Action of Black Widow Spider Venom on Quantized Release Of

Proc. Nat!. Acad. Sci. USA Vol. 76, No. 2, pp. 991-995, February 1979 Neurobiology

Action of black widow spider venom on quantized release of acetylcholine at the frog neuromuscular junction: Dependence upon external Mg2+ (neurotoxin/membrane permeability/divalent cations/potassium/osmotic pressure) STANLEY MISLER AND WILLIAM P. HURLBUT Department of Biophysics, The Rockefeller University, New York, New York 10021 Communicated by Frank Brink, Jr., November 22, 1978

ABSTRACT Black widow spider (Latrodectus tredecim- and glucosamine at pH 6.5 as a Na+ substitute), the swelling of guttatus) venom (BWSV) increases several hundredfold the the nerve terminals that usually accompanies BWSV treatment frequency of occurrence of miniature end-plate potentials was decreased but the depletion of vesicles-and apparent ex- (Fmepp) at frog neuromuscular junctions bathed in Ringer's solutions containing either Ca2+ or Mg2t, but it has little effect haustion of transmitter still occurred. These authors suggested on Fmepp at junctions bathed in modified Ringer's solution that BWSV may indeed increase the Na+ and Ca2+ perme- containing 1-2 mM ethylene glycol bis(,B-aminoethyl ether) ability of the terminals but that BWSV stimulated release "by N,N'-tetraacetic acid (EGTA) but no Ca2+ or Mg2+. When Mg2+ a mechanism which may not involve its ionophore property" is added to preparations that have been treated with BWSV in (7). the modified solution, Fme increases exponentially with time. Recent results of other workers, however, suggest that di- Fmepp falls again to low values when the Mg2+ is removed. The valent cations exert a powerful effect on transmitter release rate constant of the exponential rise is proportional to [Mg2+Jo in the range 1-4 mM, and the threshold [Mg2+J0 is 0.1-0.5 mM. induced by BWSV. Smith et al. (8) found that high concen- Increasing the K+ concentration of the bathing solution de- trations of Ca2+ increased the peak miniature end-plate po- creases the ability of Mg2+ to increase Fmepp. Addition of Ca2+, tential (mepp) frequency (Fmepp), decreased the duration of Co2+, Mn2+, or Zn2+ also leads to a large increase in Fmepp. the period of high Fmepp, and caused clumping of the residual These results are consistent with the possibility that BWSV in- population of vesicles at the frog nmj. Ornberg (9) reported that creases the permeability of the nerve terminal to divalent cat- of BWSV in the absence of both Ca2+ and did ions. BWSV can, however, increase Fmepp in hypertonic solu- application Mg2+ tions in the absence of external divalent cations. This result not increase Fmepp, but Fmepp increased when Mg2+ was re- suggests that the effects of BWSV on the nerve terminal may stored to the bathing solution. These results demonstrate that not be confined to increasing the permeability of the plasma- either Ca2+ or Mg2+ may support the BWSV increase in Fmepp lemma. and suggest that changes in the permeability of the presynaptic membrane are crucial to BWSV action. Black widow spider (Latradectus tredecimguttatus) venom We have examined the kinetics and specificity of the Mg2+ (BWSV) causes a massive release of neurotransmitter, and the dependence of the BWSV-induced increase in Fmepp at the frog depletion of synaptic vesicles, from various nerve terminals. The nmj. Most of our results are compatible with the hypothesis that effects of BWSV on vertebrate neuromuscular junctions (nmjs) BWSV increases Fmepp by increasing the permeability of the are due to an acidic protein called a-latrotoxin (Mr, 4130,000; nerve terminal membrane to various divalent ions. We also isoelectric point, pH 5.2-5.5) (1-3). The mechanism(s) of action examined the effect of BWSV on preparations soaked in a hy- of this toxin is not completely understood but two suggestions pertonic solution that contained no divalent cations and found have recently been made. that BWSV increased Fmepp. This result suggests that the effects