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1.1 Scorpion toxins: Scorpions belonging to the family Buthidae consist of most lethal and poisonous species which can be identified from non-venomous scorpions by the presence of thick tail, thin pincers and a triangular sternal plate. The poison glands are found on the tip of their long tail or telson that also delivers sting to paralyse its prey (Figure 1). The dangerous species are also known as Old World scorpions due to their presence in North Africa (Leiurus, Androctonus), Middle East (Buthus Leiurus) and India / Asia (Mesobuthus). Scorpion venom contains multiple toxins such as neurotoxins, nephrotoxins, hemolytic toxins and other compounds including polypeptides, mucus, oligopeptides, histamine, serotonin, each exhibiting different pharmacological activities (Gwee et al, 2002). Envenomation from scorpion sting presents with neuromuscular, local tissue effects and sometimes, autonomic excitation which leads to cardiopulmonary effects. Serotonin in the venom is known to contribute to the pain after sting and the toxicity to mammals is due to small polypeptide neurotoxins. These neurotoxins are the most potent and main component found in the scorpion venom (De Lima et al, 2007) and can be divided into long chain toxins composed of 60-70 amino acids residues cross-linked by 4 disulfide bridges that affect exclusively voltage-dependent Na+ channels of excitable cells. The second group has short chain toxins consisting of 30-40 amino acids residues cross-linked by 3 disulfide bridges that block several types of K+ channels in excitable cells as well as erythrocytes and lymphocytes (Legros and Martin-Eauclaire, 1997).
Figure 1 showing the poison gland from a venomous scorpion. At the end of the abdomen is the telson, a bulb-shaped structure containing a pair of venom glands and a sharp, curved stinger to deliver venom (ag.arizona.edu/pubs/insects/az1223).
1.2 Targets of scorpion toxins:
The main molecular targets of scorpion neurotoxins are the voltage- gated sodium (Na+) channels and the voltage-gated potassium (K+) channels. In other words, scorpion neurotoxins act mainly on excitable cells like nerves and muscle.
SCORPION TOXIN ACTIONS AT VOLTAGE-GATED SODIUM CHANNELS
The long-chain peptides containing neurotoxins found in the scorpion venom target the voltage gated sodium ion channels of excitable cells (Cestele and Catterall, 2000). These neurotoxins have a specific bioactivity and a high level of specificity in terms of its target; mammals, insects or crustaceans (Possani et al, 1999). These long chain peptides have a highly conserved secondary structural arrangement comprising of three strands of an antiparallel β-sheet and a stretch of α-helix, stabilised by four intramolecular disulfide bridges formed by a number of cysteine residues (Rodriguez de la Vaga and Possani, 2005). Ion channels are transmembrane proteins found in all excitable cells where they facilitate the diffusion of ions across biological membranes which is essential for the rapid electrical signalling required to maintain all physiological processes in the body. The voltage-gated sodium channels (VGSCs) conduct Na+ ions which is responsible for the initiation and propagation of action potential in excitable cells and release of neurotransmitters from nerve terminals. A slight change in sodium channel function causes significant consequences on membrane excitability, sometimes resulting in organism paralysis. The mammalian sodium channels consist of a large pore forming α subunit of 260 kDa associated with auxiliary subunits: β1, β2 and β3 (Figure 2). The α-subunit consists of four homologous domains (I-IV), each containing six transmembrane segments (S1-S6). The positively charged S4 segment acts as the voltage sensor to initiate the voltage-dependent activation of sodium channels by moving outward under the influence of membrane depolarisation. Inactivation is mediated by the short intracellular loop connecting domains III and IV. The short loop between S5 and S6 forms the ion selectivity filter and the outer region of the pore (Marban et al, 1999).
Figure 2 (A) structure of the voltage gated sodium channel, α and β subunits. The transmembrane segments (S1-S6) and homologous domains (I-IV) form the α subunit. The P regions between S5 and S6 forms the pore and the intracellular loop between domains III and IV forms the inactivation gate. (B) The three main conformational states; closed, open and inactivated of the VGSC. Non-conducting closed state is favored by hyperpolarization while open and inactivated states are favored by depolarization. (C) The central conducting pore is surrounded by four transmembrane 'gating' pores which allows the movement of the charged S4 voltage sensor containing region (Errington et al, 2005).
