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Block of locust muscle glutamate receptors by δ-philathotoxin occurs after receptor activations
Brain Research, 241 (1982) 105-114
105
Elsevier Biomedical Press
Block of Locust Muscle Glutamate Receptors by -Philanthotoxin Occurs After Receptor Activations R. B. CLARK*, P. L. DONALDSON**, K. A. F. GRATION, J. J. LAMBERT, T. PIEK, R. RAMSEY, W. SPANJER and P. N. R. USHERWOOD§
Department of Zoology, University of Nottingham, University Park, Nottingham NG7 2RD (U.K.) and (T.P. and W.S.) Pharmacological Laboratory, University of Amsterdam, Polderweg104, 1093 KP, Amsterdam (The Netherlands) (Accepted October 29th, 1981)
Key words: wasp venom - - glutamate receptors - - glutamate potential - - locust muscle - - glutamate channels - neuromuscular junctions
One component (6-philanthotoxin (6-PTX)) of the venom from the wasp Philanthus triangulum blocks transmissionpostsynaptically at excitatory synapses on locust muscle. 6-PTX depresses both the iontophoretic glutamate potential and the excitatory junctional current (e.j.c.) in a glutamate receptor activation-dependentmanner. The rate of recovery from the effects of the toxin is reduced following either prolonged application of L-glutamate or repetitive iontophoretic application of this amino acid or high frequency neural stimulation of the muscle in the presence of 6-PTX. The decay phase of the e.j.c, is shortened by 6-PTX. The effects of 6-PTX on the e.j.c, are not voltage dependent. The open-close kinetics of glutamate channels in extrajunctional muscle membrane are modified by 6-PTX as shown by patch clamp analysis. The mean life time of the glutamate channel is reduced, whilst the mean interval between single opening events is increased with the events often occurring in bursts. These data are consistent with glutamate channel blocking by this toxin. It is proposed that the toxin blocks open channels gated by both junctional and extrajunctional glutamate receptors on locust muscle. It is further proposed that 6-PTX enters a compartment of the muscle through the glutamate open channels and that it can also block the open channels from this site. INTRODUCTION Studies of glutamate receptors o n excitable cells has suffered from the absence of p o t e n t a n d p r o v e n antagonists 2°, which makes the c o n t i n u i n g search for such c o m p o u n d s i m p o r t a n t to those seeking further insight into the role of these receptors o n excitable cells of vertebrates a n d invertebrates. F o r this reason we have investigated the properties of a toxin fraction, O-philanthotoxin ( O - P T X ) i s o l a t e d from the v e n o m o f the digger wasp Philanthus triangulum, which at first sight seemed to possess some of the characteristics o f a n a n t a g o n i s t at locust glutamatergic n e r v e - m u s c l e junctions12,13.
The sting o f the female digger wasp is used to paralyze honeybees, which the wasp t h e n carries into b r o o d cells as food for its larvae 18. The v e n o m from the sting apparatus also affects other insects where it exerts a variety of effects o n peripheral a n d central nervous systems. Pick a n d his co-workers have shown that the crude v e n o m blocks transmission at excitatory a n d i n h i b i t o r y synapses o n locust leg muscle p r o b a b l y by inhibiting t r a n s m i t t e r release at these sites 14. Recently the v e n o m has been separated into a n u m b e r of active c o m p o n e n t s , one of which (O-PTX) blocks t r a n s m i s s i o n postsynaptically at excitatory synapses o n locust leg muscle 16,~7. I n this paper we report o n the effects of O-PTX
* Present address: Department of Physiology and Biophysics, The University of Texas Medical Branch, Galveston, TX 77550, U.S.A. ** Present address: Department of Physiology and Biophysics, University of Iowa, Basic Science Building, Iowa City, IA, 52242, U.S.A. § To whom correspondence should be addressed at: Department of Zoology, University of Nottingham, Nottingham NG7 2RD, U.K. 0006-8993/82/0000-0000/$92.75 O Elsevier Biomedical Press
106 on nerve-muscle preparations of locust which have been studied using a variety of techniques, including patch clamp analysis of the activity of single receptor-ionophore complexes in extrajunctional muscle membrane. We shall confirm the conclusion of May and Pick s that 6-PTX is not a glutamate receptor antagonist, and show that it blocks glutamate gated ionophores in the postsynaptic membrane of locust excitatory nerve-muscle junctions and also the ion channels gated by glutamate receptors on extrajunctional membrane of locust muscle. METHODS AND MATERIALS Metathoracic extensor tibiae muscles 2 of the locusts Schistocerea gregaria and Locusta migratoria were mainly used in these studies. The preparations were dissected in a small Perspex bath with a replaceable volume of 2 ml through which saline flowed at a rate of 5-10 ml/min. The saline contained (mM): NaC1, 180; KCI, 10; CaCI~, 2; Na2HPO4, 6; NaH2PO4, 4 (pH 6.8). L-Glutamate was applied iontophoretically to single nerve-muscle junctions through micropipettes containing 0.1 M sodium glutamate dissolved in distilled water (pipette resistance, ca. 100 M ~ ) and with constant braking currents of 2-5 nA. Excitatory junctions were located by delivering pulses of glutamate of about 2 nC onto the muscle fibre membrane. The appearance of transient depolarizations (glutamate potentials) of rapid rise time (10-50 ms) signalled the presence of a junction. Junctional glutamate potentials were readily distinguished from extrajunctional responses by their more rapid time course 3. Junctional depolarizations were recorded using conventional intracellular techniques. In some cases the muscle fibre was voltage-clamped in the region of the nerve-muscle junction using a two-electrode three point clamp and the membrane currents resulting from glutamate iontophoresis were recorded 1. Excitatory junctional currents (e.j.c.'s) resulting from nerve stimulation were measured intracellularly under voltage-clamp from fibres in 3 distal bundles of the extensor tibiae muscle, i.e. those with length constant/length ratios > 1. Extracellular excitatory junctional potentials (e.j.p.'s) were recorded differentially between an 'active' electrode located at a single nerve-muscle junction and a
'reference' electrode less than 100/~m away. Single active sites were located by trial and error during repetitive stimulation of the excitatory innervation of the muscle preparation at a frequency of 1 Hz ag. Since insect muscle fibres are muttiterminally innervated the extracellular e.j.p., unlike the e.j.c, recorded under voltage-clamp, monitors the activity of a single site on the muscle fibre. Its time course is more or less identical to that of the e.j.c, so it can be used to estimate, through their effects on decay of the e.j.p., the influence of drugs on the life-times of channels in the postjunctional membrane of superficial nerve-muscle junctions where problems of drug accessibility are minimized. The fact that recordings of extracellular e.j.p.'s are limited to single superficial junctions validates comparison between data obtained in this way and those obtained using drug iontophoresis, e.j.c.'s and extracellular e.j.p.'s were recorded in saline containing 140 mM NaC1 and 40-50 mM MgCI2. This saline reduced the amplitude of the excitatory junctional potential and thereby prevented muscle contraction without completely blocking nerve-muscle transmission 16. Currents through single glutamate-activated ion channels in the extrajunctional membrane of locust muscle fibres were recorded using the 'patch clamp' technique of Neher et al. 4,5,9,11. Muscle preparations treated with 10-6 M concanavalin A to block glutamate receptor desensitization were used in these studiesa,L Our studies of single channel activity were restricted to those sites at which only a single receptor was active, i.e. there was no indication of overlapping events. Further details of the application of the patch clamp technique to locust muscle preparations are contained in recent publications from one of our laboratories 4,5. Crude extracts of venom were obtained from abdomens of female wasps (Philanthus triangulum) and separated into a series of fractions using Sephadex chromatography and freeze-drying. The &PTX fraction was dissolved in locust saline prior to use. Since the molecular identity of d-PTX is unknown, quantities of the toxin are expressed in arbitrary 'units' (U). (See refs. 16 and 17 for a description of the separation procedure and for a quantitative definition of 'units'.)
107
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Fig. 1. Effect of 6-PTX on L-glutamate potentials evoked by glutamate iontophoresis at excitatory neuromuscular junctions of locust extensor tibiae muscle. A: i, saline containing 0.1 U/ml of 6-PTX was introduced (~) during repetitive applications of 1.5 nC iontophoric glutamate doses. Note the reduction in response amplitude. After 1.5 min exposure to 6-PTX, toxin-free saline was introduced (~); ii, following 10 rain wash with toxin-free saline, the response was restored to control amplitude. Saline containing 0.1 U/ml was reintroduced, but iontophoretic application of glutamate was discontinued for 1 min and then resumed. The amplitude of the first response at the end of the 1 min period was nearly identical to the control amplitude (cf. i). Note the decline in amplitude of response to subsequent glutamate doses. Measurements were made at the resting potential of the muscle fibre, - - 6 0 mV. Calibration bars: potential (vertical), 4 mV; times (horizontal), 20 s. B : recovery from effects of 6-PTX iontophoretic studies. i, pulses (1 nC) of L-glutamate were applied to an excitatory nerve-muscle junction at a constant frequency of 0.74 Hz. During bath application of 0.05 U/ml of 6-PTX (between arrows) the glutamate potentials were depressed and they recovered only slowly after removal of the toxin; ii, recording of glutamate potentials at same junction as in i above. 0-PTX (0.05 U/ml) was applied for the period between arrows. In this experiment application of the pulses (1 nC, 0.74 Hz) of L-glutamate was interrupted for periods of 6, 12, 24 and 40 s. Note the slight recovery of response amplitude following these brief rest periods. Calibration bars: voltage (vertical), 5 mV; time (horizontal), 20 s.
