 276, 159.
ao~t-0tot~a2/osoes~r~ sos ogro © t992 ~~ t~.. t.ta.
INDUCTION OF ION-PERMEABLE CHANNELS BY THE VENOM OF THE FANGED BLOODWORM GLYCERA DIBRANCHIATA
HRUCB L. KAaAI~ t' HAxvmt B. PoLLAxD~ and ROHBRT B. HA1v1vA3 'Department d Phydobgy and Biophysics, Albert Einstein College of Medicine, New York, 10461 and Marine Biological Laboratory, woods Hole, Massachusetts 02543, 'Clinical Hematology Branch, National Institute dArthritis, Metabolism and Digestive Diseases National Institutes d Health, Betbesda, MD 20205 and Marine Biological Laboratory, Roods Hole, Massachusetts 02543 and 'College d Fa~estry sad Faviranmental Sciemoe State University d New York at Syracuse, Syracuse, New York and Marine Biological Laboratory, Woods Hok, Massachusetts 02543 (Acceptedfor puh&scion 27 March 1982) B. L. K~aex, H.B. Pa .uttuand R B. Hexx~. Induction of ion-permeable channel by thevenom of the fanged bloodworm Glyoera dibrancMata. Taxiton 20, 887-893,1982 Venom from the poism glaads d the polydtaebe annelid Glyoera consa6rta has bees reported to dramatically increase the 6roquemcy d miaiaàue ~m pokmtiab at the Frog and aayfieh nearamusailarjuoctims, without causing detxtable ultrastructural changes. we mport here that additim d venom from the related annelid Glycera rübranchlata to one side d a lipid bilayer results in the formation dion-permeable channels in the membrane. The channel forming activity was found in the void volume of a Sephadex ß-25 comma (mot wt. > 5000 The conductance d a single channel v shoat 330pmho in 0 .1 M NaCI and is ohmia The channel exhibit moderate (but not ideal) cation selectivity in NaCI or KCI gradients. Other selectivity measurements suggest that Cas+ and Mgs+ are also permeable. The flannel show a slight voltage sensitivity. The steady state conductance at - 70 mV (side opposite venom) is about 3 times the conductance at + 70 mV. we suggest that these channel in the venom may evoketransmitterrelease at nenromusailar junctions either by (1) depolaris~g the pre-synaptic terminal and thus opening voltage~ependent Cas+ channeL~, or (2) diroctly albwing Cas+ to eater the terminal Black widow spider venom l known to produce similar effects an neuromuscular junctions and lipid bilayets. The single channel ooaductaaoes and ionic selectivities of the channel found in the vraoms dGlycera and Latrodcctw are strikingly similar. Taken together, these results suggest that d~annel formation tan explain the electrophysiologic e8'ects of these two different venom'. INTRODUCTION
Glycera dibranchiata is a segmented worm found along the coast ofthe Eastern United States and Canada. It possesses four fangs on its proboscis which can be evened with great rapidity when the worm is aggravated. This "sting may result in an itchy inflammation for a cetele~ investigator and can be bthal to a avstaoeaa (HAISreADS 1965 ; KLAWE and Dlcl~ 1957). A venom extract of the related polychaete annelid Glycera coreooluta has previously been shown to dramatically increase the fiequency of miniature end-plate potentials (m.e.p.p.s) at crustacean and frog neuromuscular junctions, without inducing concomitant ultrastructural changes (MANARANCHE et a1.,1980). A similar increase in m.e.p.p. frequenc y is observed when black widow spider venom is applied to neuromuscular preparations " To whom reprint requests should be addressed. Present addren : Department d Psychiatry, UCLA-Neuropsychiatry Lastitate, Los Angeles, CA, U.SA 887
888
BRUCE L. KAGAN, HARVEY B. POLLARD and ROBERT B. HANNA
