Daboia Russelli Research Articles

Myocardial Infarction as a Rare Consequence of a Snakebite

A healthy 28-year-old man with no history of cardiac disease and no cardiac risk factors presented to the hospital 2 hours after being bitten on his right hand by his pet snake. He was in anaphylactic shock and was rapidly resuscitated with fluids, inotropic support, intramuscular antitetanus serum, and intravenous infusion of Viper-FAB, an antidote. His vital signs normalized and he was admitted to intensive care for further observation. One hour after admission, the patient’s systolic blood pressure dropped to 40 mm Hg. Intravenous noradrenaline was started. Thirty minutes later, the patient lost consciousness in association with a rhythm change to torsade de pointes. He was defibrillated with 100 J (biphasic defibrillator) to sinus rhythm. Intravenous magnesium was started, and a repeat 12-lead ECG showed a prolonged corrected QT interval at 490 ms with no ST elevation (Figure 1). An urgent echocardiogram demonstrated normal-sized left ventricle with mild hypokinesis of the anterior wall and a global left ventricular fraction of 60%. Tissue Doppler images acquired by Vivid-7 (GE Medical) …

Crystal structure of the complex of the secretory phospholipase A 2 from Daboia russelli pulchella with an endogenic indole derivative, 2-carbamoylmethyl-5-propyl-octahydro-indol-7-yl-acetic acid at 1.8 Å resolution

Phospholipase A 2 (PLA 2) enzymes from snake venoms are approximately 14 kDa secretory proteins and catalyze the release of arachidonic acid which is the precursor of proinflammatory mediators such as prostaglandins, leukotrienes, thromboxanes and platelet-activating factors. The structure of the PLA 2 enzyme purified from the venom of Daboia russelli pulchella was determined using molecular replacement method and refined to an R value of 18.3% for all the reflections to 1.8 Å resolution. The structure contains two crystallographically independent molecules A and B which form an asymmetric homodimer. The Ca 2+ ion was not detected in the present structure, however, a characteristic non-protein high quality electron density was observed at the substrate-binding site of molecule A which allowed a clear interpretation of a natural ligand identified as a derivative of indole, 2-carbamoylmethyl-5-propyl-octahydro-indol-7-yl)-acetic acid. The corresponding substrate-binding site in molecule B was empty. The ligand present in molecule A is involved in extensive interactions with the protein atoms including important catalytic residues such as Asp-49 and His-48. The results also show that the indole derivatives act as potent inhibitors of secretory group II PLA 2 enzymes that can be further modified to be used as potential therapeutic agents.

Crystal structure of the complex of group I PLA2 with a group II-specific peptide Leu-Ala-Ile-Tyr-Ser (LAIYS) at 2.6 Å resolution

Phospholipases A2s (PLA2s) are widely distributed in mammals and snake venoms. They catalyze the production of arachidonic acid from membrane phospholipids leading to the bioynthesis of pro-inflammatory eicosanoids. A peptide Leu-Ala-Ile-Tyr-Ser (LAIYS) was designed and synthesized as a specific inhibitor of PLA2. It was shown earlier that the peptide bound to group II PLA2 specifically and had a dissociation constant (Kd) of 8.8 × 10− 9 M. In the present studies for the binding of LAIYS with a group I PLA2 from Naja naja sagittifera using surface plasmon resonance the dissociation constant was found to be 4.5 × 10− 5 M which is considerably lower than the value found for the group II PLA2. In order to determine the details of binding at the molecular level, a group I PLA2 from the venom of Naja naja sagittifera was crystallized with peptide LAIYS. The crystal structure showed the presence of LAIYS at the substrate-binding site but has fewer interactions than those observed with group II PLA2 from Daboia russelli pulchella. The observed difference in the binding affinity is caused primarily due to poor fitting of the peptide LAIYS in the binding site of group I PLA2. Apparently, the location of Trp 19 in group I PLA2 is not favourable for the binding of LAIYS. The two complexes also differ drastically in the formation of intermolecular interactions. In the present structure, the side chain of Ser (P) interacts with His 48 and Asp 49 while in the complex with group II PLA2 it was Tyr (P) OH that formed the corresponding interactions. Tyr (P) in group I PLA2 is the main contributor of the hydrophobic interactions whereas in the complex of LAIYS with group II PLA2 it was the peptide segment Leu-Ala-Ile that produced the bulk of hydrophobic forces. The structures further showed that the peptide LAIYS was fully inside the substrate-binding region of the group II PLA2 while a significant portion of the peptide LAIYS was hanging outside the surface of the group I PLA2. The buried area in the complex with group II PLA2 was 811 Å2 whereas, the corresponding area in group I PLA2 was 449 Å2. This shows that the peptide LAIYS is very compatible with the substrate-binding site of group II PLA2 and rather poorly fits into the substrate-binding site of group I PLA2. This indicates that a highly specific ligand for one form of PLA2 may be a poor partner for another form of enzyme.