As described before the scorpion venom is a mixture of 50-100 polypeptide toxins. To date only 200 distinct polypeptide toxins have been described from 30 different species of scorpions (Srinivasan et al, 2001). For example, full- length cDNA of about 370 nucleotides encoding precursor of toxin active on mammals and insects has been isolated from the scorpion, Androctonus australis. Sequence analysis of the cDNAs have shown that the precursors contain signal peptides of about 20 amino acid resides which is different in toxins targeting mammals. The latter have extensions of arg or glu-arg at their COOH-terminus (Bougis et al 1989). Such information is valuable to understand the selectivity and mode of action of toxins in terms of differences in its molecular target. A SCORPION database has been compiled to allow extracting existing information in the field of scorpion toxins and help better classify the toxins from different species. The SCORPION database contains several entries of fully referenced scorpion toxin data which includes primary sequence, three dimensional structures and other related structure-functional annotations information.
1.2.1 Modulation of sodium channels by scorpion toxins
VGSCs are the molecular targets for a broad range of neurotoxins which alter the channel function by binding to specific receptor sites. Atleast 7 distinct receptor sites have been identified on the α-subunit of the channel by pharmacological competition and mutagenesis studies (Figure 3). Of these 6 sites are known to bind neurotoxins and one site with local anaesthetics and related drugs. Site 1 binds to water-soluble toxins such as tetrodotoxin, saxitoxin and conotoxin which block the pore and hence inhibit the sodium conductance. Site 2 binds to lipid soluble alkaloid toxins such as batrachotoxin, veratridine, aconitine and grayanotoxin that are activators of the channel. These cause block of channel inactivation and shift the voltage dependence of activation to more negative potentials (Yong-Hua et al 2008). Site 3 binds to a certain group of scorpion toxins; α-toxins, spider toxins and sea anemone toxins that are known to delay or block the channel inactivation. The site 4 binds to a different group of scorpion toxins; β-toxins, which shift the voltage dependence of channel activation towards hyperpolarisation. Site 5 binds to brevetoxin and ciguatoxin that also block channel inactivation and shift the voltage dependence of activation towards negative potentials. The site 6 binds to δ- conotoxins which inhibit channel inactivation. Site 7 has been shown recently to bind pyrethroid insecticides which block the inactivation and slow down deactivation of sodium channels (Gordon, 1997). The action of these toxins is summarised in Table I.
Figure 3 showing the neurotoxins bound to their specific receptor sites on the α subunit protein of the sodium channel. Tetrodotoxin (TTX) binds to receptor site 1; veratridine (VER) and batrachotoxin (BTX) alkaloid neurotoxins with site 2; scorpion α-toxin (αScTx), sea anemone toxin II (ATX II) binds to receptor site 3; scorpion β-toxin (βScTx) at site 4; brevetoxin (Brev) at site 5; aconotoxin (δTxVI) binds to receptor site 6; DDT and pyrethroids (Pyreth) with site 7.
Table I showing the receptor sites identified by neurotoxins binding on VGSCs (Cestele and Catterall, 2000).
1.2.2 Classification of scorpion Na+ channel toxins:
As illustrated above the scorpion toxins can modulate the function of VGSCs via pharmacological manipulation of their properties. Based on the target organisms the toxins can be insect, mammalian or crustacean- specific. This specificity is due to the relative position of the C-terminal peptide in relation to the hydrophobic surface common to all scorpion toxins. The mammalian Na+ channel toxins are divided into two major classes: α and β- toxins depending on their physiological effects on the opening and closing kinetics of the channel. However another class known as α-like toxins exists. These toxins are structurally similar to α- toxins but show insect as well as mammalian toxicity. The α-toxins affect the inactivation kinetics of Na+ channels, whereas the β-toxins modify the activation kinetics (Figure 4). The α-scorpion toxins bind to a receptor site 3 in the S3-S4 extracellular loop in domain IV (IVS3-S4) of the Na+ channel α-subunit. Because of its close proximity to the voltage-sensing IVS4 segment, the toxin bound across the IVS3-S4 loop prevents the outward movement of the IVS4 segment during depolarization (Gwee et al, 2002). Thus, α-scorpion toxins trap the voltage sensor (IVS4) in the inward (not-activated) conformation during channel activation and hence slow or delay the channel activation. This is seen as prolongation of the action potential (Rodriguez de la vaga and Possani et al, 2005). These toxins are found mostly in the Old World scorpion and a few New World species. The 'anti-insect' Lqh αIT is an example of α toxins (Rodriguez de le Vega et al, 2005). The β- toxins on the other hand found exclusively in the New World scorpions (North and South America) bind to receptor site-4 in the S3-S4 extracellular loop in domain 2 of Na+ channels. During activation of the Na+ channel, the IIS4 voltage sensors move outward. The β-toxin interacts with the extracellular end of the IIS4 segment and traps it in this outward, activated position. This enhances activation of the channel in subsequent depolarization and, consequently, causes a shift in the voltage dependence of activation to more negative membrane potentials. This promotes spontaneous and repetitive firing of action potentials (Srinivasan et al, 2001). This difference in the action of these two types of toxin is due to their ability to bind on distinct receptor sites, α-toxin on site-3 and β-toxins on site-4 on the sodium channel.
