- Email: [email protected]
Concanavalin A blocks the Ca2+-dependence of crayfish muscle fiber responses to glutamate
Brain Research, 243 (1982) 165-168
165
Elsevier Biomedical Press
Concanavalin A blocks the Ca'+-dependence of crayfish muscle fiber responses to glutamate M. THIEFFRY Laboratoire de Neurobiologie Cellulaire du C.N.R.S. 91190 Gif-sur- Yvette (France)
(Accepted March llth, 1982) Key words: glutamate - - receptors - - calcium - - lanthanum - - concanavalin A - - desensitization
(1) The response of crayfish muscle fibers to bath-applied glutamate is strongly inhibited when the Ca concentration of the physiological solution is reduced. Other divalent cations cannot substitute for Ca. The trivalent impermeant cation La can at low concentration replace Ca. Moreover, decreasing the Ca concentration in the presence of La potentiates the glutamate response. (2) The time course of responses to ionophoretically applied glutamate suggests a faster desensitization in low Ca solutions. The lectin concanavalin A, which blocks desensitization, also eliminates the decrease of the glutamate response in low Ca solutions. (3)The above results are compared to available data concerning Ca-dependence, desensitization and effects of concanavalin A. Glutamate, the putative excitatory transmitter 9, triggers in crustacean muscle fibers an inward current carried mainly by N a + ions 6,16,17. A smaller but unknown part of the response is possibly due to a Ca 2+ inflUX1,6,9,16,17, but the size of the glutamateinduced calcium conductance is much too small to explain the marked decrease in the glutamate response that is observed when external calcium concentration is lowered1,2,15,17,20. This dependence on calcium remains even when calcium ions are replaced by other divalentsl, 15,17, which in fact block, rather than 'protect', the glutamate response1, ~. The results presented below show that, on the other hand, the impermeant cation, La 8+, which at concentrations of 1-20 m M also antagonizes the glutamate effect4, zl, can, at the concentration of 0.5 mM, substitute for Ca 2+, and that the lectin concartavalin A (Con A) irreversibly eliminates the decrease of the glutamate response observed in low Ca 2+ solutions. Experiments were carried out on the posterior fast abdominal flexor is of the crayfish, Procambarus clarkii, a preparation which shows no rectification over a w;de range of potential 11. The preparation was continuously superfused at 15 °C w_;th saline of composition 195 m M NaCI, 5.4 m M KCI, 13 m M CaC12, 2.8 m M MgCI2 buffered at p H 7.2 by 10 m M 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
Tris-maleate-NaOH. Solution containing lower concentrations of Ca 2+ were obtained by mixing the standard saline with a solution in which Ca z+ was isotonically replaced by N a +. Con A (Sigma, grade IV or IBF), N a glutamate and LaC13 (Merck) were added to the perfusion solution. Ionophoretic applications of glutamate were made through 150-300 M [~ pipettes filled with a 0.1 M solution at p H 8 and Ca 2+ was applied through low resistance pipettes filled with 1 M CaC19.. In both cases, constant current generators 5 were used. Bath application of glutamate induces a depolarization of the muscle fibers which, for high doses of agonist, rapidly fades due to desensitization (Fig. 1, 1A). When the external Ca ~+ concentration was reduced to 1.3 mM, the response was strongly decreased (Fig. 1, 1B) but recovered after returning to standard solution (Fig. 1, 1C). As in other crayfish preparations 15,17, Ca ~+ substitution by equimolar concentrations of Mg 2+, Sr z+, Ba z+ did not prevent the decrease of the response in low Ca 2+. When the same dose of glutamate was applied in standard saline to which 0.5 m M La 3+ had been added, the glutamate response was potentiated (Fig. 1, 2A). In addition, reducing external Ca 2+ to 1.3 m M in the presence of 0.5 m M La a+ not only prevented the
166
A
1o~iv mln :',~
C
Fig. 1. Depression of the glutamate response in low Ca 2+ is overcome by addition of 0.5 mM La 3+. Left: intracellularly recorded responses to 0.5 mM bath applied glutamate (bar) in solutions containing 13 m M Ca ~+ (A), 1.3 mM Ca 2+ (B) and after return to 13 m M Ca 2+ (C). Right: in the presence of 0.5 m M La 3+, responses to the same application in the presence of 13 mM Ca z+ (A) and 1.3 m M Ca 2+ (B). C: after return to control conditions. Input resistance monitored by an hyperpolarizing current pulse applied every 10 s.