Specific distribution of VEGF-F in Viperinae snake venoms: Isolation and characterization of a VEGF-F from the venom of Daboia russelli siamensis

Vascular endothelial growth factor exerts multiple functions through binding to two distinct receptor tyrosine kinases, Flt-1 and KDR. We previously demonstrated that snake venom VEGFs, designated VEGF-Fs, exhibit potent biological activities when compared with VEGF165 and that they selectively recognize KDR. We herein report the screening of several snake venoms for VEGF-Fs using antibodies against vammin and HF, VEGF-Fs derived from the venom of Vipera ammodytes ammodytes and Vipera aspis aspis, respectively. Specific immunoreactivity was observed in the venoms of Vipera and Daboia species. The present results suggest that VEGF-Fs are specific components of venoms from the Viperinae snakes that inhabit Europe, India, and Asia. We also report the isolation and characterization of an additional VEGF-F from Daboia russelli siamensis venom.

Practical applications of snake venom toxins in haemostasis

Snake venom toxins affecting haemostasis have facilitated extensively the routine assays of haemostatic parameters in the coagulation laboratory. Snake venom thrombin-like enzymes (SVTLE) are used for fibrinogen/fibrinogen breakdown product assay and for the detection of fibrinogen dysfunction. SVTLE are not inhibited by heparin and can thus can be used for assaying antithrombin III and other haemostatic variables in heparin-containing samples. Snake venoms are a rich source of prothrombin activators and these are utilised in prothrombin assays, for studying dysprothrombinaemias and for preparing meizothrombin and non-enzymic forms of prothrombin. Russell's viper ( Daboia russelli) venom (RVV) contains toxins which have been used to assay blood clotting factors V, VII, X, platelet factor 3 and, importantly, lupus anticoagulants (LA). Other prothrombin activators (from the taipan, Australian brown snake and saw-scaled viper) have now been used to assay LA. Protein C and activated protein C resistance can be measured by means of RVV and Protac ®, a fast acting inhibitor from Southern copperhead snake venom and von Willebrand factor can be studied with botrocetin ® from Bothrops jararaca venom. The disintegrins, a large family of Arg-Gly-Asp (RGD)-containing snake venom proteins, show potential for studying platelet glycoprotein receptors, notably, GPIIb/IIIa and Ib. Snake venom toxins affecting haemostasis are also used in the therapeutic setting: Ancrod (from the Malayan pit viper, Calloselasma rhodostoma), in particular, has been used as an anticoagulant to achieve ‘therapeutic defibrination’. Other snake venom proteins show promise in the treatment of a range of haemostatic disorders.

REVISED SYSTEMATICS OF TAIWANESE VIPERID SNAKES AND THE CORRELATION TO VENOM DIVERSITY AND EVOLUTION

The major viperid snakes present in Taiwan today are: Daboia russelli siamensis, Deinagkistrodon acutus, Protobothrops mucrosquamatus, Trimeresurus stejnegeri, Ovophis monticola and Ovophis gracilis. As suggested by the species trees deduced from mtDNA sequences of the family Viperidae, these species apparently belong to five different genera. Molecular cloning, N-terminal sequencing, and mass spectrometry have facilitated the sequence-determination of venom proteins. Previous examples of using venom proteins as characteristics for the phylogenetic and evolutionary studies are reviewed herein. Two new phylogeny trees using protein sequence datasets for the crotalid venom acidic phospholipases and the disintegrins are shown. Both trees grossly reveal phylogeographic relationships between the species, although some inconsistencies exist. Moreover, distinct protein markers in each of the Taiwanese viperid venoms and their relations to the snakebite symptoms are discussed in terms of the known snake systematics.

Improved Detection of FV Leiden Patients Using a Novel Snake-Venom-Based Activated Protein C Resistance Assay.

