Original paper
Alpha-bungarotoxin (α-Bgtx) and beta-bungarotoxin (β-Bgtx) binding activities in human cadaver brain

Introduction
The presence of nicotinic cholinergic receptors in mammalian brain has been suggested by a number of investigators with the application of nicotinic ligands [1, 2]. a-Bgtx is a selective antagonist of postsynaptic nicotinic acetylcholine receptors in vertebrate and insects. By the use of potent nicotinic antagonist 125Ia-Bgtx as a ligand, cholinergic nicotinic receptors has been identified in rat brain [3] and honey bee brain [4]. Three radio ligands have been commonly used to label putative nicotinic cholinoreceptors in the mammalian central nervous system: the agonists 3H nicotine and 3H acetylcholine in the presence of atropine to block muscranic receptors and snake venom extract 125Ia-Bgtx. Binding studies employing brain homogenates indicate that the regional distribution of both 3H nicotine and 3H ACh differ from that of 125I-Bgtx [2]. It was generally agreed that in rat brain there is only one class of sites for a-Bgtx, but there is a difference in properties and in the distribution of a-Bgtx and high affinity nicotine binding sites [18] suggest that these ligands label separate receptor population. b-Bgtx a snake venom toxin which acts solely on the presynaptic membrane of cholinergic nerves [5, 6] and blocks neuronal A-type K+-channels [7]. b-Bgtx produces an irreversible blockade of neurotransmission by acting preferentially on nerve terminals and inhibiting the release of transmitters [8]. Studies on the distribution of the b-Bgtx receptors indicate their predominance in nerve terminal rich areas of rat brain [9]. Release of acetylcholine and choline by the action of b-Bgtx has been studied in various species [10]. 125Ib-Bgtx binding sites were identified in synaptic membrane fraction of chick brain [11], synaptosomes of torpedo electric organ by autoradiography [12], in CNS of rat [13]. High affinity acceptors for 125Ib-Bgtx have been identified on the cryostat sections of the rat’s brain and mapped quantitatively using 3H-sensitive sheet film autoradiography [13]. b-Bgtx affects the release of several neurotransmitters including acetylcholine [22-24] and binds specifically to a single class of non interacting sites of high affinity on the rat’s cerebral synaptosomes [9]. Using 125Ib-Bgtx as a ligand b-Bgtx binding proteins were identified in synaptic fraction of the chick’s brain with an affinity constant of 0.47±0.14 nM [11]. The present study aims to identify the localization of a and b-Bgtx binding proteins in different regions of human cadaver brain.
Material and methods
Alpha bungarotoxin (a-Bgtx), beta bungarotoxin (b-Bgtx), Tween-20, bovine serum albumin (BSA) were from Sigma Chemical co., St. Louis Radio-Iodine 125I from Saxson laboratories, GFC filter discs from Whatman. Other reagents were of analytical grade.
Receptor solubilization
Different cadaver tissues including brain regions were obtained from mortuary of AIIMS. The details are given in Table I. All the tissues were from non smoker fellows. The membranes and their triton extracts were prepared as described earlier [14]. Briefly the tissue was homogenised in four volumes of chilled homogenising buffer (HB) (0.01 phosphate buffer, pH 7.4) containing 1 M NaCl, 0.02% NaN3, 0.001 M EDTA, 0.1 M benzethonium chloride, 0.002 M benzemidene hydrochloride, and 0.0001 M phenylmethyl sulphonyl fluoride and 0.5 mg/ml bacitracin. The homogenate was centrifuged at 20,000 γ for 60 min at 4°C. The supernatant was collected (soluble supernatant) and pellet was suspended in homogenizing buffer and stored in aliquots at –20°C as membranes or extracted for 3 hrs at 4°C in two volumes of HB containing 2% (v/v) Triton X-100. The supernatant (triton extract) obtained on centrifugation at 20,000 γ for 60 min at 4°C was filtered through glass wool, aliquoted and stored as solubilised receptor (Triton Extract) at –20°C.
Radio-iodination of toxin (Alpha-Bgtx and Beta-Bgtx)
Radio-iodination of a and b-Bgtx by Iodogen (1,3,4,6-tetrachloro-3a6a-diphenylglycouril) method was performed [15]. Dichloromethane solubilized iodogen was introduced into glass tubes and evaporated with gentle swirling by passing nitrogen gas to form a thin film. To the precoated Iodogen tube, phosphate buffer + 2-10 µg a or b-Bgtx + 0.5 mCi of 125I Na, were added in sequence and the resultant volume was made to 100 µl. The mixture was incubated for 15 min at RT followed by addition of 20 µl of 2% KI. The contents were gently mixed and transferred onto Sephadex G-25 column for separating toxin from free Iodine. Elution was done at RT with 0.01 M phosphate Buffer containing 0.1% BSA. The void volume peaks containing radio-iodinated toxins were pooled and stored aliquoted at –20°C.
