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 tredecimguttatus) venom (BWSV) increases several hundredfold the frequency of occurrence of miniature end-plate potentials (Fmepp) at frog neuromuscular junctions bathed in Ringer's solutions containing either Ca2+ or Mg2t, but it has little effect on Fmepp at junctions bathed in modified Ringer's solution containing 1-2 mM ethylene glycol bis(,B-aminoethyl ether) N,N'-tetraacetic acid (EGTA) but no Ca2+ or Mg2+. When Mg2+ is added to preparations that have been treated with BWSV in the modified solution, Fme increases exponentially with time. Fmepp falls again to low values when the Mg2+ is removed. The 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. Increasing the K+ concentration of the bathing solution decreases the ability of Mg2+ to increase Fmepp. Addition of Ca2+, Co2+, Mn2+, or Zn2+ also leads to a large increase in Fmepp. These results are consistent with the possibility that BWSV increases the permeability of the nerve terminal to divalent cations. BWSV can, however, increase Fmepp in hypertonic solutions in the absence of external divalent cations. This result suggests that the effects of BWSV on the nerve terminal may not be confined to increasing the permeability of the plasmalemma.
Black widow spider (Latradectus tredecimguttatus) venom (BWSV) causes a massive release of neurotransmitter, and the depletion of synaptic vesicles, from various nerve terminals. The effects of BWSV on vertebrate neuromuscular junctions (nmjs) are due to an acidic protein called a-latrotoxin (Mr, 4130,000; isoelectric point, pH 5.2-5.5) (1-3). The mechanism(s) of action of this toxin is not completely understood but two suggestions have recently been made. One suggestion is that the crucial step in BWSV action is a redistribution of the molecular components of the nerve terminal membrane that is modulated by a microtubular-microfilament array in the cytoplasm (4). This suggestion is derived from the observations that (i) concanavalin A inhibits BWSV action at nmjs in tissue culture and in adult frogs, and (ii) the inhibitory action of concanavalin A can be prevented by prior treatment of the junctions with colchicine (4). A second suggestion is that BWSV enhances transmitter release by increasing the ionic permeability of the nerve terminal membrane (5). This suggestion is prompted by the finding that purified a-latrotoxin creates channels in thin lipid membranes that allow monovalent and divalent cations to pass through
(6).
Several investigators have recently examined the ionic dependencies of BWSV action. Rubin et al. (4) and Gorio et al. (7) found that, when BWSV was applied to the adult frog nmjs bathed in Ca2+- and Na+-free solutions (containing 4 mM Mg2+
and glucosamine at pH 6.5 as a Na+ substitute), the swelling of the nerve terminals that usually accompanies BWSV treatment was decreased but the depletion of vesicles -and apparent exhaustion of transmitter still occurred. These authors suggested that BWSV may indeed increase the Na+ and Ca2+ permeability of the terminals but that BWSV stimulated release "by a mechanism which may not involve its ionophore property" (7). Recent results of other workers, however, suggest that divalent cations exert a powerful effect on transmitter release induced by BWSV. Smith et al. (8) found that high concentrations of Ca2+ increased the peak miniature end-plate potential (mepp) frequency (Fmepp), decreased the duration of the period of high Fmepp, and caused clumping of the residual population of vesicles at the frog nmj. Ornberg (9) reported that application of BWSV in the absence of both Ca2+ and Mg2+ did not increase Fmepp, but Fmepp increased when Mg2+ was restored to the bathing solution. These results demonstrate that either Ca2+ or Mg2+ may support the BWSV increase in Fmepp and suggest that changes in the permeability of the presynaptic membrane are crucial to BWSV action. We have examined the kinetics and specificity of the Mg2+ dependence of the BWSV-induced increase in Fmepp at the frog nmj. Most of our results are compatible with the hypothesis that BWSV increases Fmepp by increasing the permeability of the nerve terminal membrane to various divalent ions. We also examined the effect of BWSV on preparations soaked in a hypertonic solution that contained no divalent cations and found that BWSV increased Fmepp. This result suggests that the effects of BWSV on the nerve terminal may not be confined to changing its ionic permeability (7). Some of our results have been reported in abstract form (10). METHODS AND MATERIALS All experiments were done at room temperature (18-230C) with cutaneous pectoris nerve-muscle preparations freshly dissected from frogs (Rana pipiens). End-plate regions of single muscle fibers were impaled with glass micropipettes filled with 3 M KCI (resistances, 8-30 MQ) and mepps were recorded and photographed through the use of standard electrophysiological techniques (11). Solutions. The standard Ringer solution (pH 6.9) contained (in mM): Na+, 116; K+, 2.0; Ca2+, 1.8; Cl-, 117; HPO2-, 2; H2PO4, 1. When the ionic composition of the Ringer solution Abbreviations: BWSV, homogenate of venom glands of black widow
spider; mepp, miniature end-plate potential; Fmepp, frequency of occurrence of mepps; [Mg2+1o and [Mg2+li, extracellular and intracellular concentrations of Mg2+; nmj, neuromuscular junction; EGTA, ethylene glycol bis(,B-aminoethyl ether)-N,N'-tetraacetic acid; TTX, tetrodo-
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
toxin.