A new functional prothrombin-based activated protein C (APC) resistance (APC-R) test (Pefakit® APC-R Factor V Leiden, Pentapharm, Basel, Switzerland) is presented.Methods: The plasma sample is mixed with a reagent containing APC and snake venom specifically activating FV (RVV-V, Daboia russelli) and plasma that has been depleted of FV. During an incubation period of 180 sec the activated FV is inactivated by APC. Subsequently a reagent that contains a FV dependent prothrombin activator (Noscarin, Notechis scutatus) and EDTA is added. The clotting time is recorded. A second determination is performed under identical conditions, with the exception that no APC is added to the first reagent. A ratio between the two measurements is calculated. 703 samples of patients undergoing thrombophilia screening were analysed. Results were correlated to PCR based FVL testing, aPTT, PT, and to levels of Protein C, Protein S, Fibrinogen, FVIII and lupus anticoagulant index.Results: Using a predefined cut-off of a ratio of 2.5 a 100% sensitivity and specificity for the detection of a FVL mutation was found. Using a cut-off ratio of 1.2 a complete but narrow distinction of FVL heterozygous (n=192) and FVL homozygous samples (n=27) was determined. No interference by sample's INR and aPTT, PS, fibrinogen and FVIII levels and lupus anticoagulant ratio was detected.Conclusion: The new snake-venom-based APC-R assay provides an improved distinction of FV wild-type and FVL carriers compared to the data reported for the aPTT based methods. The use of a FV dependent prothrombin activator eliminates effects of FVIII concentration or lupus anticoagulants in the sample.

Russell's viper snakebite in Taiwan: differences from other Asian countries

Formosan Russell's viper ( Daboia russelli siamensis) is the sixth most frequent cause of snakebite in Taiwan. Its venom has been thought to have both neurotoxic and hematoxic properties. This viper's snakebite is rare and thus scarcely subjected to systemic studies. In this paper, we retrospectively analyzed and described 18 cases of viper snakebite from 1987 to 1999. Like that of the Russell's viper snakebite in other South East Asian areas, varied degrees of acute renal failure, incoagulable blood with bleeding diathesis and hemolysis were the major symptoms found in the systemic envenoming patients. Systemic thrombosis seems to be the distinguishing feature in Formosan Russell's viper snakebite. Neither symptoms nor signs of neuromuscular junction blocking effects were observed, which is another difference from symptoms observed after bites of some other Russell's viper subspecies, suggesting a significant geographic variation. These findings confirmed the clinical importance of Russell's viper snakebite in Taiwan.

Snake Venom Activators of Factor X: An Overview

Activators of blood coagulation factor X have been described in the venom of many snake species belonging to the genus Viperidae and Crotalidae as well as from a few Elapid species. Based on the structural and functional properties of purified activating principles, factor X activators are either metalloproteases or serine proteases. The best known activator is RVV-X from Russell’s viper (Daboia russelli), a metalloprotease consisting of a heavy chain containing the catalytic domain and two light chains which share homology with C-type lectins and which are thought to exert a regulatory function in the Ca²⁺-dependent activation of factor X. This activator is also one of the best examples of the use of exogenous activators in coagulation research and in addition it is used in many diagnostic research kits. In this paper, an overview is given of the structural and functional properties of snake venom factor X activators thus far described in the literature.

Creening of Antisnake Venom Activity of Dichrostachys cinerea W. & A

Objective : To study the antisnake venom effect of roots of Dichrostachys cinerea W. & A. Materials and methods : Antisanke venom property of D. cinerea was evaluated by using Russell's viper venom ( Daboia russelli ) and albino mice. Potency of methanolic and etherial extract was compared. Results : Methanolic extract of the roots of Dichrostachys cinerea has shown significant decrease in the mortality percentage in venom induced mice. Conclusion: D. cinerea has shown a positive effect of nullifying the viper venom toxicity in mice and can be recommended for further studies as an antidote to snakebite poisoning.

Regulation of catalytic function by molecular association: structure of phospholipase A2 from Daboia russelli pulchella (DPLA2) at 1.9 A resolution.