Binding assay for receptor activity
The activity of postsynaptic AChR and presynaptic membrane receptor in membrane preparation and triton extract of muscle and brain tissue was determined by virtue of its high affinity for a and b-Bgtx. Radio-iodinated a or b-Bgtx was used as a ligand. The free and bound ligand was separated by rapid filtration method in case of membrane preparations [16] and ammonium sulphate precipitation method in triton extracts i.e; solubilized receptor [17].
Rapid filtration method
The membrane suspension was incubated with 125Ia or b-Bgtx in 0.01 M phosphate buffer pH 7.4 containing 0.1% BSA for 30 minutes at room temperature. Incubation was followed by addition of 5 ml of ice cold 0.01 M phosphate buffer pH 7.4. The mixture was rapidly filtered through glass filter disc (2.5 cm diameter). The disc was washed 3 times with ice cold 0.01 M phosphate buffer (5 ml each) and counted for radioactivity in a g-counter [15, 16].
Ammonium sulphate precipitation method (for solubilized receptor)
125Ia or b-Bgtx was incubated with triton extract for 60 minutes at 37°C. 60% saturated solution of ammonium sulphate was added and allowed to stand for 16 hrs at 4°C. The precipitate was then filtered on GFC glass filter discs. The disc was washed 3 times with 30% ammonium sulphate solution and the radioactivity was counted on a g-counter [17]. To determine the non specific binding the samples was incubated in the presence of 100 molar excess of non radioactive toxin. The difference in total binding and non-specific binding counts represents the specific binding of the receptors.
Results Binding assays and specificity
a-Bgtx and b-Bgtx were iodinated by the iodogen method as described in the methodology [15]. The elution profile is shown in Figure 1. The peak fractions (radio-iodinated toxins) were pooled and counted in a gamma counter and their specific activity was calculated. Specific activities of three preparations of radio-iodinated a and b-Bgtx are given in Table II. The mean specific activities of 125Ia-Bgtx and 125Ib- Bgtx were 1.38±0.02×108 cpm/nmole and 5±2.6×108 cpm/nmole, respectively. After collecting the tissues from mortuary we wash it cold PBS and then proceesed for making membranes and solubilized receptor. The specific binding of a-Bgtx to membranes and triton extract of fresh muscle is 19.8±0.2 and 10.2±0.12 fmoles/mg tissue respectively. While in cadaver muscle, the specific biding is 19.4±0.15 and 9.8±0.16 fmoles/mg tissue in membranes and triton extract. For b-Bgtx, the specific binding with membranes and triton extract of fresh muscle is 10.2±0.15 and 7.2±0.12 fmoles/mg tissue. Similarly the specific binding of b-Bgtx with membranes and triton extract of cadaver muscle is 9.6±0.14 and 7.1±0.12 fmoles/mg tissue. The extent of non specific binding was calculated by adding 100 M excess of non radioactive toxin. Figure 2 shows that specific binding of radio-iodinated a and b-Bgtx to membranes/triton extract of fresh and cadaver muscle is identical. The binding is saturable and concentration dependent. Binding of radio-iodinated toxin (a or b) to triton extract of parietal lobe and cerebellum was done by ammonium sulphate precipitation method as described in methodology. Specific a and b-Bgtx binding activity was observed in parietal lobe, while as in cerebellum shows only b-Bgtx activity was observed as shown in Figure 3. In addition to parietal lobe and cerebellum, specific a and b-Bgtx binding was observed in liver and different regions of brain. Specific binding in terms of fmoles/mg tissue was studied in parietal lobe (6±0.6) occipital lobe (6.1±0.1) temporal lobe (5.2±1.4), cerebral cortex (6.2±0.3), hippocampus (5.6±0.2), hypothalamus (4.3±0.4) and brain stem (4.8±0.2). No specific binding of alpha bungarotoxin was found in cerebellum and frontal lobe. Beta bungarotoxin binding activity was present in all the above regions of brain and was in range of 4.2-4.9 fmoles/mg tissue (Figure 4). Both a and b-Bgtx was absent in liver.