One suggestion is that the crucial step in BWSV action is a of BWSV on the nerve terminal may not be confined to redistribution of the molecular components of the nerve ter- changing its ionic permeability (7). minal membrane that is modulated by a microtubular-mi- Some of our results have been reported in abstract form crofilament array in the cytoplasm (4). This suggestion is de- (10). rived from the observations that (i) concanavalin A inhibits BWSV action at nmjs in tissue culture and in adult frogs, and METHODS AND MATERIALS (ii) the inhibitory action of concanavalin A can be prevented All experiments were done at room temperature (18-230C) by prior treatment of the junctions with colchicine (4). with cutaneous pectoris nerve-muscle preparations freshly A second suggestion is that BWSV enhances transmitter re- dissected from frogs (Rana pipiens). End-plate regions of single lease by increasing the ionic permeability of the nerve terminal muscle fibers were impaled with glass micropipettes filled with membrane (5). This suggestion is prompted by the finding that 3 M KCI (resistances, 8-30 MQ) and mepps were recorded and purified a-latrotoxin creates channels in thin lipid membranes photographed through the use of standard electrophysiological that allow monovalent and divalent cations to pass through techniques (11). (6). Solutions. The standard Ringer solution (pH 6.9) contained Several investigators have recently examined the ionic (in mM): Na+, 116; K+, 2.0; Ca2+, 1.8; Cl-, 117; HPO2-, 2; dependencies of BWSV action. Rubin et al. (4) and Gorio et al. 1. When the ionic composition of the Ringer solution (7) found that, when BWSV was applied to the adult frog nmjs H2PO4, bathed in Ca2+- and Na+-free solutions (containing 4 mM Mg2+ Abbreviations: BWSV, homogenate of venom glands of black widow spider; mepp, miniature end-plate potential; Fmepp, frequency of oc- The publication costs of this article were defrayed in part by page currence of mepps; [Mg2+1o and [Mg2+li, extracellular and intracellular charge payment. This article must therefore be hereby marked "ad- concentrations of Mg2+; nmj, neuromuscular junction; EGTA, ethylene vertisement" in accordance with 18 U. S. C. §1734 solely to indicate glycol bis(,B-aminoethyl ether)-N,N'-tetraacetic acid; TTX, tetrodo- this fact. toxin. 991 Downloaded by guest on September 26, 2021 992 Neurobiology: Misler and Hurlbut Proc. Natl. Acad. Sci. USA 76 (1979) was changed, the concentration of NaCi was altered to keep the tonicity constant. In the experiments in which Mn2+, Co2+, or Zn2+ was used, all solutions were buffered with 6mM Tris-HCl adjusted to pH 7.2. Ringer's solutions were made hypertonic by the addition of analytical grade sucrose. Many test solutions a contained 1-5 mM ethylene glycol bis(f3-aminoethyl ether)- N,N'-tetraacetic acid (EGTA). In most experiments, the test solutions also contained 10-20 ng of tetrodotoxin (TTX) per ml b to prevent the spontaneous twitching that occurred when .L U w ' muscle fibers were impaled in solutions without divalent cations. 10 The BWSV was crude homogenates of venom glands obtained from frozen cephalothoraxes of L. tredecimguttatus and prepared as described (1, 2). In many experiments, the ho- mogenizing solution contained 1 mM EGTA. Procedure. The preparation was mounted in standard Ringer's solution and several surface muscle fibers were impaled to find a region rich in nmjs. The chamber (volume, -3 ml) was then flushed several times with 10-15 ml of modified Ringer's solution that contained 1 mM EGTA but no Ca2+ or Mg2+. The chamber was subsequently flushed once every 5 min during the total preliminary soaking time of 15-20 min. BWSV (50-60 ,i), containing about 2 ,gg of a-latrotoxin (2), was added to the chamber; 20 min later, the muscle was washed for 10-20 1 mMM9 t min with several flushes of modified Ringer's solution. This standard preparation, free of external divalent ions and treated with BWSV, was suitable for testing the dependence of BWSV