991
992
Neurobiology: Misler and Hurlbut
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 6 mM Tris-HCl adjusted to pH 7.2. Ringer's solutions were made hypertonic by the addition of analytical grade sucrose. Many test solutions 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 to prevent the spontaneous twitching that occurred when muscle fibers were impaled in solutions without divalent cations. 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 homogenizing 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 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 60-120 mM sucrose to the modified solution that contained no divalent cations. This hypertonic solution was applied to the muscle after the preliminary wash. BWSV was added 10-20 min later when Fmepp had reached a steady state. It was difficult to maintain impalements in fibers bathed in solutions free of divalent cations. Most of the data gathered under these conditions were obtained by impaling several fibers in each muscle. However, the data for each experiment on the kinetics of Mg2+ action were obtained from single end-plates that were impaled a few minutes before or a few minutes after the introduction of solution with 1 mM Mg2+. The membrane potentials of these fibers usually declined during the course of the impalements to levels of -40 mV. Data are reported as means + SD (number of experiments). RESULTS Mg2+ Dependence of BWSV Action. In the absence of external divalent cations, Fme p was low, 0.46 + 0.38 sec-1 (n = 18). Addition of BWSV under these conditions did not significantly increase Fmepp, the value being 0.57 + 0.67 sec-1 (n = 49) at 30-40 min after addition of BWSV. * Addition of 1 mM Mg2+ resulted in a progressive increase in Fmepp that could reach nearly 100 sec-1 in 15 min. During its initial stage, the increase in Fmepp was approximately exponential with time. When Mg2+ was removed, Fmepp declined exponentially with time to reach levels 100 sec-1 within 10 min. The effect of Mn2+ was totally reversed and the effects of Ca2+ and Zn2+ were partially reversed by washing the muscles with modified Ringer's solution free of divalent cations. The reversibility of Co2+ was not tested. The large increases in Fmepp did not occur when these ions were applied to muscles that had been soaked for 1 hr in the divalent cation-free solution but not been exposed to BWSV. Effects of Extracellular K+. If the Mg2+-dependent increase in Fmepp seen at nmjs treated with BWSV were due to Mg2+ entering the nerve terminal by diffusion, the rate of entry and extent of accumulation should be determined, in part, by the membrane potential of the terminal. To examine this possibility, we tested the effects of K+ on Fmepp. K+, at concentrations up to 40 mM, had no obvious effects on the build-up of Fmepp in 2 or 4 mM Mg2+. However, effects were observed when K+ was increased from 2 to 20 or 40 mM in the presence of 1 mM Mg2+. These experiments were difficult because mepp amplitude was t
In two of these experiments Mg2+ was decreased from 1.0 to 0.5 mM and Fmepp decreased, over 10 min, from nearly 100 sec-1 to a steady level of 40-50 sec-1. This indicates that, for these nmjs, the threshold Mg2+ was between 0.1 and 0.5 mM.
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. Increasing 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. Effects of BWSV in Hypertonic Divalent Cation-Free Solutions. Hypertonic solutions increase Fmepp at frog nmjs even in the absence of external divalent cations (9, 13). It is possible that this increase in frequency occurs because the hypertonic solutions increase the concentrations of free divalent ions within the terminals. If BWSV increases the permeability of the membrane to divalent ions, then its application to terminals bathed in hypertonic divalent cation-free solutions might decrease the concentrations of these ions within the terminals and thereby decrease Fmepp. Shimoni et al. (13) recently reported 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. Fig. 4 shows the results of an experiment in which BWSV was applied to a muscle bathed in a hypertonic solution that contained no divalent cations. Fmepp in the isotonic solution free of divalent cations was about 0.1 sec-1. Stepwise increases in tonicity, produced by addition of 60 mM and then 120 mM sucrose, led to increases in Fmepp to peak rates of 0.6 and 6 sec-, 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.
u
60
6C6.