Specificity
Using competition ELISA (Figure 5), it was observed there was a consistent decrease in bound cpm with a-cold toxin but not with b-cold toxin, when triton extract of skeletal muscle was incubated with 125Ia-Bgtx and competed with both a and b-cold toxin. Similarly triton extract of cadaver skeletal muscle was incubated with 1.5×105 cpm of 125Ib-Bgtx in the presence of different concentration of cold toxin (a or b). There was decrease in bound cpm with a cold toxin, but no competition was observed with β (Figure 5). Hence indicating that 125I-a Bgtx and 125I-b Bgtx are specific for the a-Bgtx and b-Bgtx binding proteins respectively. The binding of radiolabelled a-Bgtx was found in membranes and triton extracts of human cadaver tissue. No specific binding was observed in frontal lobe, cerebellum and liver membranes and triton extract hence indicating that a-Bgtx binding proteins are absent in these tissues. Radio-labelled b-Bgtx binding sites was also observed in the membranes and triton extract of human cadaver tissue. No specific binding was observed in liver membranes and triton extract (Figure 4). The binding was described in terms of fmoles/mg tissue. Different tissues of human cadaver were collected on different postmortem time. The details are given in Table III. Specific binding of radiolabelled a and b-Bgtx were studied in membranes and triton extract of different tissues. It was observed there is not a significant decline in specific binding to membranes and triton extract of muscle with increase in post mortem time (5-24) (Figure 6 and Table IV). There is a decline (11-24 hrs) in specific binding of radiolabelled a and b-Bgtx to membranes and triton extract of different brain regions (Figure 7). This means that a and b-Bgtx binding proteins in muscle are more stable.
Discussion
Regional distribution of 125Ia-Bgtx binding protein in rat brain has been identified by autoradiography and revealed high binding in hippocampus, hypothalamus and other regions, while as cerebellum was devoid of specific binding [3]. Using micropunched tissue homogenates 125Ia-Bgtx binding was observed in hypothalamus followed by hippocampus, cortex, globus pallidus, nucleus caudatus and nucleus accumbens of rat brain [1]. Invitro autoradiography reveals the presence of 125Ia-Bgtx binding sites, localized in the subesophageal ganglion, the optic tubercles, antennal lobes of honey bee brain [4]. Three radio ligands have been commonly used to lable putative nicotinic cholinoreceptor in the mammalian central nervous system:the agnosists 3H nicotine and 3H acetylcholine in the presence of atropine to block muscranic receptors and snake venom extract 125Ia-Bgtx. Binding studies employing brain homogenates indicate that the regional distribution of both 3H nicotine and 3H acetylcholine ACh differ from that of 125Ia-Bgtx [2]. It is generally agreed that in rat brain there is only one class of sites for a-Bgtx [7] but difference in properties and distribution of a-Bgtx and high affinity nicotine binding sites [18]. b-Bgtx blocks cholinergic transmission by presynaptic action [19]. High affinity b-Bgtx binding sites were identified in CNS of rat [9, 11, 20, 21], torpedo [12] chick [7]. Enriched areas of grey mater and synaptic regions of central nervous system of rat brain, particularly binds with 125Ib-Bgtx. Using 3H sensitive sheet film autoradiography, it has been demonstrated that b-Bgtx acceptors are voltage sensitive K channels [13]. Electron microscopic autoradiography was used for evaluation of 125Ib-Bgtx in synaptosomes of torpedo electric organ, which inhibits the release of transmitter on plasma membrane [12]. Using these radiolabelled toxins (a or b-Bgtx) as a specific ligand, we explored the regional distribution of a-Bgtx and b-Bgtx binding sites in human cadaver muscle and in different regions of brain. In most brain regions 125Ia-Bgtx binding sites are localized to neuronal cell bodies (Breese et al., 1997). Specific binding of radio-iodinated a and b-Bgtx was carried out in membranes (rapid filtration method) and triton extract (ammonium sulphate precipitation method) of fresh and cadaver muscle. The binding was saturable and concentration dependent (Figure 2). a and b-Bgtx binding proteins have been identified in the membranes and triton extract of different tissues of human cadaver (Figure 5). a-Bgtx binding proteins were absent in the cerebellum, frontal lobe and liver tissues. Radio-labelled b-Bgtx binding sites were also observed in the membranes and triton extract of human cadaver tissue. All the brain regions, skeletal muscle and heart (except liver) contain presynaptic receptor (b-Bgtx binding protein) (Figure 5). The cadaver tissues used for this study differed in postmortem as well as the age of the cadavers. There was no significant decline in specific binding to membranes and triton extract of muscle with increase in post mortem time (5-24 h), but there is a little decline (11-24 h) in specific binding of radiolabelled a and b-Bgtx to membranes and triton extract of the different brain regions (Figures 6-7). It has earlier been reported that postmortem delays (up to 96 h at 4°C) did not produce any significant changes in the density and binding properties of the nAChR [25]. We also observed that in addition to postsynaptic receptors (a-Bgtx binding proteins), presynaptic receptors (b-Bgtx binding proteins) also shows immunoreactivity with Myasthenic sera.
Conclusions
In conclusions, MG is not only due to the damage of the postsynaptic membrane, but it could concurrently be a consequence of antibody mediated damage of the presynaptic membrane, resulting particularly the damage of its b-Bgtx binding sites and could play an important role in the pathogenesis of myasthenia gravis.
Acknowledgement
We thank to Indian Council of Medical Research for providing grant.

References:
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