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NSL 08093

Neuroscience Letters, 131 ( 1991) 196-200 c~ 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50

ADONIS 030439409100577N

Quisqualate-sensitive glutamate receptors of the locust Schistocerca gregaria are antagonised by intracellularly applied philanthotoxin and

spermine

P. Brundell 1, R. Goodnow Jr. 2, C.J. Kerry 1, K. Nakanishi 2, H.L. Sudan I and P .N.R . U s h e r w o o d 1

1Department of Life Science, University of Nottingham, Nottingham ( U.K. ) and 2Department of Chemistry, Columbia University, New York, N Y (U.S.A.)

(Received 20 June 1991; Revised version received 3 July 199l; Accepted 3 July 1991)

Key words: Glutamate receptor; Philanthotoxin; Intracellular application; Voltage clamp; Patch clamp; Locust muscle

The effects of intracellularly and extracellularly applied synthetic analogues of 6-philanthotoxin (PhTX-433) and the polyamine spermine on the excitatory postsynaptic current (EPSC) of glutamatergic synapses and single channel currents gated by quisqualate-sensitive glutamate receptors (QUIS-R) on locust leg muscle have been compared. When applied extracellularly all 3 compounds reversibly antagonised the EPSC and the single channel currents. Antagonism was voltage independent, but use (agonist) dependent. Antagonism also occurred when they were injected into muscle fibres, but in this case it was not use dependent. It is proposed that spermine and the two toxins bind to the closed and open channel conformations of QUIS-R at a site near the intracellular opening of the channel gated by this receptor.

Many species of predatory arthropod subdue their invertebrate prey by injecting them with paralysing venoms. In some species of wasp and spider the active components in these venoms are polyamine-containing toxins (polyamine amides) which non-competitively antagonise ionotropic glutamate receptors (Glu-R) of vertebrates and invertebrates [8]. A major component of the venom of the wasp Philanthus triangulum is philan- thotoxin-433 (PhTX-433; the numerals denote the number of methylenes between the amino groups of the polyamine moiety) [6, 14], which contains a butyryl/ tyrosyl/spermine sequence and has a molecular weight of 435 Da. Polyamines such as spermine, PhTX-433 and synthetic analogues [4-6, 10, 14, 15, 20] and similar polyamine amide toxins present in some spider venoms [1, 11, 12] are purported reversibly to block the open channels gated by quisqualate-sensitive Glu-R (QUIS- R) which are present postjunctionally and extrajunctio- nally on arthropod skeletal muscle. However, it has also been suggested that antagonism might arise through in- teraction of these compounds with a closed channel con- formation of QUIS-R [1, 8, 11, 12]. PhTX-433 (or its

Correspondence." P.N.R. Usherwood, Department of Zoology, Univer- sity of Nottingham, Nottingham NG7 2RD, U.K.

close synthetic analogue PhTX-343) and spermine also antagonise the responses to L-kainate and N-methyl-D- aspartate (NMDA) of Xenopus oocytes injected with RNA from rat brain [3, 17]. The L-kainate responses of cat brainstem neurones in vivo are also inhibited by PhTX-433 [9].

Studies of the permeability of the channel gated by lo- cust muscle QUIS-R, allied to examination of Corey- Pauling-Koltum space-filling models of polyamines and polyamine amide toxins of wasp and spider venoms led Usherwood and Blagbrough [20] to conclude that these compounds could, in principle, pass through this chan- nel, thereby gaining access to the intracellular environ- ment of the muscle fibre. This led us to enquire whether philanthotoxins and spermine, for example, would anta- gonise QUIS-R if they were injected into a muscle fibre instead of being applied extracellularly, as has been the case for all previous studies of these compounds. In this paper, we have addressed this question by comparing the effects of extracellular application and intracellular injection of spermine and two potent, synthetic analo- gues of PhTX-433 on the excitatory postsynaptic current (EPSC) of glutamatergic synapses of locust muscle and on the single channel currents recorded from QUIS-R of extrajunctional membrane of locust muscle.