6
E
E
c
B
1Mg, 20K
40
a 20 uL
n
1CMg,40K
60C
40 20 0 Time, min
0
0.5 1.0 mepp amplitude, mV
FIG. 3. Effect of [K+], on Fmepp in 1.0 mM Mg2+. (Left) Increasing [K+10 from 2 to 20 mM slowed the rate of increase of Fmepp; increasing [K+]0 to 40 mM resulted in a decrease in Fmepp over time. Decreasing [K+]0 to 2 mM resulted in a large increase in Fmepp. When Mg2+ was removed, Fmepp fell to
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 tredecimguttatus) venom (BWSV) increases several hundredfold the frequency of occurrence of miniature end-plate potentials (Fmepp) at frog neuromuscular junctions bathed in Ringer's solutions containing either Ca2+ or Mg2t, but it has little effect on Fmepp at junctions bathed in modified Ringer's solution containing 1-2 mM ethylene glycol bis(,B-aminoethyl ether) N,N'-tetraacetic acid (EGTA) but no Ca2+ or Mg2+. When Mg2+ is added to preparations that have been treated with BWSV in the modified solution, Fme increases exponentially with time. Fmepp falls again to low values when the Mg2+ is removed. The 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. Increasing the K+ concentration of the bathing solution decreases the ability of Mg2+ to increase Fmepp. Addition of Ca2+, Co2+, Mn2+, or Zn2+ also leads to a large increase in Fmepp. These results are consistent with the possibility that BWSV increases the permeability of the nerve terminal to divalent cations. BWSV can, however, increase Fmepp in hypertonic solutions in the absence of external divalent cations. This result suggests that the effects of BWSV on the nerve terminal may not be confined to increasing the permeability of the plasmalemma.
Black widow spider (Latradectus tredecimguttatus) venom (BWSV) causes a massive release of neurotransmitter, and the depletion of synaptic vesicles, from various nerve terminals. The effects of BWSV on vertebrate neuromuscular junctions (nmjs) are due to an acidic protein called a-latrotoxin (Mr, 4130,000; isoelectric point, pH 5.2-5.5) (1-3). The mechanism(s) of action of this toxin is not completely understood but two suggestions have recently been made. One suggestion is that the crucial step in BWSV action is a redistribution of the molecular components of the nerve terminal membrane that is modulated by a microtubular-microfilament array in the cytoplasm (4). This suggestion is derived from the observations that (i) concanavalin A inhibits BWSV action at nmjs in tissue culture and in adult frogs, and (ii) the inhibitory action of concanavalin A can be prevented by prior treatment of the junctions with colchicine (4). A second suggestion is that BWSV enhances transmitter release by increasing the ionic permeability of the nerve terminal membrane (5). This suggestion is prompted by the finding that purified a-latrotoxin creates channels in thin lipid membranes that allow monovalent and divalent cations to pass through
(6).
Several investigators have recently examined the ionic dependencies of BWSV action. Rubin et al. (4) and Gorio et al. (7) found that, when BWSV was applied to the adult frog nmjs bathed in Ca2+- and Na+-free solutions (containing 4 mM Mg2+
and glucosamine at pH 6.5 as a Na+ substitute), the swelling of the nerve terminals that usually accompanies BWSV treatment was decreased but the depletion of vesicles -and apparent exhaustion of transmitter still occurred. These authors suggested that BWSV may indeed increase the Na+ and Ca2+ permeability of the terminals but that BWSV stimulated release "by a mechanism which may not involve its ionophore property" (7). Recent results of other workers, however, suggest that divalent cations exert a powerful effect on transmitter release induced by BWSV. Smith et al. (8) found that high concentrations of Ca2+ increased the peak miniature end-plate potential (mepp) frequency (Fmepp), decreased the duration of the period of high Fmepp, and caused clumping of the residual population of vesicles at the frog nmj. Ornberg (9) reported that application of BWSV in the absence of both Ca2+ and Mg2+ did not increase Fmepp, but Fmepp increased when Mg2+ was restored to the bathing solution. These results demonstrate that either Ca2+ or Mg2+ may support the BWSV increase in Fmepp and suggest that changes in the permeability of the presynaptic membrane are crucial to BWSV action. We have examined the kinetics and specificity of the Mg2+ dependence of the BWSV-induced increase in Fmepp at the frog nmj. Most of our results are compatible with the hypothesis that BWSV increases Fmepp by increasing the permeability of the nerve terminal membrane to various divalent ions. We also examined the effect of BWSV on preparations soaked in a hypertonic solution that contained no divalent cations and found that BWSV increased Fmepp. This result suggests that the effects of BWSV on the nerve terminal may not be confined to changing its ionic permeability (7). Some of our results have been reported in abstract form (10). METHODS AND MATERIALS All experiments were done at room temperature (18-230C) with cutaneous pectoris nerve-muscle preparations freshly dissected from frogs (Rana pipiens). End-plate regions of single muscle fibers were impaled with glass micropipettes filled with 3 M KCI (resistances, 8-30 MQ) and mepps were recorded and photographed through the use of standard electrophysiological techniques (11). Solutions. The standard Ringer solution (pH 6.9) contained (in mM): Na+, 116; K+, 2.0; Ca2+, 1.8; Cl-, 117; HPO2-, 2; H2PO4, 1. When the ionic composition of the Ringer solution Abbreviations: BWSV, homogenate of venom glands of black widow
spider; mepp, miniature end-plate potential; Fmepp, frequency of occurrence of mepps; [Mg2+1o and [Mg2+li, extracellular and intracellular concentrations of Mg2+; nmj, neuromuscular junction; EGTA, ethylene glycol bis(,B-aminoethyl ether)-N,N'-tetraacetic acid; TTX, tetrodo-
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
toxin.