reduction of the glutamate response, but induced a further potentiation (Fig. 1, 2B). After washing with ncrmal saline, these effects were reversed (Figs. 1, 2C). At the concentration of 0.5 mM, Sr ~÷, Ba ~+, Mn 2+ and Co 2+ were not capable of countering the antagonistic effect of low Ca ~+. A mcre detailed examination of the response in normal and low Ca 2+ was carried out using ionophoretically applied glutamate. The external Ca 2-concentration was varied either by changing the superfusion solution or by local Ca 2+ icnophoresis from a second pipette in a low Ca 2+ solution. Both techniques gave similar results; the latter was used in the experiment shown in Fig. 2. When compared to responses obtained in the presence of higher Ca z+ concentrations, the glutamate response in low Ca ~+ is characterized by a shorter time to peak and a faster decline whereas the initial slope is not modified. Since the rate of rise of the response is unaffected by the change in calcium concentration it appears that only the activated receptor-channel complex is modified by Ca 2+. Furthermore, it seems clear that neither of the cations Ca z+ and La ~+ is acting from the inside of the membrane, since lanthanum is impermeant and the action of exter-
Fig. 2. External Ca z+ concentration modifies the time course of the glutamate response. Intracellularly recorded responses to glutamate applied by 1 s, 30 nA current pulse (bar). The perfusion solution contained 2.6 mM Ca 2+, one-fifth the normal concentration. The local Ca ~+ concentration was increased by iontophoresis of Ca 2+ from a second pipette. The Ca 2+ pulse was applied 2 s before glutamate application and maintained throughout the record. The values 0, 100 and 200 nA refer to the intensity of the Ca iontophoresis current. Glutamate was applied at some distance from the sensitive spot to minimize possible artefacts due to contractions.
nally applied calcium is very rapid (acting within 1 s from the onset of ionophoresis of the cation). The means by which calcium and lanthanum keep the activated receptor-channel complex functional remain, however, obscure. They might act by permitting greater repetitive binding, or by protecting the activated receptor-channel complex from being blocked. Whatever the hypothesis proposed for explaining their actions, it must also account for the finding that low calcium solutions affect spontaneous miniature potentials in the same way s as they have been shown here to affect responses to longduration ionophoretic applications of glutamate. The fact that calcium affects both of these membrane events in the same way makes it difficult to hypothesize that it is acting by interfering with the desensitization process. Likewise, previous studies of the glutamate response of crayfish muscle in normal and low Ca 2+ solutions did not permit the detection of a role of Ca 2÷ ions in the desensitization process15, 20. Furthermore, in studies in which calcium has been shown to play a role in desensitization (e.g. in the ACh response of frog muscle), it was found that the higher the calcium concentration, the greater the desensitization (with calcium presumably acting intracellularly) 14. In spite of these earlier data, which make it difficult to explain the calcium effects observed here in
167 terms of desensitization, the fact remains that 'apparent desensitization' is faster in low Ca 2÷. Consequently, it seemed worthwhile evaluating the effects of calcium concentration on the glutamate response in preparations in which desensitization had been blocked by exposure of the muscle to the lectin, concanavalin A 1~,19. The experimental procedure consisted of: (1) recording the responses to glutamate in both normal and low Ca z+ solutions; (2) exposing the muscle for 10-30 min to Con A (100--500/~g/ml); (3) soaking the muscle for 10-15 min in lectin-free, normal calcium solutions: and (4) testing the response to the same glutamate application in low and high calcium. As can be seen in Fig. 3, the calcium dependence of the glutamate response is irreversibly eliminated by exposure of the muscle to Con A. That is to say, in a preparation in which the glutamate response does not desensitize, the removal of calcium no longer causes a diminution of the glutamate response. In fact, after a correction is made for changes in input resistance, the ~esponse of a Con A-treated muscle in low Ca 2+ appears even slightly larger than that in normal saline. The response of a Con A-treated preparation is still antagonized by divalent cations (50 ~ reduction by 10 mM
13 mM Ca
,
l!l'flfl ~i":llrr~rrrTT /~[I
1.3 mM Ca 5 mV -
~r~
ii I rll
4 ! i l l ; ' MT
i]]l
T]]]I2 H]F ,'
,_
,
,t!IHUTIkHTIIT]T
Fig. 3. Con A blocks the Ca 2+ dependence of the glutamate response, lntracellularly recorded responses to 0.1 mM bath applied glutamate (bar). Top: control responses in 13 m M Ca 2+ (left) and 1.3 m M Ca 2+ (right) before treatment by ConA. Bottom: after 20 rain treatment by 400 #g/ml Con A and 10 min washing, the responses to the same glutamate application were again recorded in 13 mM Ca ~+ (left) and 1.3 mM Ca z+ (right). Input resistance monitored every 10 s by an hyperpolarizing 200 nA current pulse.