All experiments were performed on the metathoracic

extensor tibiae muscle of adult, female Schistocerca gre- garia, 7-10 days post-fledgling, using two-electrode vol- tage clamp [2] in conjunction with the neural stimulation technique of Usherwood [19] and mega-ohm seal patch clamp [11]. The preparations were bathed in standard saline of the following composition (in mM): NaC1 180, KC1 10, CaC12 2, HEPES 3, pH 6.8 (adjusted with 1 M NaOH). In some EPSC studies, the calcium concentra- tion of this saline was reduced to 0.25 mM to diminish the amplitude of the EPSC and, thereby, to prevent non- clamped fibres from contracting during stimulation of the extensor tibiae nerve. In the patch clamp experi- ments, the muscle was pretreated with concanavalin A to block desensitization of extrajunctional QUIS-R [13]. Two synthetic analogues of PhTX-433 were used, PhTX- 343 and 3,5-dibromo-PhTX-343. They, and spermine, were dissolved in standard saline for extracellular, bath application, or, for intracellular injection, in either dis- tilled water or an intracellular saline (in mM: KCI 180, EGTA 1, HEPES 3, pH 6.8, adjusted with 1 M KOH). For intracellular injection, the toxin- and spermine-con- taining solutions were placed in micropipettes (1-2 MI2 resistance) by backfilling. The compounds were ejected from a micropipette either under pressure (20-30 Torr) for periods of 2 min using an electrically driven syringe or for periods of 10 min by applying 40 V pulses (duration 2 ms; frequency 10 Hz) to the contents of the pipette. The nerve-muscle preparations were maintained

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Fig. 1. Intracellular recordings from 3 (A-C, D-G, H-I) locust exten- sor tibiae muscle fibres showing antagonism of EPSC by Br2-PhTX- 343. Each set of records in A-J represent mean EPSCs (n=20) obtained at various holding potentials and with a nerve stimulation frequency of I Hz. A,D,H: EPSCs recorded in absence of toxin at hold- ing potentials (10 mV steps) of - 5 0 mV to - 100 mV (A--C, H-I) or --40 mV to - 100 mV (D-G). B,C: EPSCs recorded 40 min and 130 min respectively after start of 10 min period of current injection of 5 × 10 -4 M Br2-PhTX-343 (see text) showing antagonism. E~3: EPSCs recorded 10 rain, 40 min and 70 min respectively after start o f current injection of 5 x 10 -3 M Br2-PhTX-343. I: EPSCs recorded 10 min after

start of current injection of 10-2 M Br2-PhTX-343.

197

in a bath (vol. 2.5 ml) and superfused (3 ml.min -I) with standard saline throughout an experiment, except during extracellular application of spermine and philantho- toxin. All of the experiments were undertaken at room temperature, ca. 23°C.

Bath application of both toxins at concentrations as low as 10 -7 M initially potentiated and then depressed the EPSCs elicited continuously at the rate of 1 Hz. The dibromo analogue was about two times more active than PhTX-343. The effects of both compounds were fully reversible, although recovery following application of higher concentrations of these toxins was slow (e.g. time to EPSC half amplitude recovery following application for 10 min of 10 -5 M dibromo-PhTX-343 was about 2 h). If the preparation was not stimulated during extracel- lular application of 10- 5 M toxin an EPSC evoked imme- diately following 30-60 min toxin washout was similar in amplitude to its control EPSC recorded before toxin application. The use-dependent depression of the EPSC caused by these two toxins was not voltage dependent over the potential range - 30 mV to - 100 mV. Whereas depression of the EPSC was clearly use dependent, it was not possible unequivocally to determine whether this was also the case for the potentiation seen initially dur- ing extracellular application of a low concentration of

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Fig. 2. Antagonism of EPSC of locust extensor tibiae muscle fibre by 5 x 10 -3 M Br2-PhTX-343 studied under voltage clamp. A-D: Rela- tionships between mean (+ S.D., n =20) amplitude of EPSC and hold- ing potential for holding potentials between - 3 0 mV and - 1 0 0 mV (A,B) and - 4 0 mV and - 100 mV (C,D). A: data obtained in absence of toxin. B: data obtained 10 min after start of current injection of toxin (see text). C,D: data obtained 40 min (C) and 70 min (D) after

start o f toxin injection.