RESULTS
Effect of ~3-PTX on responses to glutamate iontophoresis Passage of a brief (ca. 10 ms) pulse of current through a glutamate-filled micropipette with its tip placed close to a nerve-muscle junction resulted in a transient depolarization of the muscle membrane. The amplitude of the 'glutamate' depolarization declined when 6-PTX was included in the bathing saline (Figs. 1 and 2). When 0.1 U/ml of 6-PTX was introduced the glutamate response was reduced to about 15 ~ of its control amplitude within 50 s. After 10 min wash in toxin-free saline the response was restored to its control amplitude. Next, saline containing 0.1 U/ml of 0-PTX was re-introduced, but the iontophoretic pulses were discontinued until the muscle had been exposed to 6-PTX for 60 s. The amplitude of the first glutamate response after resuming the iontophoretic pulses was almost identical to the control response, in contrast to the small responses which resulted during repetitive application of L-glutamate in the presence of O-PTX.
Subsequent responses declined rapidly, and after 10 pulses the response amplitude was about 15 ~ of the control. The response amplitude declined more rapidly when the muscle was pre-exposed to 6-PTX saline before glutamate pulses were applied than when doses were applied continuously during initial introduction of the O-PTX saline. It is possible that this difference reflects the time taken for the concentration of 6-PTX in the region of the superficial nerve-muscle junction to equilibrate with the bulk concentration in the saline. For this reason 6-PTX was usually applied to the muscle preparation for at least 60 s before glutamate pulses were applied. The rate of decline of response amplitude in the presence of O-PTX was dependent on the concentration of ~-PTX in the saline (Fig. 2) and, for a fixed pulse current, on the frequency at which glutamate pulses were applied iontophoretically to the junctional site. The amplitudes of successive responses in a train declined in an approximately exponential manner to a constant 'steady-state' level. Examples of this exponential decay of pulse train amplitude for two different concentrations of O-PTX are shown
108 in Fig. 2B. It is clear that the rate o f decline of amplitude increases with an increase in the concentration o f &PTX. The data o f Figs. 1 and 2 suggest that the action of d-PTX arises f r o m 'use-dependent' block induced by &PTX. N o t e that in the lower trace in Fig. 2A, unlike the upper 3 traces, the amplitude o f the first glutamate response in the d-PTX saline was smaller than the control amplitude. This m a y indicate that & P T X blocked a small p r o p o r t i o n o f the postsynaptic glutamate 'receptor' population prior to the first application o f glutamate either t h r o u g h antagonism at the receptor level or by blocking closed glutamate-gated m e m b r a n e channels. A more likely explanation is that there was a small steady leakage of glutamate f r o m the tip o f the iontophoretic pipette and/or f r o m nerve terminal during spontaneous transmitter release, and that this resulted in a small,
undetectable activation (and consequent block by OPTX) of a p r o p o r t i o n o f the receptors in the absence of glutamate ejection current pulses. It is unlikely that 6-PTX caused or enhanced desensitization o f the postjunctional glutamate receptors because o f the unpredictable reduction o f the first response in & P T X and also because similar data were obtained in the presence o f 10 -6 M concanavalin A, a lectin which eliminates desensitization o f glutamate receptors at locust excitatory nerve-muscle junctions6, 7. We cannot, o f course, exclude the possibility that the position o f the iontophoretic electrode vis-/t-vis that o f the junctional site sometimes changed during an experiment and, thereby, influenced the amplitude o f the glutamate potential. The decrease in amplitude o f the glutamate response did not result f r o m changes in the passive electrical properties o f the muscle fibre membrane,
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Fig. 2. A: effect of concentration of d-PTX on the amplitude of iontophoretically evoked glutamate potentials recorded from a nerve-muscle junction of locust extensor tibiae muscle fibre. Pulses of glutamate (1 nC) were applied at a frequency of 0.56 Hz. Glutamate pulses were terminated immediately prior to introduction of toxin saline (~) and recommenced 60 s later; the muscle was exposed to 6-PTX in the saline as shown at the left (U/ml). Note that the rate of decline of glutamate potential amplitude increased as the concentration of 6-PTX was increased. All recordings were made at the same nerve-muscle junction. The muscle was washed in toxin-free saline for 5-10 min after removal of 6-PTX (~,) to restore the glutamate potential to the control amplitude. The recordings were made at the resting potential of the fibre, --58 inV. Calibration bars: voltage (vertical), 10 mV; time (horizontal), 20 s. B: exponential decay of response amplitude in two of the pulse trains shown in A (0.025 U/ml and 0.1 U/ml 6-PTX). The 'steady-state' response ratio (r~o)has been subtracted from each response (rd and the difference has been normalized to the difference for the first pulse in the train frO. For the two examples shown roo = 0.54 for 0.025 U/ml 6-PTX and 0.19 for 0.1 U/ml 6-PTX. The linear relationship between ri-r~ (on log scale) and pulse number indicates an exponential decline in response amplitudes. For [6-PTX] = 0.025 U/ml, the slope of the solid line is 0.063 pulse -z, and for 0.1 U/ml the slope is 0.31 pulse--L
109 as d-PTX caused a similar decline in the amplitude of voltage-clamp currents resulting from glutamate iontophoresis (see below). The effective input conductance of muscle fibres in the presence of 0.1 U/ml of &PTX did not differ significantly from the input conductance in toxin-free saline (input conductance in d-PTX saline ~- 1.02 4- 0.05 times conductance in toxin-free saline - - 3 different fibres). Changes in the resting membrane potential ot muscle fibres following exposure to saline containing 0.1 U/ml of &PTX were not significant (i.e. < 1 mV). d-PTX at 0.05 U/ml produced a decrease in the half-decay time of the glutamate response ranging from 13 to 29 (mean 18 ~ , n = 3 sites). There was also a small ( < 5 ~ ) decrease in the time to peak of the response. Qualitatively similar changes in time course were noted for glutamate depolarizations of unclamped fibres 15. The postsynaptic block produced by &PTX persisted for many seconds when glutamate was applied in the presence of c3-PTX. This is illustrated by the records in Fig. lB. In this experiment the glutamate pulses were applied to a toxin-treated muscle until the response was reduced to approximately 75 ~ of its control amplitude. The iontophoretic application of glutamate was interrupted for various time periods during d-PTX application, then single pulses of this amino acid were applied. The amplitude of the glutamate response increased slightly under these conditions (but it did not recover to its control amplitude even when the time between the single pulses of glutamate was almost 40 s). The action of &PTX was also investigated by microperfusing 0.2 U/ml of &PTX onto a junctional area whilst applying constant iontophoretic pulses of L-glutamate at a frequency of 0.25 Hz. After 1 min of d-PTX application, the amplitude of the glutamate-induced depolarization was reduced to 15.8 45.6 ~ (mean 4- S.D., n = 5 from 3 preparations) of control. When the microperfusion pipette was removed, the amplitude of the glutamate induced depolarizations recovered exponentially with a time constant of 57.7 4- 39.5 s (mean 4- S.D.; n = 3).
Effect of &PTX on excitatory junctional currents The amplitude and time course of the e.j.c, were influenced by O-PTX in a manner dependent upon the concentration of the toxin applied in the bathing
medium, the duration of exposure of the preparation to the toxin and the frequency of stimulation during its application. &PTX at O. 1 U/ml failed to depress the e.j.c, even when the stimulation frequency was as high as 3 Hz. However, there was a ca. 6 0 ~ reduction in e.j.c, amplitude after 5 min exposure to 1 U/ml with a stimulation frequency of 1.9 Hz, together with a shortening of the decay time of this event. The shortening of the decay time, which was independent of membrane potential (range --60 mV to --120 mV), was evident with the first e.j.c, in the series, when the amplitude was unaffected. This was due mainly to a shortening of the pre-exponential phase of the e.j.c, decay (Fig. 3A). In both control and &PTX-treated preparations e.j.c, decay was biphasic, consisting of an exponential phase preceded by a non-exponential phase. The effect of 6PTX on the amplitude of the e.j.c, was also independent of membrane potential (Fig. 3B) and the extrapolated reversal potential for the e.j.c, was unaffected by the toxin (Fig. 3B, C). The extracellular e.j.p, was more like the glutamate potential and current in terms of its susceptibility to &PTX, a significant reduction in amplitude being recorded with 0.! U/ml of toxin at a stimulation frequency of 3 Hz. However, when the stimulus frequency was reduced below 1 Hz this concentration of venom failed to depress the e.j.p. Possible presynaptic action of &PTX was investigated by recording the number of e.j.p, failures to continuous (3 Hz) stimulation in the presence and absence of toxin. These studies, which were undertaken on the locust retractor unguis muscle, demonstrated that 10-15 min exposure of this muscle to 6PTX (0.3-0.8 U/ml) had no effect on evoked transmitter release. It remains to be established whether higher concentrations or longer applications of OPTX are equally ineffective presynaptically.