Co2+). Likewise, the effect of lanthanum (0.5 mM) is reversed: it now causes a reduction (of about 15-207/o) in the glutamate response. Finally, it should be noted that after Con A treatment, the repolarization of the membrane during the wash of the agonist is noticeably slower in low Ca 2+ solutions than in normal ones (see Fig. 3). As was shown above, calcium, in this preparation, does not change the rise time of the response, but acts to slow down the decay of the response. One could consider that Con A acts like a more effective and irreversible calcium-type agent and, once in place, pre-empts any similar role for calcium ions. Another interpretation of the disappearance of the calcium dependence of the glutamate response after Con A treatment might be that Con A modifies the ionic conductances triggered by glutamate, eliminating the participation of calcium ions while increasing that of others. Con A does appear to alter the glutamate-induced permability changes in molluscan neurones10 and, llke the 'Ca 2+ blocker' D-600 at frog endplates 3, it has been shown to eliminate the voltage dependence of channel life-time in arthropod muscle 7,1~. A careful analysis of the effects of varying calcium concentrations as a function of agonist dose and membrane voltage should give some insight concerning the means by which calcium acts in the normal preparation to maintain the glutamate response. On the other hand, a comparison of the ionic conductances triggered by glutamate in a Con A-treated fiber with those underlying the glutamate response in a normal fiber might help clarify the mechanism by which Con A eliminates desensitization and the calcium dependence of the glutamate response. If and when these data are explained by a unifying hypothesis, our understanding of the desensitization process in this preparation should be enhanced. This work was supported in part by Grant 387670 from the Institut National de la Sant6 et de la Recherche Mddicale. I am very indebted to J. Kehoe for discussing the above results and for her considerable help in the preparation of the manuscript.
168 1 Ashley, C. C. and Campbell, A. K., Free-calcium and tension responses in single barnacle muscle fibers following the application of L-glutamate, Biochim. biophys. Acta, 512 (1978) 429-534. 2 Barker, J. L., Divalent cations: effects on post-synaptic pharmacology of invertebrate synapses, Brain Research, 92 (1975) 307-323. 3 Bregestovski, P. D., Miledi, R. and Parker, I., Blocking of frog endplate channels by the organic calcium antagonist D 600, Proc. roy. Soc. B, 211 (1980) 15-24. 4 Colton, C. K. and Freeman, A. R., La 3+ blockade of glutamate action at the lobster neuromuscular junction, Comp. Biochem. PhysioL, 51C (1975) 285-289. 5 Dreyer, F. and Peper, K., Iontophoretic application of acetylcholine: advantages of high resistance micropipettes in connection with an electronic current pump, Pfliigers Arch. ges. Physiol., 348 (1974) 263-272. 6 Dudel, J., Nonlinear voltage dependence of excitatory synaptic current in crayfish muscle, Pfliigers Arch. ges. Physiol., 352 (1974) 227-241. 7 Dudel, J., The voltage dependence of the decay of the excitatory postsynaptic current and the effect of concanavalin A at the crayfish neuromuscular junction, J. Physiol. (Paris), 75 (1979) 601-604. 8 Dudel, J., The effect of reduced calcium on quantal unit current and release at the crayfish neuromuscular junction, Pfliigers Arch. ges. PhysioL, 391 (1981) 35-40. 9 Kawagoe, R., Onodera, K. and Takeuchi, A., Release of glutamate from the crayfish neuromuscular junction, J. Physiol. (Lond.), 312 (1981) 225-236. 10 Kehoe, J. S., Transformation by concanavalin A of the response of molluscan neurones to L-glutamate, Nature, (Lond.), 274 (1978) 866-869. 11 Lehouelleur, J., Electrical properties of phasic and tonic muscle fibers of the abdominal flexor muscles in crayfish, J. Physiol. (Paris), 74 (1978) 675-686.