The following table II (Possani et al, 1999) sums up the various groups of toxins described so far [Centruroides suffusus suffusus (Css), Leiurus quinquestriatus quinquestriatus (Lqq), Androctonus australis Hector (AaH), Leiurus quinquestriatus hebraeus (LqH)]
Class of toxin
Classical α-toxins active on mammals, New World, α-type toxin (Lqq III) and anti-mammal specific.
Toxic to insects and crustaceans.
Specific for both mammals and insects. AaH IT1 (excitatory insect toxin) and Lqh IT2 (depressant toxin) in insect neurons.
Very weakly active on insects
Competitively inhibit both AaH II and Lqh αIT. Show moderate affinities to both mammal and insect sodium channels so intermediate between α and α-like
Weakly active toxins
Poorly characterized with α-type activity and nontoxic to mammals.
Not very specific, as they are also active on mammals and inhibit sodium current inactivation in rat neuronal cells.
Competes for excitatory insect (AaH IT1) and both α (AaH II) and β (Css II) mammal toxin binding
Toxic to both mammals and insects.
Table II to illustrate the various groups of Na+ channel scorpion toxins
Figure 4 showing the α, β1 and β2 subunits of the Na channel and the binding site for scorpion toxins on VCSC. The α-scorpion toxin (α-ScTx) affects the inactivation kinetics, whereas the β-scorpion toxins (β-ScTx) modify the activation kinetics (Cestele and Catterall, 2000).
1.3 Importance of scorpion Na+ toxins: Scorpions are interesting organisms to study due to their medical importance and the presence of a variety of biologically active components in their venoms of which the polypeptides toxins are widely studied as these interact with ion channels in excitable membranes (Possani et al, 1999). The scorpion toxins have already been used as 'molecular yardsticks' (Blumenthal and Seibert, 2003) in providing important pharmacological tools for probing the structure and function of VGSCs. Hence, neurotoxins from scorpions are essential agents for identification and purification of many target molecules found in excitable tissues and to analyse their distribution, isoform patterns, and physiological function.
Another important use of scorpion toxins comes from their ability to distinguish between vertebrate (mammalian) and invertebrate (insect) sodium channels. Some neurotoxins in the scorpion are highly active against some insects like leaf-eating moths, locusts, flies and beetles but have no effect on beneficial insects like honeybees or on mammals like humans. These insect-selective scorpion toxins have the potential for use in the development of novel and safe insecticides. The insect selective excitatory β- toxins such as AahIT (from Androctonus australis hector) and LqqIT1 (from Leiurus quinquestriatus quinquestriatus) induce spastic paralysis caused by repetitive activity of motor neurons due to activation of sodium currents at more negative membrane potentials. Anti-insect depressant toxins such as LqhIT2 (from Leiurus quinquestriatus hebraeus), Bj IT2 (from Bothotus judaicus), Lqh-dprIT3 induce flaccid paralysis upon injection into blowfly larvae of insects by blocking the action potentials propagation due to a strong depolarization of the membrane (Gurevitz et al, 2007) and have a greater scope in insect pest control. Current efforts are concentrating on engineering the toxin peptides to be able to penetrate into the insect blood stream to impact the insect nervous system and not get metabolised in the insect gut.