Currents through single glutamate-activated channels
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Currents through single ion channels associated with L-glutamate receptors in extrajunctional (nonsynaptic) membrane of locust muscle fibres were measured using the 'patch clamp' technique. Single channels were recorded using patch electrodes containing L-glutamate (10 -4 M and 5 × 10-4 M 4- OPTX) dissolved in locust saline. Fresh pipettes were
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Fig. 3. A: effect of d-PTX (1 U/ml) on the e.j.c, recorded from locust extensor tibiae muscle fibre under voltage damp. e.j.c.'s were evoked by stimulation of the extensor tibiae nerve at a frequency of 3 Hz. Records i-iii are averages of 20 consecutive e.j.c.'s. Record i was obtained in the absence of 6-PTX. The preparation was then rested for 5 rain during which time it was 'equilibrated' in saline containing 1 U/ml 6-PTX. Record ii is the averaged e.j.c, obtained immediately after the 5 min rest period, whereas record iii is the averaged e.j.c, obtained after 1 min continuous stimulation at 3 Hz in &PTX. Note that increase in rate of decay of e.j.c. precedes the fall in amplitude of response. Clamp potential, --60 mV; temperature, ca. 20 °C. B: relationship between peak amplitude of e.j.c, and membrane potential (Em) in presence (0) and absence (O) of 1 U/ml 6-PTX. The preparation was stimulated continuouslyat 3 Hz. Lines drawn by eye. C: relationship between peak amplitude of e.j.c, and membrane potential in presence (0) and absence ([~) of 1 U/ml 6-PTX with amplitudes normalized at --100 mV to demonstrate lack of voltage sensitivity of 6-PTX action. (O, before toxin; [~, after toxin).
used for each c o n c e n t r a t i o n of g l u t a m a t e a n d for recordings in presence a n d absence of & P T X . The a m p l i t u d e o f currents t h r o u g h the i o n channels (and c h a n n e l conductance) was u n c h a n g e d by & P T X , b u t the open-close kinetics o f the channels were modified. This is illustrated in Fig. 4 which shows single c h a n n e l currents recorded in the presence of 10 -4 M or 5 x 10 -4 M L-glutamate ! & P T X . The same electrode was used to record channels in the absence a n d presence o f d - P T X by exchanging the contents o f the electrode. However, because it was necessary to remove the patch electrode from the recording system to change its contents, recordings in the presence a n d absence of & P T X were presum a b l y made at different, b u t nevertheless closely adjacent sites, o n the m e m b r a n e . I n the absence of O-PTX, 10 .4 M glutamate in the ' p a t c h ' electrode i n d u c e d single c h a n n e l activity at a m e a n frequency o f 14 c h a n n e l openings/s. W h e n 0.2 U / m l of d - P T X was included with 10 -4 M glutamate in the same p a t c h electrode the m e a n frequency of c h a n n e l openings was reduced to less t h a n 3 s -1 (Fig. 5).
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111
6pAl looms Fig. 5. Long-term recording of single channel activity obtained in the presence of 5 × 10 4 M glutamate, plus 1.0 U/ml 6-PTX. Note the appearance of bursts of channel activity and the occurrence of single channel currents separated by long closed intervals. Muscle membrane potential, --55 mV; Temperature, ca. 20 °C. These and other patch data were recorded at a bandwidth of 0-1 KHz. Many of the small events resulted from activity of 'rin' channels9. The activity of single channels in the presence of 6-PTX sometimes consisted of clearly defined 'bursts' of channel events separated by quiet intervals > 100 ms (Figs. 4 and 6), the duration of these 'bursts' varying between 12 and > 100 ms. At other sites 'bursts' were less evident. Possibly the variations in channel activity achieved with & P T X reflect the non-random behaviour of glutamate channels seen in the control (i.e. in absence of 6PTX) conditions 4. Although & P T X resulted in a significant (P < 0.001 Student's t-test) increase in the mean channel closed time (calculated by averaging the closed intervals between a// successive opening events, including the quiet intervals between bursts), there was no significant difference between the mean closed time within bursts and that recorded in the absence of c3-PTX. This suggests that, in contrast to the action of QX 222 on ACh channels, for example, which causes the channel to fluctuate rapidly between open and blocked states 10, the block by & P T X is relatively long lasting. 6-PTX reduces the frequency of channel openings by blocking channels either in their open or closed
conformations rather than by reducing the probability of channel opening. Other data presented in this paper suggest that it is the open conformation of the glutamate channel which is blocked. To obtain reliable determinations of the channel blocking rate it is necessary to study the effects of a range of toxin concentrations at a constant glutamate concentration. Unfortunately our very limited availability of & P T X precluded this approach. However, the blocking rate G can be roughly estimated from t0 (a ÷ G.c) -1 where t0 = M0 (mean open time) in the presence of &PTX, a = l/M0 in the absence of the toxin and c = the concentration of d-PTX. For 10 -4 M L-glutamate, and 0.2 U/ml &PTX, G _ 5 × 10 -2 s -1 U -1 ml-1. F r o m the distributions of channel closed times obtained in the presence and absence of O-PTX (Fig. 6A, B) it is possible to estimate the unblocking rates for the action of toxin on the glutamate channel. In the absence of & P T X distributions of closed times can be fitted approximately with a single exponential with a time constant of ca. 40-100 ms. In the presence of venom a second larger time constant is
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present with a value at least 7 × greater t h a n this (Fig. 6C, D), suggesting t h a t the u n b l o c k i n g rate a p p r o x i m a t e s to between 1 s -1 a n d 1.5 s -1. I f & P T X is a g l u t a m a t e channel b l o c k e r it m i g h t be a n t i c i p a t e d t h a t the presence o f this t o x i n in the p a t c h electrode w o u l d cause a r e d u c t i o n in the lifetime o f the channel. This is clearly the case, as illustrated in T a b l e I. W h e n the p a t c h electrode c o n t a i n e d 10 .4 M glutamate, the single channels h a d a m e a n o p e n time o f 2.9 -t- 0.39 (S.D.) ms (n = 7 recordings each o f 200 events f r o m 7 sites on 4 p r e p a r a t i o n s ) . W h e n 0.2 U / m l d - P T X was a d d e d to the contents o f the p a t c h electrode, m e a n channel o p e n time was r e d u c e d to 2.24 ms (n = 8 recordings each o f 200 events f r o m 8 sites on 5 p r e p a r a t i o n s ) . The difference between the m e a n life-time o f the channels r e c o r d e d in the presence a n d absence o f dP T X , a l t h o u g h slight, is significant ( P < 0.01).
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Fig. 6. Comparison of the frequency distributions of glutamate channel closed times measured in the absence (A) and presence 03) of 0.2 U/ml &PTX. Glutamate concentration, 10-4M. Each distribution represents pooled data from 3 experiments. Note the wider distribution of closed times in the presence of &PTX. Muscle membrane potential, --55 mV to --60 mV; temperature, ca. 20 °C. C: data of A plotted on semi-log axes. The solid line which was fitted to the data points by the method of least squares has a time constant (T) of 43 ms. D: data of B plotted on semi-log axes. The data points could be fitted with two exponentials. The best fits were obtained with zrast = 100 ms and rslow = 758 ms. Channels with life times > 200 ms were absent from these readings.
The depression b y d - P T X o f i o n t o p h o r e t i c glutam a t e potentials (and currents) a n d p o s t s y n a p t i c currents at locust excitatory n e u r o m u s c u l a r j u n c tions is very similar to the effects p r o d u c e d at these sites b y crude v e n o m f r o m Philanthus 12-14,18. M a y a n d Piek 8 have shown t h a t Philanthus v e n o m is a n o n - c o m p e t i t i v e a n t a g o n i s t at these sites a n d have suggested t h a t it might be a p o s t j u n c t i o n a l channel blocker, a conclusion shared b y Piek et al. 1~, w h o f o u n d t h a t the v e n o m decreased the a m p l i t u d e a n d h a l f decay time o f extracellular m i n i a t u r e e.j.c.'s r e c o r d e d f r o m locust r e t r a c t o r unguis muscle. M o d i fications b y & P T X o f the kinetic p r o p e r t i e s o f single
TABLE I Effect of 6-PTX on open and closed times of single glutamate channels (recording bandwidth, 0-1 KHz) Mean closed tittle (ms)
Concentration d-PTX (Units/ml)
Mean open time (ms) Control
+ 6-PTX
P§
Control
+ ~-PTX
P§
0.2* 1.0"*
2.9 ± 0.39 (S.D.) 4.23 ± 1.1
2.24 A_ 0.42 3.15 ± 0.75
0.009 0.001
50.8 ± 11.8 28.89 -4- 14.6
331 ~ 137.2 153 d- 78.6
0.002 <0.001
* Glutamate concentration = 1 0 - 4 M. ** Glutamate concentration = 5 × 10-4 M. § Student's t-test.