12 Mathers, D. A., The influence of concanavalin A on glutamate-induced current fluctuations in locust muscle fibres, J. PhysioL (Lond.), 312 (1981) 1-8. 13 Mathers, D. A. and Usherwood, P. N. R., Concanavalin A blocks desensitisation of glutamate receptors on insect muscle fibres Nature (Lond.), 259 (1976) 409-411. 14 Miledi, R., Intracellular calcium and desensitization of acetylcholine receptors, Proc. roy. Soc. B, 209 (1980) 447-452. 15 Nickell, W. T. and Boyarsky, L. L., The effects of calcium at the crayfish neuromuscular junction, Comp. Biochem. Physiol., 66 A (1980) 259-264. 16 Onodera, K. and Takeuchi, A. Ionic mechanism of the excitatory synaptic membrane of the crayfish neuromuscular junction, J. Physiol. (Lond.), 252 (1975) 295-318. 17 Onodera, K. and Takeuchi, A., Permability changes produced by L-glutamate at the excitatory post-synaptic membrane of the crayfish muscle, J. PhysioL (Lond.), 255 (1975) 669-685. 18 Selverston, A. 1. and Remler, M. P., Neural geometry and activation of crayfish fast flexor motoneurons, J. Neurophysiol., 35 (1972) 797-814. 19 Shinozaki, H. and Ishida, M. Prevention of desensitization of the glutamate receptor on the crayfish opener muscle by concanavalin A, J. Pharm. Dyn., 1 (1978) 246-250. 20 Thieffry, M. and Bruner, J., Sensibilitd des fibres musculaires d'Ecrevisse au glutamate et au mddiateur naturel dans des solutions pauvres en calcium, C. R. Acad. Sei. (Paris), 286 D (1978) 1813-1816. 21 Thieffry, M., Bruner, J. and Personne, B., The presynaptic action of L-glutamate at the crayfish neuromuscular junction: results obtained by intraaxonal recordings and nerve terminal damage, J. Physiol. (Paris), 75 (1979) 635-639.
165
Elsevier Biomedical Press
Concanavalin A blocks the Ca'+-dependence of crayfish muscle fiber responses to glutamate M. THIEFFRY Laboratoire de Neurobiologie Cellulaire du C.N.R.S. 91190 Gif-sur- Yvette (France)
(Accepted March llth, 1982) Key words: glutamate - - receptors - - calcium - - lanthanum - - concanavalin A - - desensitization
(1) The response of crayfish muscle fibers to bath-applied glutamate is strongly inhibited when the Ca concentration of the physiological solution is reduced. Other divalent cations cannot substitute for Ca. The trivalent impermeant cation La can at low concentration replace Ca. Moreover, decreasing the Ca concentration in the presence of La potentiates the glutamate response. (2) The time course of responses to ionophoretically applied glutamate suggests a faster desensitization in low Ca solutions. The lectin concanavalin A, which blocks desensitization, also eliminates the decrease of the glutamate response in low Ca solutions. (3)The above results are compared to available data concerning Ca-dependence, desensitization and effects of concanavalin A. Glutamate, the putative excitatory transmitter 9, triggers in crustacean muscle fibers an inward current carried mainly by N a + ions 6,16,17. A smaller but unknown part of the response is possibly due to a Ca 2+ inflUX1,6,9,16,17, but the size of the glutamateinduced calcium conductance is much too small to explain the marked decrease in the glutamate response that is observed when external calcium concentration is lowered1,2,15,17,20. This dependence on calcium remains even when calcium ions are replaced by other divalentsl, 15,17, which in fact block, rather than 'protect', the glutamate response1, ~. The results presented below show that, on the other hand, the impermeant cation, La 8+, which at concentrations of 1-20 m M also antagonizes the glutamate effect4, zl, can, at the concentration of 0.5 mM, substitute for Ca 2+, and that the lectin concartavalin A (Con A) irreversibly eliminates the decrease of the glutamate response observed in low Ca 2+ solutions. Experiments were carried out on the posterior fast abdominal flexor is of the crayfish, Procambarus clarkii, a preparation which shows no rectification over a w;de range of potential 11. The preparation was continuously superfused at 15 °C w_;th saline of composition 195 m M NaCI, 5.4 m M KCI, 13 m M CaC12, 2.8 m M MgCI2 buffered at p H 7.2 by 10 m M 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