action upon divalent ions. Hypertonic solutions were prepared by adding 60-120mM Time, min sucrose to the modified solution that contained no divalent FIG. 1. Mg2+ dependence of BWSV action. The preparation cations. This hypertonic solution was applied to the muscle after was washed for 20 min with a modified Ringer solution containing 1 the preliminary wash. BWSV was added 10-20 min later when mM EGTA and no Ca2+ or Mg2+. BWSV (50/,4) was added at zero Fmepp had reached a steady state. time, left for 20 min, and then washed away with the modified Ringer It was difficult to maintain impalements in fibers bathed in solution. A solution containing 1 mM Mg2+ was added at 40 min and solutions free of divalent cations. Most of the data gathered flushed away at 52 min. (Insets) Sample records of mepps at times under these conditions were obtained by impaling several fibers marked a, b, and c; calibration markings, 1.0 mV X 0.5 sec. in each muscle. However, the data for each experiment on the kinetics of Mg2+ action were obtained from single end-plates in the presence of Ca2 , the amplitude of the end-plate po- that were impaled a few minutes before or a few minutes after tential declined to a new steady-state value within 2 min (un- the introduction of solution with 1 mM Mg2+. The membrane published observation). This indicates that the exchange of potentials of these fibers usually declined during the course of Mg2+ in the extracellular space is complete in this time. the impalements to levels of -40 mV. Data are reported as Fig. 2 shows the effects of three concentrations of Mg2+ on means + SD (number of experiments). Fmepp at a junction treated with BWSV. Addition of 1 mM Mg2+ resulted in an exponential increase in Fmepp that reached RESULTS 80 sec-1 after 15 min. When Mg2+ was decreased to 0.1 mM, Mg2+ Dependence of BWSV Action. In the absence of ex- Fmepp declined exponentially (after a brief delay), reaching 6 ternal divalent cations, Fme p was low, 0.46 + 0.38 sec-1 (n = sec I after 15 min. Introducing 4 mM Mg2+ (with 2 mM 18). Addition of BWSV under these conditions did not signifi- EGTA) led to a more rapid increase in Fmepp, which reached cantly increase Fmepp, the value being 0.57 + 0.67 sec-1 (n = -.300 sec1 within 5 min. Decreasing Mg2+ again to 0.1 mM 49) at 30-40 min after addition of BWSV. * Addition of 1 mM led, after a 5 min delay, to an exponential decline in Fmepp. Mg2+ resulted in a progressive increase in Fmepp that could Reintroducing 1 and then 4 mM Mg2+ caused Fmep to increase reach nearly 100 sec-1 in 15 min. During its initial stage, the again, but the increases were smaller than previously observed, increase in Fmepp was approximately exponential with time. perhaps due to partial exhaustion of transmitter stores. When Mg2+ was removed, Fmepp declined exponentially with This experiment demonstrates that: (i) the rate of increase time to reach levels <1 sec-1 (Fig. 1). of Fmepp is proportional to extracellular Mg2+; (ii) the Mg2+- It is unlikely that the rate of increase of Fmepp was deter- dependent effects are reversible and a given preparation can mined by the rate of exchange of Mg2+ in the extracellular be cycled through several additions and removals of this ion; space. When the external concentration of Mg2+ was increased and (iii) the concentration of Mg2+ required to support significant increases in Fmepp is somewhere between 0.1 and 1.0 * In 7 of a total of 50 experiments, abrupt increases in Fmepp occurred mM. We performed many experiments of this type. In 4 mM 2-30 min after BWSV was added to the solution free of divalent Mg2+, Fmepp increased to rates well above 200 sec-1 in 3-5 min. cations. Three of these experiments were on successive days and the The mean rate constant for the rise was 1.7 + 0.65 min-i (n = more common results were obtained when all the stock solutions were 13). In 1 mM Mg2+, Fmepp initially increased with a rate con- replaced. These spurious results may have resulted from contami- + = nated