113 glutamate-activated ion channels described in this paper suggest that the toxin blocks the open glutamate channel of locust extrajunctional muscle membrane. It has not been possible yet to study the activity of single glutamate-activated channels in junctional membrane using the patch clamp technique, but the shortening of the e.j.c, and junctional glutamate currents by &PTX and the activationinduced reduction in amplitude of these events is consistent with the hypothesis that this toxin also blocks open glutamate-gated channels in this membrane component. The frequency of channel openings, measured under 'patch' recording conditions, was consistently reduced by the presence of 6-PTX, but it is unlikely that this reflects a direct effect of &PTX on the probability of channel opening (or glutamate receptor activation). Indeed, this explanation would be inconsistent with the results illustrated in Figs. 1 and 2. Single channel currents recorded by the 'patch clamp' technique represent the activity of a single receptor or a small population of the receptors under approximately 'steady-state' conditions. It follows that the frequency of channel openings will be influenced not only by events at the receptor and ionophore 'gate' levels, but also by the rate of recovery from block of channels which might become blocked in their open conformation. The frequency of channel openings during the 'bursts' of activity seen in the presence of &PTX was not significantly different from the control frequency recorded in the absence of the toxin. It seems most likely, therefore, that the quiet intervals between 'bursts' represent the periods during which the individual glutamate channels are blocked by the venom. Piek and Spanjer 17 have shown that whole venom from P. triangulum paralyzes locust nerve-muscle preparations and that the effect of the venom persisted for at least 2 h with venom concentrations of 0.3-1.0 U/ml. If, after removal of the venom, the preparation was not stimulated for 5-15 min, the first nerve stimulus given after a period of rest resulted in a muscle contraction of similar amplitude to that of the control response. However, subsequent stimuli were acompanied by progressively decreasing contractions which contrasted with the consistent responses of untreated muscles. Similar
results to these have been obtained with locust retractor unguis nerve-muscle preparations using 0PTX (Usherwood, unpublished data). A possible explanation for these results is that 0-PTX enters some internal compartment of locust muscle during opening of glutamate gated channels and disperses only slowly from this compartment when the toxin is removed from the bathing medium. The implication that &PTX blocks the open glutamate channel either from external or internal sources is compatible with the voltage insensitivity of the blocking process seen in the e.j.c, studies reported herein. Indeed, it is possible that the real unblocking rate for the action of the &PTX on the glutamate channel is an indica, tion of the mobility of the toxin molecule as it moves through the channel from outside to inside or vice versa. If the toxin reaches some intramembranous or intracellular compartment via the open glutamate channel and can block the channel from this compartment as well as from the extracellular compartment, it follows that the unblocking rate would be dependent not only upon the concentration of this ligand applied to the postsynaptic membrane, but also upon the duration of its application and the total number of channel openings which accompany its application. Also, the discharging of the intracellular/intramembranous component would be dependent upon the frequency of channel openings. This would explain the slow rate of recovery following bath application of &PTX and concomitant repetitive iontophoretic application of L-glutamate and the even slower recovery following prolonged neural stimulation in the presence of toxin. On the other hand, the limited exposure of extrajunctional membrane to 6-PTX and L-glutamate in the patch clamp studies would not be expected to significantly charge an internal compartment with &PTX, especially if there is lateral diffusion of this toxin in either membrane or muscle cytoplasm. Recovery rates from the &PTX block in these studies would be expected, therefore, to be much higher than in the iontophoretic e.j.p, and e.j.c, experiments and probably approximate to the 'real' channel unblocking rates. It appears from our studies that the effectiveness of &PTX as a channel blocker arises from its low unblocking rate. In conditions where a high degree of block is obtained there is only a rather modest
114 change in channel life-time (Table I), which suggests t h a t the p r o b a b i l i t y o f open channel b l o c k b y 6 - P T X is low. T h e qualitative action o f 6 - P T X is thus similar to t h a t of, for example, local anaesthetics, b u t it differs greatly in its quantitative b e h a v i o u r . A l t h o u g h these studies have n o t led to the discovery o f a g l u t a m a t e r e c e p t o r a n t a g o n i s t they have p r o d u c e d some interesting i n f o r m a t i o n on the act i o n o f a n a t u r a l toxin on ionic channels in locust muscle m e m b r a n e gated b y receptors for L-glutamate. A p a r t f r o m the usefulness o f this t o x i n in giving further insight into the p r o p e r t i e s o f these m e m b r a n e i o n o p h o r e s , O-PTX m a y also p r o v e o f value in studies a i m e d at d e t e r m i n i n g whether glutam a t e is a t r a n s m i t t e r at synapses in systems other t h a n the locust nerve-muscle. I n p a r t i c u l a r we have in m i n d those regions o f the v e r t e b r a t e central n e r v o u s system where this a m i n o acid has been i m p l i c a t e d as a n e u r o t r a n s m i t t e r . However, before REFERENCES 1 Anwyl, R. and Usherwood, P. N. R., Voltage clamp studies of glutamate synapse, Nature (Lond.), 252 (1974) 591-593. 2 Clark, R. B., Gration, K. A. F. and Usherwood, P. N. R., Desensitization of glutamate receptors on innervated and denervated locust muscle fibres, J. Physiol. (Lond.), 290 (1979) 551-568. 3 Cull-Candy, S. G. and Usherwood, P. N. R., Two populations of L-glutamate receptors on locust muscle fibres, Nature (Lond.), 246 (1973) 62-64. 4 Gration, K. A. F., Lambert, J. J., Ramsey, R. and Usherwood, P. N. R., Non-random openings and concentration-dependent life-times of glutamate gated channels in muscle membrane, Nature (Lond.), 291 (1981) 423-425. 5 Gration, K. A. F. and Usherwood, P. N. R., Interactions of glutamate with amino acid receptors on locust muscle, Verh. Dtsch. ZooL Ges., (1980) 122-132. 6 Mathers, D. and Usherwood, P. N. R., Concanavalin A blocks desensitization of glutamate receptors on insect muscle fibres, Nature (Lond.), 259 (1976) 409-411. 7 Mathers, D. and Usherwood, P. N. R., Effects of convanavalin A on junctional and extrajunctional L-glutamate receptors on locust skeletal muscle fibres, Comp. Biochem. PhysioL, 59C (1978) 151-155. 8 May, T. E. and Pick, T., Neuromuscular block in locust skeletal muscle caused by a venom preparation made from the digger wasp Philanthus triangulum F. from Egypt. J. lnsect PhysioL, 25 (1979) 685-691. 9 Neher, E., Sakmann, B. and Steinbach, J. H., The extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes, Pfliigers Arch., 375 (1978) 219-228. 10 Neher, E. and Steinbach, J. H., Local anaesthetics transiently block currents through single acetylcholine-receptor channels, J. Physiol. (Lond.), 277 (1978) 153-176.
using O-PTX for this p u r p o s e it will be necessary to establish t h a t it blocks ionic channels in excitable m e m b r a n e s other t h a n those o f insect muscle. Since v e n o m f o r m P. triangulum shortens the e n d - p l a t e current o f r a t d i a p h r a g m muscle (A. W i l g e n b u r g a n d T. Pick, in p r e p a r a t i o n ) , it is possible t h a t the action o f ~ - P T X is n o t restricted to channels g a t e d b y L-glutamate, a l t h o u g h studies with ~-PTX, r a t h e r t h a n crude venom, in n o n - g l u t a m a t e r g i c systems m u s t be u n d e r t a k e n before this can be established unequivocally. ACKNOWLEDGEMENTS The a u t h o r s wish to t h a n k Mr. A. Tivey o f the D e p a r t m e n t o f Z o o l o g y , University o f N o t t i n g h a m for his technical assistance. This w o r k was s u p p o r t ed in p a r t b y a g r a n t to P . N . R . U . f r o m the British Science R e s e a r c h Council. 11 Patlak, J., Gration, K. A. F. and Usherwood, P. N. R., Glutamate-activated channels in locust muscle, Nature (Lond.), 278 (1978) 643-645. 12 Pick, T., Site of action of the venom of the digger wasp Philanthus triangulum F. on the fast neuromuscular system of the locust, Toxicon, 3 (1966) 191-198. 13 Piek, T., The effect of the venom of the digger wasp Philanthus on the fast and slow excitatory and inhibitory system in the locust muscle, Experientia, 12 (1966) 462-463. 14 Piek, T., Mantel, P. and Engels, E., Neuromuscular block in insects caused by the venom of the digger wasp Philanthus trianguhtm F., Comp. gen. PharmacoL, 2 (1971) 317-331. 15 Piek, T., Mantel, P. and Jas, H., Ion-channel block in insect muscle fibre membrane by the venom of the digger wasp, Philanthus triangulum F.,, J. Insect PhysioL, 26 (1980) 345-349. 16 Piek, T., May, T. E. and Spanjer, W., Paralysis of insect skeletal muscle by the venom of the digger wasp Philanthus triangulum F. In Insect Neurobiology and pestieide action, Soc. Chem. Indust., London, 1980, pp. 219-226. 17 Piek, T. and Spanjer, W., Effects and chemical characterization of some paralysing venoms of solitary wasps. In D. L. Shankland, R. M. Hollingworth and T. Smyth (Eds.), Pesticide and venom neurotoxicity, Plenum, New York, 1978, pp. 211-226. 18 Rathmayer, W., Paralysis caused by the digger wasp Philanthus, Nature (Lond.), 196 (1962) 1148-1151. 19 Usherwood, P. N. R., Release of transmitter from insect excitatory motor nerve terminals, J. Physiol. (Lond.), 227 (1972) 527-551. 20 Usherwood, P. N. R., Amino acids as neurotransmitters, Advanc. Comp. PhysioL Biochem., 7 (1978) 227-309. 21 Usherwood, P. N. R. and Machili, P., Pharmacological properties of excitatory neuromuscular synapses in the locust, 3". exp. BioL, 49 (1968) 341-361.