The crystal structure of phospholipase A(2) from the venom of Daboia russelli pulchella has been refined to an R factor of 0.216 using 17,922 reflections to 1.9 A resolution. The structure contains two crystallographically independent molecules in the asymmetric unit. The overall conformations of the two molecules are essentially the same except for three regions, namely the calcium-binding loop including Trp31, the beta-wing and the C-terminal residues 119-131. Although these differences have apparently been caused by molecular packing, they seem to have functional relevance. Particularly noteworthy is the conformation of Trp31, which is favourable for substrate binding in one molecule as it is aligned with one of the side walls of the hydrophobic channel, whereas in the other molecule it is located at the mouth of the channel, thereby blocking the entry of substrates leading to loss of activity. This feature is unique to the present structure and does not occur in the dimers and trimers of other PLA(2)s.

Three-dimensional structure of a presynaptic neurotoxic phospholipase A2 from Daboia russelli pulchella at 2.4 Å resolution

The phospholipase A2 from Daboia russelli pulchella(DPLA2) is the only known member of subclass II of group IIA. The three-dimensional structure of this presynaptic neurotoxic DPLA2 enzyme has been determined at 2.4 Å resolution. The structure was determined by the molecular replacement method using the model Crotalus atrox, and refined using X-PLOR to a final R-factor of 18.8 % for all data in the resolution range 20.0 Å–2.4 Å. The final refined model comprises 1888 atoms from two crystallographically independent protein molecules and 160 water oxygen atoms. The overall folding of DPLA2, with three long helices and two short antiparallel β-strands is grossly similar to those observed for other PLA2 s. In the present structure, the calcium binding site is empty but the conformation of the calcium binding loop is similar to those observed in the calcium bound states. Two spatially adjacent regions of residues 55–61 (a typical β-turn I) and 83–94 (a well defined loop) are remarkably different in conformation, electrostatic characteristics and inter-segmental interactions from those found in non-neurotoxic PLA2 s. Yet another striking structural feature in DPLA2 pertains to the stretch of residues 53–77, which has a series of positively charged residues protruding outwardly. The above segment is presumed to be involved in the anticoagulant activity. A unique hydrophobic patch including residues Leu17, Ala18, Ile19, Pro20, Phe106 and Leu110 is found on the surface together with an equally emphatic region of -OH groups containing residues such as Ser21, Tyr22, Ser23, Ser24, Tyr25 and Tyr28. The interactions between two molecules of DPLA2 in the asymmetric unit are remarkably different from those observed in the standard dimers and trimers of PLA2 s, leaving the enzyme’s active site fully exposed for enzyme-substrate reactions, it makes this structure one of the most favourable examples for structure-based drug design through soaking experiments.

Some biochemical properties of Russell’s viper ( Daboia russelli) venom from Eastern India: correlation with clinico-pathological manifestation in Russell’s viper bite

In the present study, some biochemical properties and pathological effects of Daboia russelli venom from Burdwan district of West Bengal, eastern India are presented. The clinical features of Russell’s viper envenomation observed in patients admitted to Burdwan Medical College & Hospital are also reported. In vitro, whole venom exerts strong trypsin inhibitory, phospholipase A 2 and procoagulant activities in addition to moderate adenosine monophosphatase and adenosine triphosphatase activities. Lethality (LD 50) of this venom sample is 0.7 mg/kg (i.v.) of mice. Significant local tissue damaging effects including edema, hemorrhage and necrosis are observed in experimental animal models. An increase in the level of serum enzymes, such as aspartate transaminase, alkaline phosphatase, creatine phosphokinase, lactate dehydrogenase after D. russelli venom injection in albino rats is indicative of cell or tissue damage. High incidence of intravascular hemolysis in addition to hemostasis, haemoptysis and haematuria are observed as the most prominent features of RVV envenomation from this part of India. The present study reinforces the hypothesis that variation in the venom composition of RVV from eastern India with respect to venom samples of Russell’s vipers from other parts of India is responsible for the differences in the clinical manifestation in patients from eastern India.

Purification, crystallization and preliminary x-ray crystallographic analysis of a phospholipase A2 from Daboia russelli pulchella.

Phospholipases are esterolytic enzymes which hydrolyze glycerophospholipids. The pharmacological efficiency of phospholipase A2 (PLA2) enzymes is reflected by their specificity towards a tissue or organ. The Russell's viper has been classified into two classes. Class 1 contains Viper russelli russelli, Viper russelli siamensis and Viper russelli formosensis, whereas class 2 contains Daboia russelli pulchella. The sequence identity between the PLA2s from these two classes is 47%. The novel PLA2 from Daboia russelli pulchella has been crystallized using the hanging-drop vapour-diffusion method with ammonium sulfate as precipitating agent. Crystals belong to the orthorhombic space group C2221 with unit-cell parameters a = 77.01, b = 92.29, c = 76.90 A and two molecules in the asymmetric unit. These crystals diffract to about 2. 49 A resolution using a rotating-anode source.

