Excitatory effects of Buthus C56 toxin on Drosophila larval neuromuscular junction

ORIGINAL PAPER

Excitatory effects of Buthus C56 toxin on Drosophila larval neuromuscular junction

S. P. Gawade

Department of Pharmacology, Al-Ameen College of Pharmacy, Hosur Road, Near Lalbagh main gate Bangalore, India

^{Address to correspondence}

ABSTRACT

Buthus C56 toxin from venom of the Indian red scorpion Mesobuthus tamulus was studied for its effects on spontaneous miniature excitatory junctional potentials (MEJP) on Drosophila larval neuromuscular junctions. C56 toxin was isolated on CM-Cellulose with linear gradient of ammonium acetate buffer, pH 6.0. Toxin purity was determined on SDS slab gel electrophoresis. Effective concentration of C56 toxin was based on contraction paralysis units (CPU) in Drosophila 3rd instar larvae by microinjection (0.1 CPU/ml = 2 x 10^–6 g/ml). The toxin-induced excitatory junctional potentials were studied for calcium dependency (0.2 mM to 1.2 mM Ca²⁺) in Drosophila Ringer. Excitatory junctional potential amplitude was increased with increasing calcium concentration; maximum increase in the frequency at 0.4 mM Ca²⁺/4 mM Mg²⁺Drosophila Ringer. It was suggested that while amplitude of excitatory junctional potentials was increased with concentration, maximum frequency increase at 0.4 mMCa²⁺/4 mM Mg²⁺Drosophila Ringer may be due to augmented Ca²⁺ influx in 0.4 mM Ca²⁺, when NMDA receptors were maximally activated in C56 toxin-treated Drosophila larval neuromuscular junction.

Keywords: Mesobuthus tamulus, C56 toxin, Drosophila melanogaster, neuromuscular, miniature excitatory junctional potentials (MEJPs).

INTRODUCTION

Indian red scorpion Mesobuthus tamulus is usually found under rocks and in crevices at the base of shrubs and bushes, abundant in the Konkan region of Western Maharashtra, especially in winter. Scorpions are vigorous diggers with front legs and exhibit negative geotaxis. Venom is stored in the telson and is used primarily for defense and paralyzing the prey. Scorpion venom is a heterogeneous collection of pharmacologically active constituents. Bioassays such as mice lethality, fly larvae contraction paralysis, and isopod paralysis have been used to detect and to isolate of the mammal, insect, and crustacean scorpion toxins. In this paper, the major toxic component from the Indian red scorpion Mesobuthus tamulus venom causing reversible contraction paralysis in 3rd instar Drosophila larvae was studied for its effects by intracellular recordings on Drosophila larval nerve muscle preparation.

MATERIALS AND METHODS

Mesobuthus tamulus venom

Mesobuthus tamulus venom was obtained from the Haffkine Institute, Mumbai, by electrical milking method. The lyophilized milked venom was a matrix containing glycoprotein, cellular debris, and mucus material. The venom was reconstituted in distilled water and centrifuged at 10 KDa for 15 min to remove insoluble matrix.

Potency determination

Venom paralyzing potency and Buthus C56 toxin was quantitatively determined by injecting samples in the posterior abdominal segments of 3rd late instar Drosophila larvae cultured at 22°C. The quantity required for minimal duration of contraction of 10 seconds in 50% of test larvae (12) was defined as contraction paralysis unit (CPU).

Fractionation of Buthus C56 toxin

Buthus C56 toxin was isolated by CM-cellulose column chromatography using linear increase in the concentration of ammonium acetate buffer (0.05M, 0.2M,0.5M) at pH 6.0 (Figure 1). Protein concentration was determined by the Hatree method (10), using bovine serum albumin (Sigma) as a standard. Homogeneity test was performed on polyacrylamide gel electrophoresis (PAGE) in 12% acrylamide gel and b-alanine acetic acid buffer at pH 4.3 (13). Molecular weight of C56 toxin was determined on 15% sodium dodecyl sulphate (SDS)-PAGE using standard markers.

Figure 1
. Chromatographic and electrophoretic separation of Buthus C56 toxin.

Intracellular recordings

Electrophysiological studies were performed using the conventional procedure of intracellular recordings (8,11) with glass microelectrodes of resistance 5 to 12 mW filled with 4 M potassium acetate. Drosophila Ringer solution (DR) of composition (mM) NaCl 120, KCl 2, MgCl2 4, CaCl₂ 0.6, sucrose 7.2 was buffered with HEPES 1, at pH 7. The 2^nd dorso-lateral tubular muscle of Drosophila melanogaster (C-S canton) was used for recording miniature excitatory junctional potentials (MEJPs). The test sample was injected into the bathing solution after 5 min stabilization. The electrical events were monitored on the oscilloscope and recorded on a continuous film connected to an oscillographic camera. Resting membrane potential remained stable for at least 150 min and each observation was independently repeated on five different preparations.

Statistical analysis

Two tailed P values were analyzed statistically from the Mean ± S.E.M.(n=5) of excitatory activity with the control using unpaired Welch corrected “t” test.