Studying the scorpion toxins is also useful in managing severe scorpion envenomation by developing new antidotes to scorpion venom. Scorpion envenomation is public health problem in tropical and subtropical countries where lethal species especially Mesobuthus tamulus flourishes. The venom from Mesobuthus is a powerful sodium channel activator which results in overactivty of the autonomic nervous system. The toxins in scorpion venoms mediate selective actions on voltage-gated Na+ and K+ channels resulting in the enormous release
of autonomic neurotransmitters which are mainly responsible in the pathophysiolgy of scorpion envenomation. The symptoms include vomiting, sweating, priapism in males, cool extremities, pulmonary oedema and shock. Fatality occurs due to the cardiovascular manifestations of the venom. Scorpion antivenom (SAV) is a specific antivenin used in scorpion stings but it has no major beneficial effects in reversing cardiovascular effects of the venom (Bawaskar et al, 2007). Immunological studies of neurotoxins proposed by Delori et al, 1981 obtained antitoxins from rabbits injected with purified neurotoxins from Androctonous and Buthus species. These antitoxins neutralised the toxins belonging to the same group as the antigen. Such immunotherapy techniques could enable developing anti-sera from animals that would be effective against most species of scorpions (Gazarian et al, 2005).
Because VGSCs play an important in pain signal conduction, many neurotoxins targeting VGSCs could also produce potent anti-nociceptive effects. The knowledge of scorpion toxin action on the VGSCs can enable manipulating the toxin activity by computational analyses at the gene sequence level to make them either more potent or specific for certain pain mediating sodium channels (Errington et al, 2005). Engineering the chemical derivatives which mimic these scorpion toxins could provide for novel pain killers with high specificity and minimal side effects. For many such reasons, scorpion venoms are a valuable source for new and potential analgesic drugs. Infact, Chinese traditional medicine system have been using whole scorpions, tails or the venom extracts to treat epilepsy, facial paralysis and chronic pain for since hundreds of years (Yong-Hua and Tong, 2008).
1.4 Known facts about venoms to establish the assay
Various studies involving the isolation, purification and pharmacological screening and characterization of venoms and toxins from various animal sources have taken place.
The venom of the African scorpion Leiurus quinquestriatus has been studied in detail to understand its mechanism of modulation of the kinetics of the voltage-sensitive sodium channels in excitable membranes. Scorpion α-toxins LqhαIT, Lqh-2, and Lqh-3 have been purified and characterised from the venom of Leirus quinquestriatus hebraeus. These toxins can be classified on the basis of their selectivity to mammalian and insect Na+ channels (Gordon et al., 2002). Lqh-2 is a classic α-toxin highly active in mammalian Na channels. It is also called anti-mammalian toxin. LqhαIT is α-insect toxin, active in insects while Lqh-3 is a α-like toxin active on both the mammalian and insect central nervous system. A β- toxin that acts preferentially on the activation of Na+ currents of nerve has also been identified and purified from Lq venom (Rack et al, 1987).
The binding of scorpion α-toxins to mammalian sodium channels is voltage dependent as their affinity decreases with membrane depolarization which suggests that the receptor site 3 undergoes conformational changes during depolarisation leading to changes in affinity properties of various toxins (Leipold et al, 2004). Although these three toxins induce similar modifications of the channel properties in slowing down channel inactivation and increasing the peak inward sodium current, their kinetics of association and dissociation are very different (Figure 5). Such an action of Lq venom is consistent with its expected actions on nerve transmission, because it contains toxins that can modify neuronal Na+ channel activity to enhance transmitter release (Gwee et al, 2002).
Figure 5 (left) showing the effect of Lqh-2 and Lqh-3 toxins on the sodium channels in the rat brain (rB) expressed in HEK 293 cells. After depolarising the cells, 5 nM Lqh-2 and 2 µM Lqh-3 toxin was added to see the effect of channel kinetics and current-voltage relationship. Each toxin increased the peak inward sodium current and slowed the time for inactivation of the Na+ channel, with Lqh-2 showing more potency than Lqh-3 in modulating channel activity (Gilles et al, 2000). Lqh-3 shows lower (1000 fold) affinity of binding to its receptor site 3 and shows effect at a higher concentration than Lqh-2 to affect sodium current inactivation in this rB II channel subtype. Na+ currents from whole cell recordings of HEK-293 cells expressing Na channels in the absence (control) and presence of 5 nM Lqh-2 Lqh-3 and LqhαIT toxin respectively (Right). Under control conditions, the sodium channels inactivate rapidly but in the presence of toxins, the inactivation is slowed down (Chen et al, 2000).