991
992
Neurobiology: Misler and Hurlbut
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 6 mM Tris-HCl adjusted to pH 7.2. Ringer's solutions were made hypertonic by the addition of analytical grade sucrose. Many test solutions 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 to prevent the spontaneous twitching that occurred when muscle fibers were impaled in solutions without divalent cations. 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 homogenizing 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 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 60-120 mM sucrose to the modified solution that contained no divalent cations. This hypertonic solution was applied to the muscle after the preliminary wash. BWSV was added 10-20 min later when Fmepp had reached a steady state. It was difficult to maintain impalements in fibers bathed in solutions free of divalent cations. Most of the data gathered under these conditions were obtained by impaling several fibers in each muscle. However, the data for each experiment on the kinetics of Mg2+ action were obtained from single end-plates that were impaled a few minutes before or a few minutes after the introduction of solution with 1 mM Mg2+. The membrane potentials of these fibers usually declined during the course of the impalements to levels of -40 mV. Data are reported as means + SD (number of experiments). RESULTS Mg2+ Dependence of BWSV Action. In the absence of external divalent cations, Fme p was low, 0.46 + 0.38 sec-1 (n = 18). Addition of BWSV under these conditions did not significantly increase Fmepp, the value being 0.57 + 0.67 sec-1 (n = 49) at 30-40 min after addition of BWSV. * Addition of 1 mM Mg2+ resulted in a progressive increase in Fmepp that could reach nearly 100 sec-1 in 15 min. During its initial stage, the increase in Fmepp was approximately exponential with time. When Mg2+ was removed, Fmepp declined exponentially with time to reach levels 100 sec-1 within 10 min. The effect of Mn2+ was totally reversed and the effects of Ca2+ and Zn2+ were partially reversed by washing the muscles with modified Ringer's solution free of divalent cations. The reversibility of Co2+ was not tested. The large increases in Fmepp did not occur when these ions were applied to muscles that had been soaked for 1 hr in the divalent cation-free solution but not been exposed to BWSV. Effects of Extracellular K+. If the Mg2+-dependent increase in Fmepp seen at nmjs treated with BWSV were due to Mg2+ entering the nerve terminal by diffusion, the rate of entry and extent of accumulation should be determined, in part, by the membrane potential of the terminal. To examine this possibility, we tested the effects of K+ on Fmepp. K+, at concentrations up to 40 mM, had no obvious effects on the build-up of Fmepp in 2 or 4 mM Mg2+. However, effects were observed when K+ was increased from 2 to 20 or 40 mM in the presence of 1 mM Mg2+. These experiments were difficult because mepp amplitude was t
In two of these experiments Mg2+ was decreased from 1.0 to 0.5 mM and Fmepp decreased, over 10 min, from nearly 100 sec-1 to a steady level of 40-50 sec-1. This indicates that, for these nmjs, the threshold Mg2+ was between 0.1 and 0.5 mM.
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. Increasing 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. Effects of BWSV in Hypertonic Divalent Cation-Free Solutions. Hypertonic solutions increase Fmepp at frog nmjs even in the absence of external divalent cations (9, 13). It is possible that this increase in frequency occurs because the hypertonic solutions increase the concentrations of free divalent ions within the terminals. If BWSV increases the permeability of the membrane to divalent ions, then its application to terminals bathed in hypertonic divalent cation-free solutions might decrease the concentrations of these ions within the terminals and thereby decrease Fmepp. Shimoni et al. (13) recently reported 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. Fig. 4 shows the results of an experiment in which BWSV was applied to a muscle bathed in a hypertonic solution that contained no divalent cations. Fmepp in the isotonic solution free of divalent cations was about 0.1 sec-1. Stepwise increases in tonicity, produced by addition of 60 mM and then 120 mM sucrose, led to increases in Fmepp to peak rates of 0.6 and 6 sec-, 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.
u
60
6C6.
6
E
E
c
B
1Mg, 20K
40
a 20 uL
n
1CMg,40K
60C
40 20 0 Time, min
0
0.5 1.0 mepp amplitude, mV
FIG. 3. Effect of [K+], on Fmepp in 1.0 mM Mg2+. (Left) Increasing [K+10 from 2 to 20 mM slowed the rate of increase of Fmepp; increasing [K+]0 to 40 mM resulted in a decrease in Fmepp over time. Decreasing [K+]0 to 2 mM resulted in a large increase in Fmepp. When Mg2+ was removed, Fmepp fell to