solutions. The other four instances are unexplained, but their stant of 0.41 0.14 min-1 (n 21), but this increase did not occurrence suggests that BWSV may do more to nerve terminals than always continue. In 10 experiments, Fmepp reached an apparent increase the permeability to divalent ions. steady state of 3-38 sec-I by 15 min, whereas in 11 experiments, Downloaded by guest on September 26, 2021 Neurobiology: Misler and Hurlbut Proc. Natl. Acad. Sci. USA 76 (1979) 993 small (t100 AtV) in solutions with 40 mM K+, and it is possible that we failed to detect some of the mepps. Fig. 3 shows the results of one of our clearest experiments. When 1 mM Mg2+ was addd in the presence of 2 mM K+, Fmepp increased exponentially. Fmepp continued to increase, although more slowly, when K+ was increased to 20 mM. In- creasing K+ to 40 mM, however, resulted in a clear decrease in Fmepp over the course of 10 min. Returning the preparation to 2 mM K+ resulted in a rapid increase in Fmepp. u) I Effects of BWSV in Hypertonic Divalent Cation-Free 6 Solutions. Hypertonic solutions increase Fmepp at frog nmjs C even in the absence of external divalent cations (9, 13). It is CD44 mM Mg possible that this increase in frequency occurs because the hy- E pertonic solutions increase the concentrations of free divalent U. 10 ions within the terminals. If BWSV increases the permeability of the membrane to divalent ions, then its application to ter- j' lMMMg minals bathed in hypertonic divalent cation-free solutions might 4 mM Mg decrease the concentrations of these ions within the terminals and thereby decrease Fmepp. Shimoni et al. (13) recently re- Li MM Mg ported that high concentrations of K+ decreased Fmepp at nmjs treated with Ca-free hypertonic solutions, and they suggested a similar explanation to account for the decrease. 1 p I I I I I I I 10 30 50 70 Fig. 4 shows the results of an experiment in which BWSV was Time, min applied to a muscle bathed in a hypertonic solution that con- in free FIG. 2. Effects of varying Mg2+ concentration on Fmepp after tained no divalent cations. Fmepp the isotonic solution treatment with BWSV in a solution containing 1 mM EGTA and no of divalent cations was about 0.1 sec-1. Stepwise increases in Ca2+ or Mg2+. The end-plate was impaled about 1 min after addition tonicity, produced by addition of 60 mM and then 120 mM of 1 mM Mg2+; 0.1 mM Mg2+ or 4 mM Mg2+ (with 2 mM EGTA) was sucrose, led to increases in Fmepp to peak rates of 0.6 and 6 sec-, added as indicated. respectively. Addition of 50 Al of BWSV resulted in an increase in Fmepp to a steady-state level of nearly 100 sec-I within 5 min. it continued to increase exponentially to rates of 50-200 sec-'.t On return to solutions containing 0.1 mM Mg2+ or less, Fmepp declined exponentially, after a variable lag period, with a rate constant of 0.74 + 0.23 min-' (n = 24). The value of this rate constant was independent of the prior level of Mg2+. None of the rate constants was affected by varying the EGTA concentration from 0 to 5 mM. Effects of Other Divalent Cations. We investigated the effects of Ca2+, Mn2+, co2+, and Zn2+ on Fmepp at nmjs treated with BWSV. These ions were applied in the absence of EGTA u 60 because they are strongly complexed by this substance (12). The B 1Mg, 20K 6 6 40 introduction of 0.1 mM Ca2+ (three experiments), 4 mM Co2+ C6. c (two experiments), 4 mM Zn2+ (two experiments), or 4 mM a 20 Mn2+ (one experiment) caused Fmepp to increase to >100 sec-1 E E within 10 min. The effect of Mn2+ was totally reversed and the uL n effects of Ca2+ and Zn2+ were partially reversed by washing the muscles with modified Ringer's solution free of divalent 60C1CMg,40K cations. The reversibility of Co2+ was not tested. The large increases in Fmepp did not occur when these ions were applied to 40 muscles that had been soaked for 1 hr in the divalent cation-free solution but not been exposed to BWSV. 20 Effects of Extracellular K+. If the increase 0 Mg2+-dependent 0 0.5 1.0 in Fmepp seen at nmjs