(Lor>c~nmc~it et al., 1970} Since black widow spider venom is known to contain an ionpermeable channel which may account for its electrophysiologic effect (FiNKELSTEIN et al., 1976), we decided to test Glycera dibranchiata venom for the ability to produce ionpermeable channels in phospholipid bilayer membranes. We report here that addition of Glycera venom to one side of a phospholipid bilayer results in the formation of ion-permeable channels strikingly similar to those produced by black widow spider venom, suggesting that channel formation is responsible for the electrophysiologic effects of both venoms. MATERIALS AND METHODS from Maine were immobilized on ice and provored into evening the proboscis which we rapidlyseverod five. Poison glands were dissectedaway from other tissue.Glands were then homogenized in 10 mM sodium phosphatebuRer (pH 7.2) and centrifuged at 48,000 pfor 30 min.The supernatant was passed over Sephadex G-25 and samplesfrom thevoid vohune (mol. wt. > 5000) were added directly to one aide of aplanar phoapholipid bilayermembrane. Membranes were formed at room temperature from theunion oftwomonolayera of lipideases a hole (0.1-0.2 mm diameter) in a Saranwrap partition separating twoaqueous phases of about S ml (Moxr~r.,1974} T~ hob was prewated with a solution of vaseline in petrobnm ether. In some experiments, 1 Kl of squabne was added to the monolayer to improve membrane stability, but this did not affect the properties of the d~aanela Monolayers wero sproad using 15 p1 of a 1% solution of phoapholipid. All ion sebdivity meaaurementa were performed on membranes formed from diphantaaoylphosphatidylcboline, which has no net surface char8e. Bilayer experiments wero performed under "Voltage lamp" conditions, as desalted elsewhere (Scru?nv et aL, 1976} Two calomel ebctrades, connected to thebath through saturatedKCljunctions, were used for both applying voltages and measuring the resulting current, which was recorded on ea oacillosoope and a chart recorder . The conductance (G) of the membrane is defined by G = I/V, where I is the measured currant and V is the applied voltage. The chamber to which venom was added is virtual ground Thus V is the sigo of the potential of the side opposite the venom (corresponding to the inside of a all} The ability ofvenom to robaae K* from unilamellar phospholipid veaicbs was studied in thefollowing manner. Single walled vesicbsof approximauly 1000 A diameter were made in theprawns of 100mM KG,10mM dimethyl glutaric acid, 0.2 mM EDTA,pH 5.5,by thefreeze-thaw technique(K~serut~+aad Htxttr~,1977} After sanitation of a 3% solution of uapurified aaobctin to clarity, the mixturo was subjectedto 1 or 2 cycles of rapid-fretting in dry ioe/ETOH, followed by thawing at room temperature. Twenty ~ of thin veaicb solution was added to 2 ml of a .1 mM KCI, pH 5.5. A K* solution containing 100mM LiCI, 10 mM dimethyl glutaric acid, 0.2 mM EDTA, 0 selective electrode (Orion Research) was connected to a pH meter and a chart recorder . Rebase of K* from inside the vesicba was manitorod as a change in [K*] of the solution, which the K* ebctrode signalled as a change in millivolta. Controlexperiments with gramicidin A indicated that the vesicbs hadan internal vohmre of about 10'/ of the volume of the original lipid solution. Glycera dibranchiata
RESULTS
Addition of a small amount of Glycera venom to one side of a lipid bilayer (to a final concentration of0.001-0.OS glands/ml) resultsin adramatic increase in the conductance of a bilayer (Fig. 1). Conductances of 10,000-SO,000pmho are commonly observed. In the absence of Glyceravenom, the conductance of a lipid bilayer is about 10 pmbo and is ohmic. At shorttimes aftervenom addition, discrete fluctuations ofthe current can be observed (Fig. 2) corresponding to an apparent single channel conductance of about 350 pmho in 100 mM NaCL Occasionally, conductance jumps of other sizes have been observed and, although these jumps are probably due to multiple conductance states of the channel, the possibility that more than one channel type is presentcannot yet be rigorously excluded. The magnitude of these conductance steps is much too large to be produced by a shuttling ion carrier (LAUGER, 1972 ; HLADKY and HAYDON, 1972) and must result from the formation of large aqueous channels in the membrane. The instantaneous conductance ofa single channel in the open state is ohmic (Fig. 3aß However, the steady-state I-V relation of a membrane containing many channels is not ohmic (Fig. 3bß The conductance at - 70 mV (a value close to the resting potential ofmost excitab~ cells) is about 6 timesgreater than the conductance at + 30 mV (a value corresponding to a highly depolarized all} Taken together, these two results imply that the probability ofa channel being open is a function ofthe transmembrane