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either toxin. Spermine also initially potentiated and then antagonised the EPSC but at a higher concentration (10 -5 M) than the two toxins.

For the intracellular injection studies, the effects on the EPSC of 4 concentrations of 3,5-dibromo-PhTX-343 (10 -4 M (n= 1,), 5 x 10 -4 M (n=2), 5 x 10 -3 M (n=2)

and 10 -2 M (n = 2)) and one concentration of PhTX-343 (2 x 10 -3 M (n=2)) were tested. The muscle prepara- tions were superfused continuously with standard locust saline throughout the experiments. The effect on the EPSC of intracellular injection of spermine was not investigated. With the lowest concentration of 3,5- dibromo-PhTX-343, the EPSC amplitude was unaffect- ed 2 h post-injection; with 5 x 10 -4 M, the EPSC ampli- tude was substantially reduced, but only after about 2 h post-injection (Fig. 1). Marked inhibition of the EPSC was obvious immediately after injection of 5 x 10 -3 M 3,5-dibromo-PhTX-343 and 10 2 M PhTX-343. Cur- rent-voltage relationships determined at this time showed that the reduction in amplitude of the EPSC was not voltage dependent (Fig. 2). Also, the reduction in EPSC amplitude was not use dependent. Inhibition of the EPSC increased with time, such that, for example, by about 1 h post-injection of 5 x 10 -3 M PhTX-343 the amplitude of the EPSC had fallen to zero. The concen- tration dependence of the EPSC inhibition was further emphasised following injection of 10 -2 M 3,5-dibromo- PhTX-343, when the EPSC was almost completely abol- ished within 10 rain post-injection (Fig. 1). Recovery of the EPSC was not seen in any of these toxin injection studies, even 6 h post-injection. Intracellular injection of toxin did not potentiate the EPSC.

Control experiments involved (a) impalement of clamped muscle fibres with micropipettes containing dis- tilled water only, but with the current ejection regime de- scribed above; (b) current ejection of toxin from micro- pipettes containing 10 - 2 M toxin (either 3,5-dibromo- PhTX-343 or PhTX-343) which were located extracellu- larly in close vicinity to clamped muscle fibres; and (c) impalement of muscle fibres with micropipettes contain- ing 10 -2 M toxin for 2 h or more without current ejec- tion of toxin. In the first two cases the amplitude of the EPSC was unaffected, which indicates that neither the current ejection regime (a) nor leakage of toxin into the extracellular environment (b) were responsible for the inhibition of the EPSC observed post-injection of toxin. In (c) a small (ca. 15%) reduction in EPSC amplitude was observed in one case out of three, possibly because toxin had diffused out of the micropipette into the myo- plasm even in the absence of current pulses. As a further check, [3H]spermine (10 -3 M in the injection pipettes) was current injected into muscle fibres and its subse- quent intracellular and extracellular distributions were

determined. In two experiments, 10 min current injection of 10 -3 M [3H]spermine resulted in the accumulation in the muscle fibre of 3 x 10 -5 M and 8 x 10 -5 M polya- mine respectively. Only negligible amounts of the radio- label escaped from the injected muscle fibres.

For single channel studies, a total of 40 patches were studied, in which either PhTX-343 or spermine was ap- plied either extracellularly (n = 10) (in the bath saline or in the patch pipette at 10-7-10 -4 M) or intracellularly (n = 30) (at 10 -4 M via pressure injection) into a muscle fibre. All single channel recordings were made at a hold- ing potential of - 110 mV to optimise the signal to noise ratio (> 3:1). The effects of extracellular application of

