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 so- lutions 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 cat- ions. 10 The BWSV was crude homogenates of venom glands ob- tained 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 im- paled 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.