Channel Formation by Glyoera Venom
889
500 pAI
60er V(mV)
-70 0~ +70
FIG. 1 . CURRENT RESPONSE OF GIyCera VENOM-TREAT® MEMBRANE TO VOLTAGE When a step of - 70mV is applied to the membrane, the current (and therdore conductance) rises rapidly to a steady-state level of about 1800pA (26,000pmho~ When the voltage is reversed to + 70 mV the current instantaneously has the same magnitude but opposite sign) andthen decays to a steady-state value of about 300pA (4300 pmho)or about 1/6 thevalueof thecurrent at - 70 mV . The membrane was formed from DPPC (diphantanoylphoaphatidylcholine} The solutions were symmetric 2(10 mM NaCI, 5 n>IVi CaCl s, 5 mM Hepea (pH 7.Oj, 0.2 mM F.DTA. Venom was added 15 min prior to the recording to a final concentration of 0.05 glands/mL
electrical field. Experiments with membranes containing a single channel confirm the expectation that a channel is more likely to be open at - 70 than at + 30 mV. Venom-treated membranes show moderate, but not ideal, cation selectivity in gradients of KCl or NaCL For NaCI a diffusion potential ofabout 33 mV/10-fold gradient and for KCl a diffusion potential of about 42 mV/10-fold gradient is observed (ideal selectivity = 58 mV/10-fold gradient). Thus, the channel admits Cl - in addition to Na+ and K +. IfCaC12 or MgCl 2 is added to one side of a venom-treated membrane separating symmetricNaCI or KCl solutions, the result is complicated Immediately after addition ofthe divalent cation, the reversal potential [E, determined from I = G(V-E)] of the membrane takes on a value indicative of anion selectivity (divalent side positive). However, this potential decays and changes sign over the course of several min. A steady-state value i8 reached which indicates
v
IOpA I 500 porno
12 s~e FIG. 2. SINGIE CHANNEL CURRENT FLUCTUATIONS INDUCED BY Glycera vENOM. The membrane voäa8e was hdd cmetaat at -20mV. The membrane was termed from diphsataaoylphosphatidylcholiae and separated symmetrical solutions containing 100mM NaCI, 5 mM CaC13, 5 mM Hepea, 0 .2 mM i3DTA all adjusted to pH 7.0 Glyaera venom was added to ace side to a concentration of about 0.005 glandsy/mL The rgaosd was obtained 5 min after Glyoera sddiäeu, when many channels were catering the film. Note that channel closures (arrow) as well as opeainga occur, eves at this voltage where channels are usually open . Note also that the channel ftuduatiooa are all approximately (but not precisely) the same amplitude.
BRUCE L. KAGAN, HARVEY B . 1?OLLARD and ROBERT B. HANNA
890
I(PA)
V(mV)
38. INSTANTANEOUS CURRENT-VOLTAGE P1.0'r OF A SINGLE CHANNEL PRODUCED HY Glycaa VENOM. The open c~lannel is essentially ohmic in this range. The asolectin (soybean phospholipids ; KAGAWA and RACICER,1971) membrane separated symmetrical solutions of 100 mM KCI, S mM Hepes, S mM CaCl z, 0 .2mM EDTA, all adjusted to pH 7.0 . The slope of the line gives a conductance of about 350 pmho. FIG.
I(PA)
V(mV) -IÔO
-g0
-20
t20
+60
t100
-40 -80 -120 -160 -200 FLG. 3b. STEADY-STATE CURRENT-VOLTAGE PLOT FOR A MEMHRANB CONTAINING MANY CHANNELS The membrane lipid was diphentanoylphaphatidylchaJine . The aqueous phases were both 200mM NaCI, 5 mM CaCl 3 , S mM Hepes (pH 7.0) 0.2 mM EDTA. While the instantaneous I-V plot for this membrane was ohmic (Fig. 3aß the steady-stau plot shows significant recti5cation . Note that conductance dxreases with increasing "depolariTStion" of the membrane tslative to the typical excitable sell resting potential. This is due to a reduced probability of the channel being in the open state (data not shown) .