RESULTS

Mesobuthus tamulus venom in concentrations from 0.5 to 1 X 10^–7 g/larva caused an instant longitudinal contraction of Drosophila larvae followed by complete paralysis. The venom was resolved into seven fractions by CMC column chromatography. The contraction paralysis of Drosophila larvae by microinjection was used as an activity assay. Only two fractions, C35 and C56, showed contraction paralysis. C56 toxin was five times more potent in producing contraction paralysis and revealed single band on SDS-PAGE (Figure 1). The apparent molecular weight by SDS-PAGE was found to be about 8000 Da. At the concentration of 2 x 10^-6 g/ml (0.1CPU/ml = 50 larval CPU) in 0.6 mM Ca²⁺/4.0 mM Mg²⁺ DR, C56 toxin replaced MEJPs by EJPs, the frequency and amplitude of which was increased. Buthus C56 toxin-induced spontaneous excitatory activity in 0.6 mMCa²⁺/4 mM Mg²⁺ Ringer amplitude distribution histogram showed single peak gaussian distribution with right shift (7). Control MEJP_f and MEJP_amp in 0.6 mM Ca²⁺/4 mM Mg²⁺ DR was 2.38 ± 1.59 (/sec) and 0.8 + 0.13 (mV), respectively. The amplitude of these potentials increased with calcium concentration (0.2 mM to 1.2 mM in DR from 0.22 to 3.97 times), whereas frequency was maximum [(18.95 ± 0.45 per sec.(***p), (8.42 times higher)] in 0.4 mM DR solution. Extracellular calcium played a critical role in inducing the spontaneous excitatory activity that was eliminated in 2 mM Co²⁺ and 0 Ca²⁺ DR. The transformation of MEJPs into an EJPs following C56 toxin treatment (t) was initiated at 0.2 mM Ca²⁺ [0.45 ±0 mV (c) to 0.55 mV ± 0.13 (t)] progressively increased with calcium concentration compared to controls (c). Table 1 shows the results of Buthus C56 toxin on MEJP_f and MEJP_amp before and after application in Ca²⁺ modified DR. The excitatory effects of C56 toxin on Drosophila larval n-m junction are shown in Figure 2.

Figure 2.
The effects of Buthus C56 toxin on MEJP(f) and MEJP(amp.) on Drosophila larval n-m junction.

Table 1
. The effects of Buthus C56 toxin on MEJPf/sec and MEJPamp. (mV) on Drosophila larval n-m junction.

DISCUSSION

We studied Buthus C56 toxin-induced calcium dependent spontaneous excitatory activity on Drosophila larval n-m junction. Calcium dependency was also observed in inducing tetrodotoxin insensitive vesicular release of acetylcholine due to Buthus C56 toxin from Torpedo presynaptic synaptosomal preparation (9). The excitatory activity was primarily dependent on extracellular Ca²⁺ as a Ca²⁺ channel blocker; Co²⁺ (2 mM) or (0) Ca²⁺ eliminated this activity. Inotropic glutamate N-methyl-D-aspartate (NMDA) receptors activation have been shown to result in an increase in Ca²⁺ influx (15). It is possible that the sudden activation of a large pool of glutamate NMDA receptors by Buthus C56 toxin resulted in increased calcium availability with resultant augmented interaction of excitatory neurotransmitter L-glutamate with its receptors at the Drosophila larval n-m junction, leading to generation of EJPs. Amplitude of these EJPs was increased with Ca²⁺ concentration. Concomitant occurrences of EJPs of two amplitudes at the concentration of 0.4 mM Ca²⁺ resulted in an increase in the frequency of EJPs, which was statistically significance. Muscular twitching, spasms in muscle, and respiratory paralysis were typical peripheral symptoms of scorpion envenomation. Toxin XIII-3-a of molecular size 8000 Da. (LD50 I.V mice mg/kg) isolated from Buthus tamulus venom showed protease inhibitory activity, which resembles the kazal type pancreatic inhibitor (2). Neurotoxic peptides Tamulotoxin (TmTX) and Iberiotoxin (IbTX) with 37 amino acid residues and 3 disulfide bridges isolated from Mesobuthus tamulus have been shown to cause calcium activated K⁺ channel blockade (1,4-6), whereas Tamulustoxin [35-3], blocked selectively activated K⁺ channel (16). Toxin isolated from Androctonus australis scorpion venom showed similar contraction paralysis in blow fly larvae (18,19) and induced spontaneous excitatory activity followed by a blockade of transynaptically evoked response from cockroach 6^th abdominal ganglion preparation (3). Increased sodium conductance was suggested to be a presynaptic effect of Androctonus toxin on locust n-m preparation (17). NMDA activation type of glutamate receptors at the glutaminergic synapses have been suggested as causing an opening of large number of calcium channels with consequent intracellular calcium excess involved in the excitotoxicity in degenerative diseases (14). When EJP amplitude was increased with concentration, maximum increase in EJP frequency in 0.4 mM Ca²⁺/4 mM Mg²⁺ Ringer was suggested to be due to maximum NMDA activation type of glutamate inotropic receptors for the augmented Ca²⁺ influx in C56 toxin-exposed Drosophila larval n-m junction.

ACKNOWLEDGEMENTS

This work was carried out at Molecular biology unit of the Tata Institute of Fundamental Research during 1981-1982 under ICMR Post-Doctoral fellowship awarded to the author. Help of Prof. O. Siddiqi is acknowledged for supporting the application to ICMR to financial support. Help and encouragement of Prof. B. G. Shivananda, Principal of Al-Ameen College of Pharmacy, Bangalore, is also acknowledged.

Received September 27, 2001

Accepted December 12, 2001

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Publication Dates

• Publication in this collection
  09 Dec 2003
• Date of issue
  2003

History

• Accepted
  12 Dec 2001
• Received
  27 Sept 2001