105
Elsevier Biomedical Press
Block of Locust Muscle Glutamate Receptors by -Philanthotoxin Occurs After Receptor Activations R. B. CLARK*, P. L. DONALDSON**, K. A. F. GRATION, J. J. LAMBERT, T. PIEK, R. RAMSEY, W. SPANJER and P. N. R. USHERWOOD§
Department of Zoology, University of Nottingham, University Park, Nottingham NG7 2RD (U.K.) and (T.P. and W.S.) Pharmacological Laboratory, University of Amsterdam, Polderweg104, 1093 KP, Amsterdam (The Netherlands) (Accepted October 29th, 1981)
Key words: wasp venom - - glutamate receptors - - glutamate potential - - locust muscle - - glutamate channels - neuromuscular junctions
One component (6-philanthotoxin (6-PTX)) of the venom from the wasp Philanthus triangulum blocks transmissionpostsynaptically at excitatory synapses on locust muscle. 6-PTX depresses both the iontophoretic glutamate potential and the excitatory junctional current (e.j.c.) in a glutamate receptor activation-dependentmanner. The rate of recovery from the effects of the toxin is reduced following either prolonged application of L-glutamate or repetitive iontophoretic application of this amino acid or high frequency neural stimulation of the muscle in the presence of 6-PTX. The decay phase of the e.j.c, is shortened by 6-PTX. The effects of 6-PTX on the e.j.c, are not voltage dependent. The open-close kinetics of glutamate channels in extrajunctional muscle membrane are modified by 6-PTX as shown by patch clamp analysis. The mean life time of the glutamate channel is reduced, whilst the mean interval between single opening events is increased with the events often occurring in bursts. These data are consistent with glutamate channel blocking by this toxin. It is proposed that the toxin blocks open channels gated by both junctional and extrajunctional glutamate receptors on locust muscle. It is further proposed that 6-PTX enters a compartment of the muscle through the glutamate open channels and that it can also block the open channels from this site. INTRODUCTION Studies of glutamate receptors o n excitable cells has suffered from the absence of p o t e n t a n d p r o v e n antagonists 2°, which makes the c o n t i n u i n g search for such c o m p o u n d s i m p o r t a n t to those seeking further insight into the role of these receptors o n excitable cells of vertebrates a n d invertebrates. F o r this reason we have investigated the properties of a toxin fraction, O-philanthotoxin ( O - P T X ) i s o l a t e d from the v e n o m o f the digger wasp Philanthus triangulum, which at first sight seemed to possess some of the characteristics o f a n a n t a g o n i s t at locust glutamatergic n e r v e - m u s c l e junctions12,13.
The sting o f the female digger wasp is used to paralyze honeybees, which the wasp t h e n carries into b r o o d cells as food for its larvae 18. The v e n o m from the sting apparatus also affects other insects where it exerts a variety of effects o n peripheral a n d central nervous systems. Pick a n d his co-workers have shown that the crude v e n o m blocks transmission at excitatory a n d i n h i b i t o r y synapses o n locust leg muscle p r o b a b l y by inhibiting t r a n s m i t t e r release at these sites 14. Recently the v e n o m has been separated into a n u m b e r of active c o m p o n e n t s , one of which (O-PTX) blocks t r a n s m i s s i o n postsynaptically at excitatory synapses o n locust leg muscle 16,~7. I n this paper we report o n the effects of O-PTX
* Present address: Department of Physiology and Biophysics, The University of Texas Medical Branch, Galveston, TX 77550, U.S.A. ** Present address: Department of Physiology and Biophysics, University of Iowa, Basic Science Building, Iowa City, IA, 52242, U.S.A. § To whom correspondence should be addressed at: Department of Zoology, University of Nottingham, Nottingham NG7 2RD, U.K. 0006-8993/82/0000-0000/$92.75 O Elsevier Biomedical Press
106 on nerve-muscle preparations of locust which have been studied using a variety of techniques, including patch clamp analysis of the activity of single receptor-ionophore complexes in extrajunctional muscle membrane. We shall confirm the conclusion of May and Pick s that 6-PTX is not a glutamate receptor antagonist, and show that it blocks glutamate gated ionophores in the postsynaptic membrane of locust excitatory nerve-muscle junctions and also the ion channels gated by glutamate receptors on extrajunctional membrane of locust muscle. METHODS AND MATERIALS Metathoracic extensor tibiae muscles 2 of the locusts Schistocerea gregaria and Locusta migratoria were mainly used in these studies. The preparations were dissected in a small Perspex bath with a replaceable volume of 2 ml through which saline flowed at a rate of 5-10 ml/min. The saline contained (mM): NaC1, 180; KCI, 10; CaCI~, 2; Na2HPO4, 6; NaH2PO4, 4 (pH 6.8). L-Glutamate was applied iontophoretically to single nerve-muscle junctions through micropipettes containing 0.1 M sodium glutamate dissolved in distilled water (pipette resistance, ca. 100 M ~ ) and with constant braking currents of 2-5 nA. Excitatory junctions were located by delivering pulses of glutamate of about 2 nC onto the muscle fibre membrane. The appearance of transient depolarizations (glutamate potentials) of rapid rise time (10-50 ms) signalled the presence of a junction. Junctional glutamate potentials were readily distinguished from extrajunctional responses by their more rapid time course 3. Junctional depolarizations were recorded using conventional intracellular techniques. In some cases the muscle fibre was voltage-clamped in the region of the nerve-muscle junction using a two-electrode three point clamp and the membrane currents resulting from glutamate iontophoresis were recorded 1. Excitatory junctional currents (e.j.c.'s) resulting from nerve stimulation were measured intracellularly under voltage-clamp from fibres in 3 distal bundles of the extensor tibiae muscle, i.e. those with length constant/length ratios > 1. Extracellular excitatory junctional potentials (e.j.p.'s) were recorded differentially between an 'active' electrode located at a single nerve-muscle junction and a
'reference' electrode less than 100/~m away. Single active sites were located by trial and error during repetitive stimulation of the excitatory innervation of the muscle preparation at a frequency of 1 Hz ag. Since insect muscle fibres are muttiterminally innervated the extracellular e.j.p., unlike the e.j.c, recorded under voltage-clamp, monitors the activity of a single site on the muscle fibre. Its time course is more or less identical to that of the e.j.c, so it can be used to estimate, through their effects on decay of the e.j.p., the influence of drugs on the life-times of channels in the postjunctional membrane of superficial nerve-muscle junctions where problems of drug accessibility are minimized. The fact that recordings of extracellular e.j.p.'s are limited to single superficial junctions validates comparison between data obtained in this way and those obtained using drug iontophoresis, e.j.c.'s and extracellular e.j.p.'s were recorded in saline containing 140 mM NaC1 and 40-50 mM MgCI2. This saline reduced the amplitude of the excitatory junctional potential and thereby prevented muscle contraction without completely blocking nerve-muscle transmission 16. Currents through single glutamate-activated ion channels in the extrajunctional membrane of locust muscle fibres were recorded using the 'patch clamp' technique of Neher et al. 4,5,9,11. Muscle preparations treated with 10-6 M concanavalin A to block glutamate receptor desensitization were used in these studiesa,L Our studies of single channel activity were restricted to those sites at which only a single receptor was active, i.e. there was no indication of overlapping events. Further details of the application of the patch clamp technique to locust muscle preparations are contained in recent publications from one of our laboratories 4,5. Crude extracts of venom were obtained from abdomens of female wasps (Philanthus triangulum) and separated into a series of fractions using Sephadex chromatography and freeze-drying. The &PTX fraction was dissolved in locust saline prior to use. Since the molecular identity of d-PTX is unknown, quantities of the toxin are expressed in arbitrary 'units' (U). (See refs. 16 and 17 for a description of the separation procedure and for a quantitative definition of 'units'.)
107
A
(i)
.,
.
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Fig. 1. Effect of 6-PTX on L-glutamate potentials evoked by glutamate iontophoresis at excitatory neuromuscular junctions of locust extensor tibiae muscle. A: i, saline containing 0.1 U/ml of 6-PTX was introduced (~) during repetitive applications of 1.5 nC iontophoric glutamate doses. Note the reduction in response amplitude. After 1.5 min exposure to 6-PTX, toxin-free saline was introduced (~); ii, following 10 rain wash with toxin-free saline, the response was restored to control amplitude. Saline containing 0.1 U/ml was reintroduced, but iontophoretic application of glutamate was discontinued for 1 min and then resumed. The amplitude of the first response at the end of the 1 min period was nearly identical to the control amplitude (cf. i). Note the decline in amplitude of response to subsequent glutamate doses. Measurements were made at the resting potential of the muscle fibre, - - 6 0 mV. Calibration bars: potential (vertical), 4 mV; times (horizontal), 20 s. B : recovery from effects of 6-PTX iontophoretic studies. i, pulses (1 nC) of L-glutamate were applied to an excitatory nerve-muscle junction at a constant frequency of 0.74 Hz. During bath application of 0.05 U/ml of 6-PTX (between arrows) the glutamate potentials were depressed and they recovered only slowly after removal of the toxin; ii, recording of glutamate potentials at same junction as in i above. 0-PTX (0.05 U/ml) was applied for the period between arrows. In this experiment application of the pulses (1 nC, 0.74 Hz) of L-glutamate was interrupted for periods of 6, 12, 24 and 40 s. Note the slight recovery of response amplitude following these brief rest periods. Calibration bars: voltage (vertical), 5 mV; time (horizontal), 20 s.