Veratridine is a naturally occurring toxin derived from plants. It has been widely used as an activator of VGSCs in high throughput fluorescent based assays for evaluating the efficacy of sodium channel blockers (Farrag et al, 2008). It binds on the neurotoxin site 2 on the VGSCs, which incorporates residues on the S6 region of the D1 domain of the channel. Veratridine binds preferentially to open or activated state of sodium channels and locks them in this open conformation (Cestele and Catterall, 2000). It not only alters the gating process but also affects the structure of the open channel. In addition to stabilizing the active states, veratridine-induced deplorisation increases the scorpion α-toxin binding to receptor site 3. Thus it is able to cooperatively increase the binding by shifting more channels to the open conformation. Toxin binding is therefore in addition to being state-dependent and induced by conformational changes due to other toxins binding at distinct receptor sites on the Na+ channel (Cestele and Gordon,1998). At saturating concentration of veratridine, the sodium channels do not exhibit fast inactivation and so modified open channels conduct persistently at resting membrane potential via an allosteric mechanism that leads to block of sodium channel inactivation and shift of the voltage dependence of activation to more negative potentials (Farrag et al, 2008).
The Indian red scorpion Mesobuthus tamulus is the most dangerous Old World scorpion species prevalent on the Indian subcontinent known to cause fatal envenoming, particularly affecting children. The main effects of Mesobuthus tamulus venom are likely to be due to toxins that affect the opening of Na+ channels in nerves and muscles (Rowan et al, 1992) which causes an increase in the release of neurotransmitters in the peripheral nervous system but the mechanism in not yet clear. So far only a partial N-terminal sequence of a toxin protein Bt-II, a mammalian-specific toxin acting on Na+ channels, isolated from its venom has been determined (Lala and Narayan, 1994). Active fractions from the Mesobuthus tamulus venom have been characterised that are known to act on other ion channels (Ca2+ and K+), histamine releasers and protease inhibitors (Badhe et al, 2007). For example, iberiotoxin is a blocker of calcium -activated potassium channel and tumulus toxin is active on K+ channel (Dhawan et al, 2002).
1.5 Assays for Channel modulators
High Throughput Screening (HTS) assays have been established for ion channel as the target classes in which chemical libraries of hundreds of compounds are screened for activity (Xu et al, 2001). The scorpion venoms make attractive peptide libraries of pharmacologically active compounds due to the extent and diversity of their gene-encoded peptide neurotoxins (Sollod et al, 2005) and therefore represent an ideal candidate for HTS assays. The detection of changes in membrane potential is the industry standard method for the identification of Ion Channel ligands. Nowadays, the drug discovery efforts in the sodium channel area are being dominated by the high-throughput methods based on fluorescence detection of membrane potential changes. This requires robust instrumentation, ease of use and high signal:background assays (Gill et al, 2003). Fluorescence measurements are widely used both to monitor intracellular ion concentrations and to measure membrane potentials (Molokanova and Savchenko, 2008). The following section describes fluorescent based methods in details.
1.5.3 Fluorescence-Based Assays
Fluorescence detection of cell membrane potentials is a widely used technique in neurobiology, cell physiology and pharmaceutical screening. Fluorescence-based assays are rapidly becoming established as the method of choice in high-throughput screening settings. The high sensitivity and ability to measure true equilibrium conditions provides a level of information content unavailable in other screening techniques. Also these methods are easy to set up and achieve a high throughput as they give robust and homogeneous cell population measurement. The application of this technology to ion channel studies has two main forms: measurement of membrane potentials with voltage-sensitive dyes and measurement of the concentration of particular ions with ion-selective fluorescent dyes (Xu et al, 2001).
220.127.116.11 Ion-selective fluorescent dyes
These probes measure intracellular ionic concentrations. Examples include: calcium indicator dyes such as Fura-2 and Fluo-3 which show change in fluorescence emission on binding to calcium and sodium-sensitive benzofuran isophthalate (SBFI) dye used for sodium channel selectively (Gill et al, 2003). These dyes show different temporal resolution and accuracy for each range of ion concentrations but so far only calcium dyes are known to give robust performance suitable for HTS of Ca2+ channels (Xu et al, 2001).
18.104.22.168 Voltage-sensitive dyes are suitable for imaging voltage in living cells (Chang and Jackson, 2003). Membrane excitability in cell-based assays is a dynamic process that requires accurate measurements to gain information. In these cell based functional assays, cells are loaded with fluorescent dyes and compound addition results in a rapid change in the fluorescence which can be measured by a suitable instrumentation.