treated with BWSV were due to Mg2+ Time, min mepp amplitude, mV entering the nerve terminal by diffusion, the rate of entry and FIG. 3. Effect of [K+], on Fmepp in 1.0 mM Mg2+. (Left) In- extent of accumulation should be determined, in part, by the creasing [K+10 from 2 to 20 mM slowed the rate of increase of Fmepp; membrane potential of the terminal. To examine this possibility, increasing [K+]0 to 40 mM resulted in a decrease in Fmepp over time. we tested the effects of K+ on Fmepp. K+, at concentrations up Decreasing [K+]0 to 2 mM resulted in a large increase in Fmepp. When to 40 mM, had no obvious effects on the build-up of Fmepp in Mg2+ was removed, Fmepp fell to <10 sec-1 (not shown). (Insets) 2 or 4 mM Mg2+. However, effects were observed when K+ was Sample traces of mepps recorded at times a, b, c, and d; calibration increased from 2 to 20 or 40mM in the presence of 1 mM Mg2+. pulse (in a) is 1 mV X 10 msec. (Right): (A, B, and C) Amplitude histograms of mepps recorded near times a, b, and c, respectively. These experiments were difficult because mepp amplitude was Mean mepp amplitudes and average resting potentials were as follows: A, 0.67 ± 0.21 mV (n = 186), -55 mV; B, 0.33 + 0.09 mV (n = 154), t In two of these experiments Mg2+ was decreased from 1.0 to 0.5 mM -45 mV; C, 0.25 + 0.07 mV (n = 132); -30 mV. Note that mepp am- and Fmepp decreased, over 10 min, from nearly 100 sec-1 to a steady plitudes show a nearly Gaussian distribution in all three cases. level of 40-50 sec-1. This indicates that, for these nmjs, the threshold Analyses of these histograms suggest that it is unlikely that the Mg2+ was between 0.1 and 0.5 mM. number of mepps lost in the noise is greater than 10% of the total. Downloaded by guest on September 26, 2021 994 Neurobiology: Misler and Hurlbut Proc. Natl. Acad. Sci. USA 76 (1979) cations were added and because BWSV can enhance osmotic pressure-dependent transmitter release in the absence of external divalent cations, it seems unlikely that these ions are required for the binding of BWSV to the nerve terminal membrane. (ii) Because the effects of Mg2+ are totally reversible and the effects of the other divalent cations are at least partially reversible, it seems unlikely that these ions are required as co- factors for enzymatic activity that causes permanent changes in the structure of the nerve terminal membrane. (iii) Because similar quantitative effects of Mg2+ occur in the presence of 5 mM EGTA or in the complete absence of EGTA, it seems a,~~~~~~ likely that our results with a whole series of divalent cations are directly due to these ions and not to contaminant traces of 6 ~~~~50 gg aBWSV 120mM Ca2+. Our suggestion of the intracellular accumulation and site of E added y sucrose aL A action of Mg2+ in particular and of other divalent cations in E general is supported by two general features of the kinetics of 160 mM Ei the Mg2+-dependent increase in Fmepp. sucrose (i) Fmepp increases exponentially with time after the addition of Mg2+ to preparations exposed to BWSV. Prolonged repetitive indirect stimulation of frog nmjs also causes a Mg2+-dependent increase in Fmepp that is exponential with time (11). The rate constants of the exponentials (0.1-2 min-') and the concen- 60 mm 4 mM trations of Mg2+ required (1-10 sucrose Mg mM) are similar for the stim- 120 mm/ ulated and BWSV-treated preparations. This similarity in kinetics suggests that the cause of the increase in Fmepp is the same 0.1 0 30 60 90 120 in both cases. Because tetanic stimulation increases Mg2+ influx Time, min in squid giant axon (20) and a-latrotoxin increases Mg2+ con- FIG. 4. Effect of BWSV on an osmotic pressure-induced increase ductance of thin lipid films (6), we suggest that the common in Fmepp in the absence of divalent cations. See text for details. (Insets) cause of the increases in Fmepp in the two cases is the accumu- Sample traces of mepps at times a and b; calibration markings, 1.0 lation of Mg2+ in the nerve terminals. mV X 0.5 