RESULTS
Effect of ~3-PTX on responses to glutamate iontophoresis Passage of a brief (ca. 10 ms) pulse of current through a glutamate-filled micropipette with its tip placed close to a nerve-muscle junction resulted in a transient depolarization of the muscle membrane. The amplitude of the 'glutamate' depolarization declined when 6-PTX was included in the bathing saline (Figs. 1 and 2). When 0.1 U/ml of 6-PTX was introduced the glutamate response was reduced to about 15 ~ of its control amplitude within 50 s. After 10 min wash in toxin-free saline the response was restored to its control amplitude. Next, saline containing 0.1 U/ml of 0-PTX was re-introduced, but the iontophoretic pulses were discontinued until the muscle had been exposed to 6-PTX for 60 s. The amplitude of the first glutamate response after resuming the iontophoretic pulses was almost identical to the control response, in contrast to the small responses which resulted during repetitive application of L-glutamate in the presence of O-PTX.
Subsequent responses declined rapidly, and after 10 pulses the response amplitude was about 15 ~ of the control. The response amplitude declined more rapidly when the muscle was pre-exposed to 6-PTX saline before glutamate pulses were applied than when doses were applied continuously during initial introduction of the O-PTX saline. It is possible that this difference reflects the time taken for the concentration of 6-PTX in the region of the superficial nerve-muscle junction to equilibrate with the bulk concentration in the saline. For this reason 6-PTX was usually applied to the muscle preparation for at least 60 s before glutamate pulses were applied. The rate of decline of response amplitude in the presence of O-PTX was dependent on the concentration of ~-PTX in the saline (Fig. 2) and, for a fixed pulse current, on the frequency at which glutamate pulses were applied iontophoretically to the junctional site. The amplitudes of successive responses in a train declined in an approximately exponential manner to a constant 'steady-state' level. Examples of this exponential decay of pulse train amplitude for two different concentrations of O-PTX are shown
108 in Fig. 2B. It is clear that the rate o f decline of amplitude increases with an increase in the concentration o f &PTX. The data o f Figs. 1 and 2 suggest that the action of d-PTX arises f r o m 'use-dependent' block induced by &PTX. N o t e that in the lower trace in Fig. 2A, unlike the upper 3 traces, the amplitude o f the first glutamate response in the d-PTX saline was smaller than the control amplitude. This m a y indicate that & P T X blocked a small p r o p o r t i o n o f the postsynaptic glutamate 'receptor' population prior to the first application o f glutamate either t h r o u g h antagonism at the receptor level or by blocking closed glutamate-gated m e m b r a n e channels. A more likely explanation is that there was a small steady leakage of glutamate f r o m the tip o f the iontophoretic pipette and/or f r o m nerve terminal during spontaneous transmitter release, and that this resulted in a small,
undetectable activation (and consequent block by OPTX) of a p r o p o r t i o n o f the receptors in the absence of glutamate ejection current pulses. It is unlikely that 6-PTX caused or enhanced desensitization o f the postjunctional glutamate receptors because o f the unpredictable reduction o f the first response in & P T X and also because similar data were obtained in the presence o f 10 -6 M concanavalin A, a lectin which eliminates desensitization o f glutamate receptors at locust excitatory nerve-muscle junctions6, 7. We cannot, o f course, exclude the possibility that the position o f the iontophoretic electrode vis-/t-vis that o f the junctional site sometimes changed during an experiment and, thereby, influenced the amplitude o f the glutamate potential. The decrease in amplitude o f the glutamate response did not result f r o m changes in the passive electrical properties o f the muscle fibre membrane,
A 0"01 ~
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t
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Fig. 2. A: effect of concentration of d-PTX on the amplitude of iontophoretically evoked glutamate potentials recorded from a nerve-muscle junction of locust extensor tibiae muscle fibre. Pulses of glutamate (1 nC) were applied at a frequency of 0.56 Hz. Glutamate pulses were terminated immediately prior to introduction of toxin saline (~) and recommenced 60 s later; the muscle was exposed to 6-PTX in the saline as shown at the left (U/ml). Note that the rate of decline of glutamate potential amplitude increased as the concentration of 6-PTX was increased. All recordings were made at the same nerve-muscle junction. The muscle was washed in toxin-free saline for 5-10 min after removal of 6-PTX (~,) to restore the glutamate potential to the control amplitude. The recordings were made at the resting potential of the fibre, --58 inV. Calibration bars: voltage (vertical), 10 mV; time (horizontal), 20 s. B: exponential decay of response amplitude in two of the pulse trains shown in A (0.025 U/ml and 0.1 U/ml 6-PTX). The 'steady-state' response ratio (r~o)has been subtracted from each response (rd and the difference has been normalized to the difference for the first pulse in the train frO. For the two examples shown roo = 0.54 for 0.025 U/ml 6-PTX and 0.19 for 0.1 U/ml 6-PTX. The linear relationship between ri-r~ (on log scale) and pulse number indicates an exponential decline in response amplitudes. For [6-PTX] = 0.025 U/ml, the slope of the solid line is 0.063 pulse -z, and for 0.1 U/ml the slope is 0.31 pulse--L
109 as d-PTX caused a similar decline in the amplitude of voltage-clamp currents resulting from glutamate iontophoresis (see below). The effective input conductance of muscle fibres in the presence of 0.1 U/ml of &PTX did not differ significantly from the input conductance in toxin-free saline (input conductance in d-PTX saline ~- 1.02 4- 0.05 times conductance in toxin-free saline - - 3 different fibres). Changes in the resting membrane potential ot muscle fibres following exposure to saline containing 0.1 U/ml of &PTX were not significant (i.e. < 1 mV). d-PTX at 0.05 U/ml produced a decrease in the half-decay time of the glutamate response ranging from 13 to 29 (mean 18 ~ , n = 3 sites). There was also a small ( < 5 ~ ) decrease in the time to peak of the response. Qualitatively similar changes in time course were noted for glutamate depolarizations of unclamped fibres 15. The postsynaptic block produced by &PTX persisted for many seconds when glutamate was applied in the presence of c3-PTX. This is illustrated by the records in Fig. lB. In this experiment the glutamate pulses were applied to a toxin-treated muscle until the response was reduced to approximately 75 ~ of its control amplitude. The iontophoretic application of glutamate was interrupted for various time periods during d-PTX application, then single pulses of this amino acid were applied. The amplitude of the glutamate response increased slightly under these conditions (but it did not recover to its control amplitude even when the time between the single pulses of glutamate was almost 40 s). The action of &PTX was also investigated by microperfusing 0.2 U/ml of &PTX onto a junctional area whilst applying constant iontophoretic pulses of L-glutamate at a frequency of 0.25 Hz. After 1 min of d-PTX application, the amplitude of the glutamate-induced depolarization was reduced to 15.8 45.6 ~ (mean 4- S.D., n = 5 from 3 preparations) of control. When the microperfusion pipette was removed, the amplitude of the glutamate induced depolarizations recovered exponentially with a time constant of 57.7 4- 39.5 s (mean 4- S.D.; n = 3).
Effect of &PTX on excitatory junctional currents The amplitude and time course of the e.j.c, were influenced by O-PTX in a manner dependent upon the concentration of the toxin applied in the bathing
medium, the duration of exposure of the preparation to the toxin and the frequency of stimulation during its application. &PTX at O. 1 U/ml failed to depress the e.j.c, even when the stimulation frequency was as high as 3 Hz. However, there was a ca. 6 0 ~ reduction in e.j.c, amplitude after 5 min exposure to 1 U/ml with a stimulation frequency of 1.9 Hz, together with a shortening of the decay time of this event. The shortening of the decay time, which was independent of membrane potential (range --60 mV to --120 mV), was evident with the first e.j.c, in the series, when the amplitude was unaffected. This was due mainly to a shortening of the pre-exponential phase of the e.j.c, decay (Fig. 3A). In both control and &PTX-treated preparations e.j.c, decay was biphasic, consisting of an exponential phase preceded by a non-exponential phase. The effect of 6PTX on the amplitude of the e.j.c, was also independent of membrane potential (Fig. 3B) and the extrapolated reversal potential for the e.j.c, was unaffected by the toxin (Fig. 3B, C). The extracellular e.j.p, was more like the glutamate potential and current in terms of its susceptibility to &PTX, a significant reduction in amplitude being recorded with 0.! U/ml of toxin at a stimulation frequency of 3 Hz. However, when the stimulus frequency was reduced below 1 Hz this concentration of venom failed to depress the e.j.p. Possible presynaptic action of &PTX was investigated by recording the number of e.j.p, failures to continuous (3 Hz) stimulation in the presence and absence of toxin. These studies, which were undertaken on the locust retractor unguis muscle, demonstrated that 10-15 min exposure of this muscle to 6PTX (0.3-0.8 U/ml) had no effect on evoked transmitter release. It remains to be established whether higher concentrations or longer applications of OPTX are equally ineffective presynaptically.