Tris-maleate-NaOH. Solution containing lower concentrations of Ca 2+ were obtained by mixing the standard saline with a solution in which Ca z+ was isotonically replaced by N a +. Con A (Sigma, grade IV or IBF), N a glutamate and LaC13 (Merck) were added to the perfusion solution. Ionophoretic applications of glutamate were made through 150-300 M [~ pipettes filled with a 0.1 M solution at p H 8 and Ca 2+ was applied through low resistance pipettes filled with 1 M CaC19.. In both cases, constant current generators 5 were used. Bath application of glutamate induces a depolarization of the muscle fibers which, for high doses of agonist, rapidly fades due to desensitization (Fig. 1, 1A). When the external Ca ~+ concentration was reduced to 1.3 mM, the response was strongly decreased (Fig. 1, 1B) but recovered after returning to standard solution (Fig. 1, 1C). As in other crayfish preparations 15,17, Ca ~+ substitution by equimolar concentrations of Mg 2+, Sr z+, Ba z+ did not prevent the decrease of the response in low Ca 2+. When the same dose of glutamate was applied in standard saline to which 0.5 m M La 3+ had been added, the glutamate response was potentiated (Fig. 1, 2A). In addition, reducing external Ca 2+ to 1.3 m M in the presence of 0.5 m M La a+ not only prevented the
166
A
1o~iv mln :',~
C
Fig. 1. Depression of the glutamate response in low Ca 2+ is overcome by addition of 0.5 mM La 3+. Left: intracellularly recorded responses to 0.5 mM bath applied glutamate (bar) in solutions containing 13 m M Ca ~+ (A), 1.3 mM Ca 2+ (B) and after return to 13 m M Ca 2+ (C). Right: in the presence of 0.5 m M La 3+, responses to the same application in the presence of 13 mM Ca z+ (A) and 1.3 m M Ca 2+ (B). C: after return to control conditions. Input resistance monitored by an hyperpolarizing current pulse applied every 10 s.
reduction of the glutamate response, but induced a further potentiation (Fig. 1, 2B). After washing with ncrmal saline, these effects were reversed (Figs. 1, 2C). At the concentration of 0.5 mM, Sr ~÷, Ba ~+, Mn 2+ and Co 2+ were not capable of countering the antagonistic effect of low Ca ~+. A mcre detailed examination of the response in normal and low Ca 2+ was carried out using ionophoretically applied glutamate. The external Ca 2-concentration was varied either by changing the superfusion solution or by local Ca 2+ icnophoresis from a second pipette in a low Ca 2+ solution. Both techniques gave similar results; the latter was used in the experiment shown in Fig. 2. When compared to responses obtained in the presence of higher Ca z+ concentrations, the glutamate response in low Ca ~+ is characterized by a shorter time to peak and a faster decline whereas the initial slope is not modified. Since the rate of rise of the response is unaffected by the change in calcium concentration it appears that only the activated receptor-channel complex is modified by Ca 2+. Furthermore, it seems clear that neither of the cations Ca z+ and La ~+ is acting from the inside of the membrane, since lanthanum is impermeant and the action of exter-
Fig. 2. External Ca z+ concentration modifies the time course of the glutamate response. Intracellularly recorded responses to glutamate applied by 1 s, 30 nA current pulse (bar). The perfusion solution contained 2.6 mM Ca 2+, one-fifth the normal concentration. The local Ca ~+ concentration was increased by iontophoresis of Ca 2+ from a second pipette. The Ca 2+ pulse was applied 2 s before glutamate application and maintained throughout the record. The values 0, 100 and 200 nA refer to the intensity of the Ca iontophoresis current. Glutamate was applied at some distance from the sensitive spot to minimize possible artefacts due to contractions.