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Fig. 3. Representative single channel recording of the effects of injected PhTX-343 on extrajunctional QUIS-R of locust metathoracic extensor tibiae muscle fibre. Mega-ohm seal recording with patch pipette con- taining 10 -4 M L-glutamate. Successive 1 s traces (A) prior to injection of toxin, and (B) 3 rain and (C) 7 rain after commencement of injection of 10 -4 M PhTX-343. Channel openings are upwards. Recordings made with muscle fibre clamped at - 110 mV (2). Data were recorded using a List EPC7 amplifier, stored on video tape using a PCM (Sony Corp., Tokyo, Japan) and low-pass filtered at 3 kHz on playback. Note in (B) the presence of bursts of channel openings and in (C) complete loss of channel openings. Although a further 10 min of recording time followed the traces illustrated in (C), channel openings were not

observed.

PhTX-343 (and spermine) were similar to those de- scribed in previous studies by Clark et al. [5] and Karst et al. [10]. Initially, during extracellular application of the lower concentration of toxin or polyamine, there was an increase in channel open time (seen also with the spi- der toxin, argiotoxin-636 (ArgTX-636) [10, 14]. This was followed by a decline in the frequency of channel open- ings, which was reflected in a gradual fall to zero in the channel open probabili ty (Po). Reductions of about 50 % in the mean channel open time (mo) were also observed prior to loss of channel activity. If, following loss of channel activity, the patch pipette was moved to an adja- cent extrajunctional site on the injected muscle fibre, QUIS-R channel openings were recorded for a brief peri- od after ' forming' a patch. In other words antagonism of QUIS-R channel activity by PhTX-343 and spermine was agonist dependent.

Po also decreased during either toxin or spermine injection; declining to zero 1-8 min post-injection (Fig. 3). Increases in mo, such as those recorded during the early stages of extracellular application of these com- pounds were not observed. Patches were monitored for about 10 min after injection of toxin or polyamine, but channel activity never returned. When the patch pipette was then moved to adjacent sites on the injected muscle fibre, QUIS-R channel activity was usually absent, although at a few sites brief bursts of channel openings

interposed between long closed periods were observed. This could imply that when either PhTX-343 or sper- mine is applied intracellularly agonist binding to QUIS- R is not essential for antagonism, which would contrast with the situation prevailing for their extracellular appli- cation. Pressure injection of intracellular saline alone had no effect on QUIS-R channel activity. When 10 -3 M [3H]spermine was pressure injected into a locust mus- cle fibre using the same procedure as that employed for toxin injection, the muscle fibre accumulated 10 -4 M of the radiolabel.

It is clear that philanthotoxins and spermine antago- nise QUIS-R when they are injected into locust muscle fibres and that this antagonism is, in some respects, qua- litatively similar to that observed during their extracellu- lar application. However, there is one major difference. Antagonism is use dependent for extracellular applica- tion only. In both instances, antagonism of QUIS-R is voltage independent which suggests that these com- pounds must be binding to a site on QUIS-R which is outside the membrane potential field [7]. The injection data suggest that this site is on the intracellular domain of QUIS-R. The philanthotoxins used in these studies, like spermine, have hydrophilic properties, so they are unlikely to diffuse across the muscle membrane. This implies that during their extracellular application sper-

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mine, PhTX-343 and 3,5-dibromo-PhTX-347 can proba- bly only gain entry to the binding site on QUIS-R via the open channel gated by this receptor. Such a mecha- nism would account for the observed use dependence of antagonism seen during extracellular application of these toxins. Since antagonism by injected toxin or sper-

mine is independent of QUIS-R Po, it follows that this binding site is available when the QUIS-R channel is both closed and open. Since it has already been estab- lished that PhTX-343 competes with spermine for a polyamine binding site on the N M D A receptor (NMDA-R) of vertebrate CNS, is there any evidence that this site is also intracellular? According to Sacaan and Johnston [18] the polyamine binding site on this receptor is on the intracellular surface of neuronal mem- brane. However, ArgTX-636 antagonism of N M D A - R is voltage dependent [16], which suggests a binding site in the receptor channel for this toxin.

This work was supported by a grant f rom the U.K. Agriculture and Food Research Council to P.N.R.U. and by Grant N I H AI10187 to K.N.

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