Currents through single glutamate-activated channels
ion
Currents through single ion channels associated with L-glutamate receptors in extrajunctional (nonsynaptic) membrane of locust muscle fibres were measured using the 'patch clamp' technique. Single channels were recorded using patch electrodes containing L-glutamate (10 -4 M and 5 × 10-4 M 4- OPTX) dissolved in locust saline. Fresh pipettes were
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Fig. 3. A: effect of d-PTX (1 U/ml) on the e.j.c, recorded from locust extensor tibiae muscle fibre under voltage damp. e.j.c.'s were evoked by stimulation of the extensor tibiae nerve at a frequency of 3 Hz. Records i-iii are averages of 20 consecutive e.j.c.'s. Record i was obtained in the absence of 6-PTX. The preparation was then rested for 5 rain during which time it was 'equilibrated' in saline containing 1 U/ml 6-PTX. Record ii is the averaged e.j.c, obtained immediately after the 5 min rest period, whereas record iii is the averaged e.j.c, obtained after 1 min continuous stimulation at 3 Hz in &PTX. Note that increase in rate of decay of e.j.c. precedes the fall in amplitude of response. Clamp potential, --60 mV; temperature, ca. 20 °C. B: relationship between peak amplitude of e.j.c, and membrane potential (Em) in presence (0) and absence (O) of 1 U/ml 6-PTX. The preparation was stimulated continuouslyat 3 Hz. Lines drawn by eye. C: relationship between peak amplitude of e.j.c, and membrane potential in presence (0) and absence ([~) of 1 U/ml 6-PTX with amplitudes normalized at --100 mV to demonstrate lack of voltage sensitivity of 6-PTX action. (O, before toxin; [~, after toxin).
used for each c o n c e n t r a t i o n of g l u t a m a t e a n d for recordings in presence a n d absence of & P T X . The a m p l i t u d e o f currents t h r o u g h the i o n channels (and c h a n n e l conductance) was u n c h a n g e d by & P T X , b u t the open-close kinetics o f the channels were modified. This is illustrated in Fig. 4 which shows single c h a n n e l currents recorded in the presence of 10 -4 M or 5 x 10 -4 M L-glutamate ! & P T X . The same electrode was used to record channels in the absence a n d presence o f d - P T X by exchanging the contents o f the electrode. However, because it was necessary to remove the patch electrode from the recording system to change its contents, recordings in the presence a n d absence of & P T X were presum a b l y made at different, b u t nevertheless closely adjacent sites, o n the m e m b r a n e . I n the absence of O-PTX, 10 .4 M glutamate in the ' p a t c h ' electrode i n d u c e d single c h a n n e l activity at a m e a n frequency o f 14 c h a n n e l openings/s. W h e n 0.2 U / m l of d - P T X was included with 10 -4 M glutamate in the same p a t c h electrode the m e a n frequency of c h a n n e l openings was reduced to less t h a n 3 s -1 (Fig. 5).
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10pA ] 200ms Fig. 4. Effect of 6-PTX on glutamate channel activity. A and B: single channel activity recorded in the absence (A) and presence (B) of 0.2 U/ml 6-PTX. Glutamate concentration 10-4 M. C and D: channel activity recorded with 5 x 10-~ M glutamate in the patch electrode, in the absence (C) and presence (D) of 1.0 U/ml 6-PTX. All records were obtained at the muscle resting potential (ca. --60 mV). A and B were obtained from different sites on the same muscle fibre; C and D from a second fibre. The two traces in both B and D are contiguous. Temperature, ca. 20 °C. Note that the seal resistance in A and B was lower (N 50 %) than that in C and D, resulting in larger 'background' current noise. Note also that channel in C was mainly in open state, with transient ._closingsduring the period of the trace.
111
6pAl looms Fig. 5. Long-term recording of single channel activity obtained in the presence of 5 × 10 4 M glutamate, plus 1.0 U/ml 6-PTX. Note the appearance of bursts of channel activity and the occurrence of single channel currents separated by long closed intervals. Muscle membrane potential, --55 mV; Temperature, ca. 20 °C. These and other patch data were recorded at a bandwidth of 0-1 KHz. Many of the small events resulted from activity of 'rin' channels9. The activity of single channels in the presence of 6-PTX sometimes consisted of clearly defined 'bursts' of channel events separated by quiet intervals > 100 ms (Figs. 4 and 6), the duration of these 'bursts' varying between 12 and > 100 ms. At other sites 'bursts' were less evident. Possibly the variations in channel activity achieved with & P T X reflect the non-random behaviour of glutamate channels seen in the control (i.e. in absence of 6PTX) conditions 4. Although & P T X resulted in a significant (P < 0.001 Student's t-test) increase in the mean channel closed time (calculated by averaging the closed intervals between a// successive opening events, including the quiet intervals between bursts), there was no significant difference between the mean closed time within bursts and that recorded in the absence of c3-PTX. This suggests that, in contrast to the action of QX 222 on ACh channels, for example, which causes the channel to fluctuate rapidly between open and blocked states 10, the block by & P T X is relatively long lasting. 6-PTX reduces the frequency of channel openings by blocking channels either in their open or closed
conformations rather than by reducing the probability of channel opening. Other data presented in this paper suggest that it is the open conformation of the glutamate channel which is blocked. To obtain reliable determinations of the channel blocking rate it is necessary to study the effects of a range of toxin concentrations at a constant glutamate concentration. Unfortunately our very limited availability of & P T X precluded this approach. However, the blocking rate G can be roughly estimated from t0 (a ÷ G.c) -1 where t0 = M0 (mean open time) in the presence of &PTX, a = l/M0 in the absence of the toxin and c = the concentration of d-PTX. For 10 -4 M L-glutamate, and 0.2 U/ml &PTX, G _ 5 × 10 -2 s -1 U -1 ml-1. F r o m the distributions of channel closed times obtained in the presence and absence of O-PTX (Fig. 6A, B) it is possible to estimate the unblocking rates for the action of toxin on the glutamate channel. In the absence of & P T X distributions of closed times can be fitted approximately with a single exponential with a time constant of ca. 40-100 ms. In the presence of venom a second larger time constant is
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present with a value at least 7 × greater t h a n this (Fig. 6C, D), suggesting t h a t the u n b l o c k i n g rate a p p r o x i m a t e s to between 1 s -1 a n d 1.5 s -1. I f & P T X is a g l u t a m a t e channel b l o c k e r it m i g h t be a n t i c i p a t e d t h a t the presence o f this t o x i n in the p a t c h electrode w o u l d cause a r e d u c t i o n in the lifetime o f the channel. This is clearly the case, as illustrated in T a b l e I. W h e n the p a t c h electrode c o n t a i n e d 10 .4 M glutamate, the single channels h a d a m e a n o p e n time o f 2.9 -t- 0.39 (S.D.) ms (n = 7 recordings each o f 200 events f r o m 7 sites on 4 p r e p a r a t i o n s ) . W h e n 0.2 U / m l d - P T X was a d d e d to the contents o f the p a t c h electrode, m e a n channel o p e n time was r e d u c e d to 2.24 ms (n = 8 recordings each o f 200 events f r o m 8 sites on 5 p r e p a r a t i o n s ) . The difference between the m e a n life-time o f the channels r e c o r d e d in the presence a n d absence o f dP T X , a l t h o u g h slight, is significant ( P < 0.01).
0
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ms
Fig. 6. Comparison of the frequency distributions of glutamate channel closed times measured in the absence (A) and presence 03) of 0.2 U/ml &PTX. Glutamate concentration, 10-4M. Each distribution represents pooled data from 3 experiments. Note the wider distribution of closed times in the presence of &PTX. Muscle membrane potential, --55 mV to --60 mV; temperature, ca. 20 °C. C: data of A plotted on semi-log axes. The solid line which was fitted to the data points by the method of least squares has a time constant (T) of 43 ms. D: data of B plotted on semi-log axes. The data points could be fitted with two exponentials. The best fits were obtained with zrast = 100 ms and rslow = 758 ms. Channels with life times > 200 ms were absent from these readings.
The depression b y d - P T X o f i o n t o p h o r e t i c glutam a t e potentials (and currents) a n d p o s t s y n a p t i c currents at locust excitatory n e u r o m u s c u l a r j u n c tions is very similar to the effects p r o d u c e d at these sites b y crude v e n o m f r o m Philanthus 12-14,18. M a y a n d Piek 8 have shown t h a t Philanthus v e n o m is a n o n - c o m p e t i t i v e a n t a g o n i s t at these sites a n d have suggested t h a t it might be a p o s t j u n c t i o n a l channel blocker, a conclusion shared b y Piek et al. 1~, w h o f o u n d t h a t the v e n o m decreased the a m p l i t u d e a n d h a l f decay time o f extracellular m i n i a t u r e e.j.c.'s r e c o r d e d f r o m locust r e t r a c t o r unguis muscle. M o d i fications b y & P T X o f the kinetic p r o p e r t i e s o f single
TABLE I Effect of 6-PTX on open and closed times of single glutamate channels (recording bandwidth, 0-1 KHz) Mean closed tittle (ms)
Concentration d-PTX (Units/ml)
Mean open time (ms) Control
+ 6-PTX
P§
Control
+ ~-PTX
P§
0.2* 1.0"*
2.9 ± 0.39 (S.D.) 4.23 ± 1.1
2.24 A_ 0.42 3.15 ± 0.75
0.009 0.001
50.8 ± 11.8 28.89 -4- 14.6
331 ~ 137.2 153 d- 78.6
0.002 <0.001
* Glutamate concentration = 1 0 - 4 M. ** Glutamate concentration = 5 × 10-4 M. § Student's t-test.