nally applied calcium is very rapid (acting within 1 s from the onset of ionophoresis of the cation). The means by which calcium and lanthanum keep the activated receptor-channel complex functional remain, however, obscure. They might act by permitting greater repetitive binding, or by protecting the activated receptor-channel complex from being blocked. Whatever the hypothesis proposed for explaining their actions, it must also account for the finding that low calcium solutions affect spontaneous miniature potentials in the same way s as they have been shown here to affect responses to longduration ionophoretic applications of glutamate. The fact that calcium affects both of these membrane events in the same way makes it difficult to hypothesize that it is acting by interfering with the desensitization process. Likewise, previous studies of the glutamate response of crayfish muscle in normal and low Ca 2+ solutions did not permit the detection of a role of Ca 2÷ ions in the desensitization process15, 20. Furthermore, in studies in which calcium has been shown to play a role in desensitization (e.g. in the ACh response of frog muscle), it was found that the higher the calcium concentration, the greater the desensitization (with calcium presumably acting intracellularly) 14. In spite of these earlier data, which make it difficult to explain the calcium effects observed here in
167 terms of desensitization, the fact remains that 'apparent desensitization' is faster in low Ca 2÷. Consequently, it seemed worthwhile evaluating the effects of calcium concentration on the glutamate response in preparations in which desensitization had been blocked by exposure of the muscle to the lectin, concanavalin A 1~,19. The experimental procedure consisted of: (1) recording the responses to glutamate in both normal and low Ca z+ solutions; (2) exposing the muscle for 10-30 min to Con A (100--500/~g/ml); (3) soaking the muscle for 10-15 min in lectin-free, normal calcium solutions: and (4) testing the response to the same glutamate application in low and high calcium. As can be seen in Fig. 3, the calcium dependence of the glutamate response is irreversibly eliminated by exposure of the muscle to Con A. That is to say, in a preparation in which the glutamate response does not desensitize, the removal of calcium no longer causes a diminution of the glutamate response. In fact, after a correction is made for changes in input resistance, the ~esponse of a Con A-treated muscle in low Ca 2+ appears even slightly larger than that in normal saline. The response of a Con A-treated preparation is still antagonized by divalent cations (50 ~ reduction by 10 mM
13 mM Ca
,
l!l'flfl ~i":llrr~rrrTT /~[I
1.3 mM Ca 5 mV -
~r~
ii I rll
4 ! i l l ; ' MT
i]]l
T]]]I2 H]F ,'
,_
,
,t!IHUTIkHTIIT]T
Fig. 3. Con A blocks the Ca 2+ dependence of the glutamate response, lntracellularly recorded responses to 0.1 mM bath applied glutamate (bar). Top: control responses in 13 m M Ca 2+ (left) and 1.3 m M Ca 2+ (right) before treatment by ConA. Bottom: after 20 rain treatment by 400 #g/ml Con A and 10 min washing, the responses to the same glutamate application were again recorded in 13 mM Ca ~+ (left) and 1.3 mM Ca z+ (right). Input resistance monitored every 10 s by an hyperpolarizing 200 nA current pulse.
Co2+). Likewise, the effect of lanthanum (0.5 mM) is reversed: it now causes a reduction (of about 15-207/o) in the glutamate response. Finally, it should be noted that after Con A treatment, the repolarization of the membrane during the wash of the agonist is noticeably slower in low Ca 2+ solutions than in normal ones (see Fig. 3). As was shown above, calcium, in this preparation, does not change the rise time of the response, but acts to slow down the decay of the response. One could consider that Con A acts like a more effective and irreversible calcium-type agent and, once in place, pre-empts any similar role for calcium ions. Another interpretation of the disappearance of the calcium dependence of the glutamate response after Con A treatment might be that Con A modifies the ionic conductances triggered by glutamate, eliminating the participation of calcium ions while increasing that of others. Con A does appear to alter the glutamate-induced permability changes in molluscan neurones10 and, llke the 'Ca 2+ blocker' D-600 at frog endplates 3, it has been shown to eliminate the voltage dependence of channel life-time in arthropod muscle 7,1~. A careful analysis of the effects of varying calcium concentrations as a function of agonist dose and membrane voltage should give some insight concerning the means by which calcium acts in the normal preparation to maintain the glutamate response. On the other hand, a comparison of the ionic conductances triggered by glutamate in a Con A-treated fiber with those underlying the glutamate response in a normal fiber might help clarify the mechanism by which Con A eliminates desensitization and the calcium dependence of the glutamate response. If and when these data are explained by a unifying hypothesis, our understanding of the desensitization process in this preparation should be enhanced. This work was supported in part by Grant 387670 from the Institut National de la Sant6 et de la Recherche Mddicale. I am very indebted to J. Kehoe for discussing the above results and for her considerable help in the preparation of the manuscript.