sec. (ii) There is a threshold concentration of extracellular Mg2+ required to increase Fmepp. If BWSV makes channels in the Removal of sucrose returned Fmepp to about 0.1 sece1. Fmepv membrane of the nerve terminal through which Mg2+ diffuses, rapidly increased again to about 6 sec-1 and then 100 sec- then a net influx of Mg2+ should occur whenever the ratio of when the hypertonic solutions were again added. Similar results the external to the internal concentrations exceeds exp- were obtained in four other experiments. [2FV/RT], in which V is the membrane potential (outside In these experiments, the increase in Fmepp induced by minus inside) and F, R, and T have their usual meanings. If V BWSV in the presence of hypertonic solutions and the enhanced is -60 mV and free [Mg2+l] is 1-4 mM (20, 21), a net Mg2+ effect of hypertonic solutions on Fmepp in the presence of BWSV influx through the channel should occur when [Mg2+]. exceeds occurred in the virtual absence of extracellular divalent cations 0.01-0.04 mM. If the nerve terminal were depolarized to -30 and must hence be independent of the entry of external divalent mV, as might well occur if BWSV enhances Na permeability cations into the axoplasm by way of BWSV-induced changes (6), a net Mg2+ flux would occur at [Mg2+] above 0.1-0.4 mM. in permeability. These levels are close to the observed levels of 0.1-1.0 mM at which increases in Fmepp occur in solutions containing 2 mM DISCUSSION K+. Decreasing Vm by increasing [K+1o should increase the This paper confirms the dependence of BWSV-induced threshold [Mg2+], required to produce net accumulation. This transmitter release in isotonic solution upon external Mg2+ (9). prediction is in qualitative agreement with the observation that We found, in addition, that Mn2 Zn2 and Co2+ in millimolar increasing [K+10 from 2 to 40 mM prevents a further increase concentrations also cause large increases in Fmepp when they in Fmepp in 1 mM Mg2+ and can even lead to a significant de- are applied to BWSV-treated muscles. Because each of these crease in Fmepp. This ability of high [K+]. to decrease the ions causes at least a small increase in Fmepp when added to a Mg2+-dependent increase in Fmepp may account for the failure standard Ringer solution (14-20), it would appear that BWSV of BWSV to cause the usual almost complete depletion of syn- does not endow these ions with new properties but rather en- aptic vesicles when applied to frog nmjs bathed in isotonic so- hances existing ones. We suggest that it does this by increasing lutions of K2SO4 with 4 mM Mg2+ (4). the permeability of the terminal to all of these ions so that un- Because Fmepp at rest is approximately 1 sec-1 in the presence usually large quantities enter the axoplasm to increase Fmepp of free [Mg2+]i at 1-4 mM, significant changes in Fmepp should by acting directly on the release-activating sites or, in the case require millimolar changes in [Mg2+],. If BWSV formed ionic of divalent cations other than calcium, by displacing Ca2+ from channels in the membrane of the nerve terminal similar to those intracellular binding sites. Blioch et al. (18) and Kita and van formed in lecithin/cholesterol bilayers by a-latrotoxin, they der Kloot (19) have previously suggested that any multivalent would have enough conductance to cause large changes in cation that enters the nerve terminal can increase Fmepp. [Mg2+]j in a few minutes. The conductance of a single channel We can rule out several alternative explanations for the di- bathed in a solution containing 10mM MgCl2 is approximately valent cation dependence of BWSV-induced quantal release. 