Channel Formation by Glyoaa Venom
89 1
cation selectivity (divalent side negative} Although further investigation of this effect is warranted, the result suggests that (a) divalent canons interactwith thechannel, perhapswith negative fixed charges, and (b) divalent canons are permeable through the channel Addition ofsmall amounts (final concentrations 0.005-0.010 glands/ml) of Glycera venom to a solution ofK+-loaded asolectin vesicles causes release ofmore than 70'/ of the internal K+ within 1 min. The remaining K+ can be released by gramicidin A and is probably contained in small (250 A diameter)vesicles ofvery high curvature. This failure ofa channel to release K + from a population of smallervesicles hasvoen observed with aaother largeprotein channel, the B,s fragment of diphtheria toxin (B. L. Kagan and A. Finkelstein, unpublished observations This K+ release assay is simple and rapid enough to use to follow the purification of the channel forming molecule from Glycera venom. DISC[JSSION
Thevenom of Glycera dibranchiata contains an ion-permeable channel quite similar to the black widow spider venom channel, which causes an increased frequency of m.e.p.p.s. at the frog neuromuscularjunction. Since thevenom ofthe closely related spocies Glycera convolute also causes an increase in the frequency of m.e.p.p.s. at the frog neuromuscularjunction, it is not unreasonable to suspect that a similar diannel in Glyoera convolute v~om may be responsible for the observed ei;octs at the neuromusailar junction. ReoeatlY, the validity of the comparison between Glyoera dibranchiata aad Glyoera convoluta has bcen called into question by Israel, IVIanaranche and Thieffry (pa~sonal oommunication~ who report that the venom of Glycera dibranchiata does not cause an increase in the frequency of m.e.p.p.s. at the frog or crayfish neuromuscular junctions, although it does appear to depolarize the axonal membrane. There are several possible reasons for this discrepaacy. (1) The crayfish axonal membrane may be the biological target of Glycera dibranchiata venom, while thefunctional membrane may be the target for the Glycera convolute venom. (2) Glycera convolute and Glycera dibranchiata toxins may have different~a.~nities for phospholipids and thus different abilities to insert into bilayer membranes. Since we used lipids which are quite different from those found in the frog and crayfish neuromuscularjunction, it is quite conceivable that the toxin may form channels in our bilayers while not being able to form channels in frog or crayfish membranes. Since these preparations are not necessarily the natural biologic targets of the Glycera venom, the channel may be effective on the membranes of the true biologic targets of the bloodworm. There is in fact a precedent for this type of behavior. We have previously found (Sct~Net a1.,1978) that bilayer membranesformed from asolectin are more sensitive to a channel forming toxin than bilayer membranes formed from the lipids of the target cell. (3) Thevoltage-dependent behavior ofthe channel formed in Glycera dibranchiata venom may be different in neuromuscular junction membranes and asolectin membranes. Thus the channel might be closed more often in the neuromuscularjunction membrane and the "lealvness" caused by the channel could be limited (see abave~ and thus more difficult to detect . In a true biologic target membrane, the channel might exhibit voltage~ependent behavior more in keeping with its biologic action. (4) Thechannelforming activity on bilayer membranes might be unrelated to the action of Giycera convolute venom on neuromuscular junctions. Although this is possible, we feel that the effects of Glycera dibranchiata venom and black widow spider venom on bilayer membranes are so similar qualitatively and quantitatively that these two venoms probably work in a similar fashion. (5) Finally we should note the case ofblack widow spider venom, a channel forming toxin which is effective on the frog and crayfish neuromuscular junctions, but is ineffective on the crayfish stretch
892
BRUCE L. KAGAN, HARVEY B. POLLARD and ROBERT B. HANNA
receptor. As FIx1cEISTaIx et al., (1976) noted, the effectiveness of the channel in depolarizing the cell membrane depends on the amount of electrical shunt the channel introduces relative to the resistance of the entire membrane. Both the size of the single channel conductance and the number of channels actually inserted in the membrane are important in this regard. The results described here for Glycera venom are remarkably reminiscent of the results described by FiNKELSTEIx et al. (1976) for the BS fraction of black widow spider venom (alatrotoxin). Both venoms contain ion-permeable channels which are water-soluble and yet are capable of inserting spontaneously into lipid bilayers. Even more remarkable is the similarity of the sizes of the single channel