168 1 Ashley, C. C. and Campbell, A. K., Free-calcium and tension responses in single barnacle muscle fibers following the application of L-glutamate, Biochim. biophys. Acta, 512 (1978) 429-534. 2 Barker, J. L., Divalent cations: effects on post-synaptic pharmacology of invertebrate synapses, Brain Research, 92 (1975) 307-323. 3 Bregestovski, P. D., Miledi, R. and Parker, I., Blocking of frog endplate channels by the organic calcium antagonist D 600, Proc. roy. Soc. B, 211 (1980) 15-24. 4 Colton, C. K. and Freeman, A. R., La 3+ blockade of glutamate action at the lobster neuromuscular junction, Comp. Biochem. PhysioL, 51C (1975) 285-289. 5 Dreyer, F. and Peper, K., Iontophoretic application of acetylcholine: advantages of high resistance micropipettes in connection with an electronic current pump, Pfliigers Arch. ges. Physiol., 348 (1974) 263-272. 6 Dudel, J., Nonlinear voltage dependence of excitatory synaptic current in crayfish muscle, Pfliigers Arch. ges. Physiol., 352 (1974) 227-241. 7 Dudel, J., The voltage dependence of the decay of the excitatory postsynaptic current and the effect of concanavalin A at the crayfish neuromuscular junction, J. Physiol. (Paris), 75 (1979) 601-604. 8 Dudel, J., The effect of reduced calcium on quantal unit current and release at the crayfish neuromuscular junction, Pfliigers Arch. ges. PhysioL, 391 (1981) 35-40. 9 Kawagoe, R., Onodera, K. and Takeuchi, A., Release of glutamate from the crayfish neuromuscular junction, J. Physiol. (Lond.), 312 (1981) 225-236. 10 Kehoe, J. S., Transformation by concanavalin A of the response of molluscan neurones to L-glutamate, Nature, (Lond.), 274 (1978) 866-869. 11 Lehouelleur, J., Electrical properties of phasic and tonic muscle fibers of the abdominal flexor muscles in crayfish, J. Physiol. (Paris), 74 (1978) 675-686.
12 Mathers, D. A., The influence of concanavalin A on glutamate-induced current fluctuations in locust muscle fibres, J. PhysioL (Lond.), 312 (1981) 1-8. 13 Mathers, D. A. and Usherwood, P. N. R., Concanavalin A blocks desensitisation of glutamate receptors on insect muscle fibres Nature (Lond.), 259 (1976) 409-411. 14 Miledi, R., Intracellular calcium and desensitization of acetylcholine receptors, Proc. roy. Soc. B, 209 (1980) 447-452. 15 Nickell, W. T. and Boyarsky, L. L., The effects of calcium at the crayfish neuromuscular junction, Comp. Biochem. Physiol., 66 A (1980) 259-264. 16 Onodera, K. and Takeuchi, A. Ionic mechanism of the excitatory synaptic membrane of the crayfish neuromuscular junction, J. Physiol. (Lond.), 252 (1975) 295-318. 17 Onodera, K. and Takeuchi, A., Permability changes produced by L-glutamate at the excitatory post-synaptic membrane of the crayfish muscle, J. PhysioL (Lond.), 255 (1975) 669-685. 18 Selverston, A. 1. and Remler, M. P., Neural geometry and activation of crayfish fast flexor motoneurons, J. Neurophysiol., 35 (1972) 797-814. 19 Shinozaki, H. and Ishida, M. Prevention of desensitization of the glutamate receptor on the crayfish opener muscle by concanavalin A, J. Pharm. Dyn., 1 (1978) 246-250. 20 Thieffry, M. and Bruner, J., Sensibilitd des fibres musculaires d'Ecrevisse au glutamate et au mddiateur naturel dans des solutions pauvres en calcium, C. R. Acad. Sei. (Paris), 286 D (1978) 1813-1816. 21 Thieffry, M., Bruner, J. and Personne, B., The presynaptic action of L-glutamate at the crayfish neuromuscular junction: results obtained by intraaxonal recordings and nerve terminal damage, J. Physiol. (Paris), 75 (1979) 635-639.