0.5 X 10-10 mhos (A. Finkelstein, personal communication). (i) Because BWSV was added to the bath for 20 min and then If BWSV created one channel per active zone of the nerve flushed from the bath 10-20 min before the external divalent terminal [an active zone being a cylinder of volume 0.8,um3 Downloaded by guest on September 26, 2021 Neurobiology: Misler and Hurlbut Proc. Natl. Acad. Sci. USA 76 (1979) 995 (22)], then the influx of Mg2+ for each mV of deviation from 1. Longenecker, H. E., Jr., Hurlbut, W. P., Mauro, A. & Clark, A. electrochemical equilibrium would change [Mg2+]i at the rate (1970) Nature (London) 225,701-703. of 18 mM/min. We have no independent estimate of Mg2+ 2. Frontali, N., Ceccarelli, B., Gorio, A., Mauro, A., Siekevitz, P., influx or intracellular accumulation. Direct measurements of Tzeng, M-C. & Hurlbut, W. P. (1976) J. Cell Biol. 68, 462- Mg2+ in nerve terminals are obviously needed. 479. 3. Tzeng, M-C. & Siekevitz, P. (1978) Brain Res. 139, 190-196. Most of our results are consistent with the hypothesis that 4. Rubin, L. L., Gorio, A. & Mauro, A. (1978) Brain Res. 143, BWSV causes large increases in the permeability of the nerve 107-124. terminal membrane to divalent ions and that the intracellular 5. Pumplin, D. W. & Reese, T. (1977) J. Physiol. (London) 273, accumulation of these ions can cause a massive increase in 443-457. Fmepp. Our experiments also demonstrate that, in the presence 6. Finkelstein, A., Rubin, L. L. & Tzeng, M-C. (1976) Science 193, of hypertonic solution, BWSV can enhance Fme p in a manner 1009-1011. independent of extracellular divalent cations. The cause of this 7. Gorio, A., Rubin, L. L. & Mauro, A. (1978) J. Neurocytol. 7, effect is unknown. This effect may suggest that BWSV stimu- 193-205. lates Fmepp in ways independent of channel formation in gen- 8. Smith, J. E., Clark, A. W. & Kuster, T. A. (1977) J. Neurocytol. eral or the facilitation of divalent cation entry in particular 6,519-539. 9. Ornberg, R. L. (1977) Neurosci. Abstr. 3, 1200. (7). 10. Misler, S. & Hurlbut, W. P. (1977) Biophys. J. 21, 178 (abstr.). There is, as yet, no evidence that a-latrotoxin forms con- 11. Hurlbut, W. P., Longenecker, H. E., Jr. & Mauro, A. (1971) J. ductance channels in other biological membranes, as might be Physiol. (London) 219, 17-38. expected for a general channel-forming protein. Most workers 12. Schwartenbach, G. & Flaschka, H. (1969) Complexometric Ti- have only reported presynaptic effects of BWSV, and initial trations (Methuen, London). biochemical experiments demonstrate that a-latrotoxin binds 13. Shimoni, Y., Alnaes, E. & Rahamimoff, R. (1977) Nature (Lon- to dog brain synaptic membranes but not to rat liver plasma don) 267, 170-172. membrane (23). If BWSV increases the ionic permeability of 14. Benoit, P. & Mambrini, J. (1970) J. Physiol. (London) 210, nerve terminal membrane, it may do so only after binding to 681-695. a specific molecular component of the membrane. The binding 15. Hubbard, J. I., Jones, S. F. & Landau, E. M. (1968) J. Physiol. (London) 194,355-380. may cause changes in ionic permeability and, in addition, affect 16. Balnave, J. & Gage, P. (1973) Br. J. Pharmacol. 47,339-352. the transmitter release system in ways that could account for 17. Weakly, J. (1973) J. Physiol. (London) 234, 597-612. the results we obtained with hypertonic solutions (7). 18. Blioch, A. I., Glagoleva, I. M., Liberman, E. A. & Nenashev, V. A. (1968) J. Physiol. (London) 199, 11-35. We thank Dr. Alan Finkelstein for allowing us to quote his unpub- 19. Kita, H. & van der Kloot, W. G. (1976) J. Physiol. (London) 259, lished data on the Mg2+ conductance of the a-latrotoxin channel in 177-198. thin lipid membranes and both he and Dr. Frank Brink, Jr. for stim- 20. Crawford, A. C. & Baker, P. F. (1972) J. Physiol. (London) 227, ulating discussions. This work was supported by National Science 855-874. Foundation Grant BNS 7715808 T to W.P.H. and was begun during 21. Nanninga, L. B. (1961) Biochim. Biophys. Acta 54,330-345. an elective period in S.M.'s internship (Department of Medicine, New 22. Peper, K., Dreyer, F., Sandri, F. C., Akert, K. & Moor, H. (1974) York Veterans Administration Hospital, New York University Medical Cell Tissue Res. 149, 437-455. Center, New York, NY). 23. Tzeng, M-C. & Siekevitz, P. (1979) J. Neurochem., in press. Downloaded by guest on September 26, 2021