conductance, a value which is a unique characteristic of a given channel. The similarity between the conductances of these two channels may reflect similarities in function and in structure. Both channels exhibit cation selectivity, although file black widow spider venom shows ideal selectivity while the Glycera channel is somewhat less selective. Both channels also exhibit behaviors suggestive of the presence of negative fixed charges. As FINKELSTEIN et al. (1976) noted, there are at least two possible mechanisms by which the channels could cause the observed increase in m.e.p.p . frequency. (1) The insertion of channels in the presynaptic membrane could lead to depolarization of the membrane, opening endogenous votage~ependent Caz+ channels, allowing Caz+ entry, and thus stimulating transmitter release. (2) The channels could directly allow Caz+ to pass into the presynaptic terminal . Mechanism (1) seems unlikely in view of the results of MISLER and HURLHUT (1979 showing that the action of black widow spider venom depends on the presence of divalent rations (Caz+, Mgz+ , Co z+~ ~z+ or Z~z + ) in the bathing solution. Since all these ions except Caz + are relatively impermeable to the endogenous voltage-dependent Ca z+ channels of the presynaptic terminal, it seems likely that the increased levels of intracellular divalents are due to direct passage of divalent rations through the black widow spider venom channel The same reasoning applies to Glycera venom and explains the Caz+ requirement for venom action observed by MANARANCHE et al. (1980 since their Ringer's solution contained no Mgz+ . The voltage dependence of the Glycera dibranchiata channel may also be relevant to the mode of action of Glycera convoluta venom at neuromuscular junctions. Since the channel tends to turn off as the cell becomesdepolarized, the amount of "leak" induced by the Glycera channel may be self-regulating. This may account for the observed "bursts" in m.e.p .p . frequency ob®emd by MAxARANCH>Eet al. The alternation of "bursts" and quiet periods could be due to a channel opening and closing in the membrane. In lipid bilayers, channels from Glyceravenom are commonly observed to open and close in this fashion. Black widow spider venom channels close only rarely (FINKELSTEIN et al., (1976), and the "bursting" phenomenon is not seen on neuromuscularjunctions treated with black widow spider venom (LONGLINECr x>:R et al., 1970 Acknowledprnrmts-We gratefully acknowledge the wit and wisdom of ALAN Fltv~ceis~t~rN and SrANr..eY M~st.ea. We thank M. Isw~t., R McNtiewxct~ and M . TrmrrrRY for sharing their unpublished data with us. (Supported by NIH grant ST32 GM 7288 to B.L.K . and SUNY Research Foundation 210-7142 to R.B.H .) REFERENCES Fttv~cetsrwrq A., RUH~N, L. L., and Tzexc, M.-C. (1976) Hlack widow spidsr venom: Eliect of puriûed toxin on lipid bilayer membranes. Scknce, Wash. 193, 1009. H~tsrnwq B. W . (1965) Poiaonow and Yenonaus Morine Animals of flee World, VoL 1 . Washington, D.C. : U .S. Government Printing O®oe .
Channel Formation by Glyoera Venom
89 3
Ht.wutcv, S. B. aad Hwvnotv, D. A. (1972) Ion transfer sacs lipid membranes in the presence of granicidin A. I. Studies of the unit conductance chsnneL Biochim. biophys. Acts 274, 294. Kwcwww, Y. and Rwcs:Ert, E (1971) Partialresolution of theenzymes catalyting oxidative phasphorylation : XXV Reconstruction of vesicles cataly2ing 3= P adenosine triphoephate exchange. J. biol. Chem. 246, 5477. Kwswxwxw, M. and Htxt~t~, P. C. (1977) Reconstitution and purification of the n-glucose transporter from human erythrocytes. J. btoL Chem. 252, 7384. Kt,wws,W. L. and DtcrcrE, L. H. (1957) Biology of the bloodworm Glycera dtbranchiata Ehkrsanditsrelation to the bloodworm üshery of the maritime provinces. Bu1L Fish. Res. Bd Can. 115, 1 . Lwuc~x, P. (1972) Carrier-mediated ion transport. Science, N.Y. 178, 24. Loxc.~~xea, H. E, Hutu.avr, W. P., Mwuxor A. andCt.wax, A. W. (1970) Effects of black widow spider venom on the frog neuromuswlar junction. Natroe, load 225ti 701. MANARANCHF, R, Ttm~tev, M. and Isw+Er, M. (1980) E&ct of the venomof Glycaa conoohua as the spontaneous quantal release of transmitters . J. Cell Biol. 85, 446. Mut es, S.and Huxi .aur, W. P. (1979) Action of black widow spider venomon quantized release of acetykholine at the frog neuromuscular junction : Dependence upon external Mg=+ . Proc . natn. Acad. Sci., U.S.A . 76, 991 . Moxrwt, M. (1974) Formation of bimolecular membranes from lipid monolayers Meth. Enzym. 328, 545. Sc~N, S J. Cow~xt, M. and Frxtcersrettr, A. (1976) Reconstitution in planar lipid bilayers of a vohagedependent anionaelective channel obtained from Paranrciam mitochondria. J . Membrane BtoL 30, 99. Sc~tx, S. J., Kwcwx, B. L. and Ftxrc~srax, A. (1978) Colicin K acts by forming voltage~peadent ~annels in phospholipid bilayer membranes Natm, f ond . 276, 159.