Membrane potential probes mainly belong to two classes: the slow responding and the fast responding dyes (Figure 6). Bis-(1,3-dibutylbarbituric acid)-trimethine oxonol [DiBAC4(3)], an anionic oxonol is a slow response dye with a relatively low to moderate potential dependent fluorescence change sensitivity of 1% per mV. It enters depolarized cells and upon binding to intracellular proteins or hydrophobic groups of the lipid membranes of the cells, exhibits enhanced fluorescence and red spectral shift (Xu et al, 2001). Increasing concentration of dyes in the cytosol causes increased binding of the dye to the cell membranes resulting in increasing fluorescence quantum yield and emission (Wolff et al, 2003). DiBAC4(3) partitions across the intracellular and extracellular compartments in a voltage dependent manner and its slow response time is caused by the slow migration of the oxonol across the lipid bilayer. Due to this slow response, DiBAC4 is not useful for probing fast inactivating or activating channels.
Figure 6 showing the two classes of membrane-potential probes- slow and fast response dyes which differ in their speed, size and mechanism of potential-dependent optical change. The fast-response probes undergo changes of intramolecular charge distribution due to changes in electric field which produces corresponding rapid changes in their fluorescence properties due to fast intramolecular redistribution of electrons. The slow-response probes on the other hand are lipophilic ions that show slow fluorescence changes associated with the relative redistribution of the dye molecules in the intracellular and extracellular environments (Molecular Probes, Invitrogen Corp, 2010)
Although patch clamping is the gold standard for electrophysiological measurements in detecting rapid changes in membrane potential, its low throughput and labour intensiveness limits its use in primary screening assays. Faster methods such as voltage sensitive dyes using the FLIPR (Fluorometric imaging plate reader) system have evolved. Due to slow response times and temperature sensitivity of oxonols, novel membrane potential dyes such as FMP (FLIPR membrane potential dye) have found increased use in HTS environments. Not only do the FMP dyes show faster response time but can also detect rapid opening as well as closing of channels. These also show an additional benefit of data comparability with whole-cell patch-clamp studies (see Figure 7) involving ligand evoked channel activation (Whiteaker et al, 2001). The fast-response dyes are usually styrylpyridinium molecular probes that show a 2-10% fluorescence change per 100 mV which is sufficiently fast to detect transient potential changes of the order of milliseconds in excitable cells (Trivedi et al, 2008). These perform by means of a change in their electronic structure, and hence their fluorescence properties, in response to changes in the surrounding electric field.
The conventional one-fluorophore indicator dyes such as DiBAC4 either respond too slowly or have limited sensitivity. Recently, a two-component FRET sensor has been designed that utilizes the transfer of fluorescence resonance energy from fluorescent donor bound on one side of the membrane and acceptor molecules in response to changes in membrane potential. A FRET voltage sensor dye with coumarin labelled phosphatidylethanolamine donor and a bis(l,3-dihexyl-2-thiobarbiturate) trimethine oxonoI as the acceptor has been reported by Gonzalez and Tsien, 1997 to give the largest sensitivity of fluorescence ratio (> 50% per 100 mV). Thus FRET-based voltage sensitive dyes give high performance and stable signals suitable for HTS in cellular assays (Dumas and Stoltz, 2005).
Figure 7 showing the correlation between manual patch clamp (mV) and FLIPR (fluorescence) assays (Molecular Devices).
1.6 Purpose of the Study
The aim of this study is to develop an assay which can identify toxic fractions from the venom of the scorpion- Leiurus quinquestriatus (Lq). The effects of Lq venom have been studied widely and known to interact with the voltage gated sodium channels found in the excitable cells. The assay utilises fluorescent membrane -potential sensitive dyes to establish the effect of toxin on Na+ channel modulation by characterising the way different toxins found in the Lq venom alter the channel kinetics. These results when compared with the known effects of this venom can help in setting up and defining assay parameters comparable with other methods already in place such as voltage clamp studies of toxins. We tried to demonstrate this ability of the assay to pick up known effects of Na+ channel activators (eg.; veratridine) by optimising various assay conditions. Once the assay is robust enough to identify venom components with an acceptable reproducibility and assay sensitivity, we proposed using it to test the fractions purified from Mesobuthus tamulus scorpion. Various toxins have been isolated from Mesobuthus venom as mentioned above, however not much is known about the Na+ channel toxins present in its venom. The purified fractions from the venom of Mesobuthus and toxin proteins can be run through this assay which could evaluate the effect of its toxin and help classify the toxins into subgroups already known (α, β, α- like, insect- selective, mammalian- selective etc) and possibly a different group of toxin altogether.