113 glutamate-activated ion channels described in this paper suggest that the toxin blocks the open glutamate channel of locust extrajunctional muscle membrane. It has not been possible yet to study the activity of single glutamate-activated channels in junctional membrane using the patch clamp technique, but the shortening of the e.j.c, and junctional glutamate currents by &PTX and the activationinduced reduction in amplitude of these events is consistent with the hypothesis that this toxin also blocks open glutamate-gated channels in this membrane component. The frequency of channel openings, measured under 'patch' recording conditions, was consistently reduced by the presence of 6-PTX, but it is unlikely that this reflects a direct effect of &PTX on the probability of channel opening (or glutamate receptor activation). Indeed, this explanation would be inconsistent with the results illustrated in Figs. 1 and 2. Single channel currents recorded by the 'patch clamp' technique represent the activity of a single receptor or a small population of the receptors under approximately 'steady-state' conditions. It follows that the frequency of channel openings will be influenced not only by events at the receptor and ionophore 'gate' levels, but also by the rate of recovery from block of channels which might become blocked in their open conformation. The frequency of channel openings during the 'bursts' of activity seen in the presence of &PTX was not significantly different from the control frequency recorded in the absence of the toxin. It seems most likely, therefore, that the quiet intervals between 'bursts' represent the periods during which the individual glutamate channels are blocked by the venom. Piek and Spanjer 17 have shown that whole venom from P. triangulum paralyzes locust nerve-muscle preparations and that the effect of the venom persisted for at least 2 h with venom concentrations of 0.3-1.0 U/ml. If, after removal of the venom, the preparation was not stimulated for 5-15 min, the first nerve stimulus given after a period of rest resulted in a muscle contraction of similar amplitude to that of the control response. However, subsequent stimuli were acompanied by progressively decreasing contractions which contrasted with the consistent responses of untreated muscles. Similar
results to these have been obtained with locust retractor unguis nerve-muscle preparations using 0PTX (Usherwood, unpublished data). A possible explanation for these results is that 0-PTX enters some internal compartment of locust muscle during opening of glutamate gated channels and disperses only slowly from this compartment when the toxin is removed from the bathing medium. The implication that &PTX blocks the open glutamate channel either from external or internal sources is compatible with the voltage insensitivity of the blocking process seen in the e.j.c, studies reported herein. Indeed, it is possible that the real unblocking rate for the action of the &PTX on the glutamate channel is an indica, tion of the mobility of the toxin molecule as it moves through the channel from outside to inside or vice versa. If the toxin reaches some intramembranous or intracellular compartment via the open glutamate channel and can block the channel from this compartment as well as from the extracellular compartment, it follows that the unblocking rate would be dependent not only upon the concentration of this ligand applied to the postsynaptic membrane, but also upon the duration of its application and the total number of channel openings which accompany its application. Also, the discharging of the intracellular/intramembranous component would be dependent upon the frequency of channel openings. This would explain the slow rate of recovery following bath application of &PTX and concomitant repetitive iontophoretic application of L-glutamate and the even slower recovery following prolonged neural stimulation in the presence of toxin. On the other hand, the limited exposure of extrajunctional membrane to 6-PTX and L-glutamate in the patch clamp studies would not be expected to significantly charge an internal compartment with &PTX, especially if there is lateral diffusion of this toxin in either membrane or muscle cytoplasm. Recovery rates from the &PTX block in these studies would be expected, therefore, to be much higher than in the iontophoretic e.j.p, and e.j.c, experiments and probably approximate to the 'real' channel unblocking rates. It appears from our studies that the effectiveness of &PTX as a channel blocker arises from its low unblocking rate. In conditions where a high degree of block is obtained there is only a rather modest
114 change in channel life-time (Table I), which suggests t h a t the p r o b a b i l i t y o f open channel b l o c k b y 6 - P T X is low. T h e qualitative action o f 6 - P T X is thus similar to t h a t of, for example, local anaesthetics, b u t it differs greatly in its quantitative b e h a v i o u r . A l t h o u g h these studies have n o t led to the discovery o f a g l u t a m a t e r e c e p t o r a n t a g o n i s t they have p r o d u c e d some interesting i n f o r m a t i o n on the act i o n o f a n a t u r a l toxin on ionic channels in locust muscle m e m b r a n e gated b y receptors for L-glutamate. A p a r t f r o m the usefulness o f this t o x i n in giving further insight into the p r o p e r t i e s o f these m e m b r a n e i o n o p h o r e s , O-PTX m a y also p r o v e o f value in studies a i m e d at d e t e r m i n i n g whether glutam a t e is a t r a n s m i t t e r at synapses in systems other t h a n the locust nerve-muscle. I n p a r t i c u l a r we have in m i n d those regions o f the v e r t e b r a t e central n e r v o u s system where this a m i n o acid has been i m p l i c a t e d as a n e u r o t r a n s m i t t e r . However, before REFERENCES 1 Anwyl, R. and Usherwood, P. N. R., Voltage clamp studies of glutamate synapse, Nature (Lond.), 252 (1974) 591-593. 2 Clark, R. B., Gration, K. A. F. and Usherwood, P. N. R., Desensitization of glutamate receptors on innervated and denervated locust muscle fibres, J. Physiol. (Lond.), 290 (1979) 551-568. 3 Cull-Candy, S. G. and Usherwood, P. N. R., Two populations of L-glutamate receptors on locust muscle fibres, Nature (Lond.), 246 (1973) 62-64. 4 Gration, K. A. F., Lambert, J. J., Ramsey, R. and Usherwood, P. N. R., Non-random openings and concentration-dependent life-times of glutamate gated channels in muscle membrane, Nature (Lond.), 291 (1981) 423-425. 5 Gration, K. A. F. and Usherwood, P. N. R., Interactions of glutamate with amino acid receptors on locust muscle, Verh. Dtsch. ZooL Ges., (1980) 122-132. 6 Mathers, D. and Usherwood, P. N. R., Concanavalin A blocks desensitization of glutamate receptors on insect muscle fibres, Nature (Lond.), 259 (1976) 409-411. 7 Mathers, D. and Usherwood, P. N. R., Effects of convanavalin A on junctional and extrajunctional L-glutamate receptors on locust skeletal muscle fibres, Comp. Biochem. PhysioL, 59C (1978) 151-155. 8 May, T. E. and Pick, T., Neuromuscular block in locust skeletal muscle caused by a venom preparation made from the digger wasp Philanthus triangulum F. from Egypt. J. lnsect PhysioL, 25 (1979) 685-691. 9 Neher, E., Sakmann, B. and Steinbach, J. H., The extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes, Pfliigers Arch., 375 (1978) 219-228. 10 Neher, E. and Steinbach, J. H., Local anaesthetics transiently block currents through single acetylcholine-receptor channels, J. Physiol. (Lond.), 277 (1978) 153-176.
using O-PTX for this p u r p o s e it will be necessary to establish t h a t it blocks ionic channels in excitable m e m b r a n e s other t h a n those o f insect muscle. Since v e n o m f o r m P. triangulum shortens the e n d - p l a t e current o f r a t d i a p h r a g m muscle (A. W i l g e n b u r g a n d T. Pick, in p r e p a r a t i o n ) , it is possible t h a t the action o f ~ - P T X is n o t restricted to channels g a t e d b y L-glutamate, a l t h o u g h studies with ~-PTX, r a t h e r t h a n crude venom, in n o n - g l u t a m a t e r g i c systems m u s t be u n d e r t a k e n before this can be established unequivocally. ACKNOWLEDGEMENTS The a u t h o r s wish to t h a n k Mr. A. Tivey o f the D e p a r t m e n t o f Z o o l o g y , University o f N o t t i n g h a m for his technical assistance. This w o r k was s u p p o r t ed in p a r t b y a g r a n t to P . N . R . U . f r o m the British Science R e s e a r c h Council. 11 Patlak, J., Gration, K. A. F. and Usherwood, P. N. R., Glutamate-activated channels in locust muscle, Nature (Lond.), 278 (1978) 643-645. 12 Pick, T., Site of action of the venom of the digger wasp Philanthus triangulum F. on the fast neuromuscular system of the locust, Toxicon, 3 (1966) 191-198. 13 Piek, T., The effect of the venom of the digger wasp Philanthus on the fast and slow excitatory and inhibitory system in the locust muscle, Experientia, 12 (1966) 462-463. 14 Piek, T., Mantel, P. and Engels, E., Neuromuscular block in insects caused by the venom of the digger wasp Philanthus trianguhtm F., Comp. gen. PharmacoL, 2 (1971) 317-331. 15 Piek, T., Mantel, P. and Jas, H., Ion-channel block in insect muscle fibre membrane by the venom of the digger wasp, Philanthus triangulum F.,, J. Insect PhysioL, 26 (1980) 345-349. 16 Piek, T., May, T. E. and Spanjer, W., Paralysis of insect skeletal muscle by the venom of the digger wasp Philanthus triangulum F. In Insect Neurobiology and pestieide action, Soc. Chem. Indust., London, 1980, pp. 219-226. 17 Piek, T. and Spanjer, W., Effects and chemical characterization of some paralysing venoms of solitary wasps. In D. L. Shankland, R. M. Hollingworth and T. Smyth (Eds.), Pesticide and venom neurotoxicity, Plenum, New York, 1978, pp. 211-226. 18 Rathmayer, W., Paralysis caused by the digger wasp Philanthus, Nature (Lond.), 196 (1962) 1148-1151. 19 Usherwood, P. N. R., Release of transmitter from insect excitatory motor nerve terminals, J. Physiol. (Lond.), 227 (1972) 527-551. 20 Usherwood, P. N. R., Amino acids as neurotransmitters, Advanc. Comp. PhysioL Biochem., 7 (1978) 227-309. 21 Usherwood, P. N. R. and Machili, P., Pharmacological properties of excitatory neuromuscular synapses in the locust, 3". exp. BioL, 49 (1968) 341-361.