The effects of green pit viper ( Trimeresurus albolabris and Trimeresurus macrops) venom on the fibrinolytic system in human

Green pit viper ( Trimeresurus albolabris and Trimeresurus macrops) venom was found to have a thrombin-like effect in vitro but cause a defibrination syndrome in vivo. The effects of venom on fibrinolytic system have not been well characterized. This knowledge can help to define the roles of antifibrinolytic therapy, give insights in fibrinolytic system regulation and potentially lead to identification of a new profibrinolytic agent from this venom. Forty-six cases of green pit viper bites were studied for various coagulation and fibrinolytic parameters and correlated with serum venom levels measured by ELISA. Fibrinolytic system activation is very common as indicated by low plasminogen (50%), low antiplasmin (56.5%) and elevated fibrin–fibrinogen degradation products (FDPs, 97.4%) levels. FDP test is very sensitive and a normal level is useful for exclusion of systemic envenomation. In contrast to some other models of defibrination syndrome, such as Russell viper ( Daboia russelli siamensis), elevation of plasminogen activator activity (PA) was found indicating a hyperfibrinolytic state. Definite increase in tissue-type plasminogen activator (t-PA) antigen ( p=0.00075) with a modest elevation of its inhibitor plasminogen activator inhibitor-1 (PAI-1) ( p=0.27) probably contributes to this effect. This supports the idea that the balance between plasminogen activators and inhibitors can determine fibrinolytic responses in pathologic states. Fibrinopeptide A levels were markedly elevated (68.43±51.57 ng/ml in cases and 2.83±3.80 ng/ml in control, p<0.0001) and correlated well with clinical severity suggesting that the fibrin deposition from the thrombin-like effect is the main mechanism of fibrinolysis. Therefore, antifibrinolytic agents probably have no role in treatment. However, the components of green pit viper venom that have these profibrinolytic effects in human are interesting and should be further identified.

ELISA for the detection of venoms from four medically important snakes of India

A double antibody sandwich enzyme linked immunosorbent assay (ELISA) was developed to detect Echis carinatus venom in various organs (brain, heart, lungs, liver, spleen and kidneys) as well as tissue at the site of injection of mice, at various time intervals (1, 6, 12, 18, 24 h and 12 h intervals up to 72 h) after death. The assay could detect E. carinatus venom levels up to 2.5 ng/ml of tissue homogenate and the venom was detected up to 72 h after death. A highly sensitive and species-specific avidin–biotin microtitre ELISA was also developed to detect venoms of four medically important Indian snakes ( Bungarus caeruleus, Naja naja, E. carinatus and Daboia russelli russelli) in autopsy specimens of human victims of snake bite. The assay could detect venom levels as low as 100 pg/ml of tissue homogenate. Venoms were detected in brain, heart, lungs, liver, spleen, kidneys, tissue at the bite area and postmortem blood. In all 12 human victim cadavers tested the culprit species were identified. As observed in mice, tissue at the site of bite area showed the highest concentration of venom and the brain showed the least. Moderate amounts of venoms were found in liver, spleen, kidneys, heart and lungs. Development of a simple, rapid and species-specific diagnostic kit based on this ELISA technique useful to clinicians is discussed.

Use of snake venom fractions in the coagulation laboratory.

Snake venom toxins are now regularly used in the coagulation laboratory for assaying haemostatic parameters and as coagulation reagents. Snake venom thrombin-like enzymes (SVTLE) are used for fibrinogen and fibrinogen breakdown product assay as well as detecting dysfibrinogenaemias. Significantly, because SVTLE are not inhibited by heparin, they can be used for defibrinating samples that contain the anticoagulant before assay of haemostatic variables. Prothrombin activators are found in many snake venoms and are used in prothrombin assays, for studying dysprothrombinaemias and preparing meizothrombin and non-enzymic prothrombin. Russell's viper (Daboia russelli) venom (RVV) contains a number of compounds useful in the assay of factors V, VII, X, platelet factor 3 and lupus anticoagulants. Activators from the taipan, Australian brown snake and saw-scaled viper have been used to assay lupus anticoagulants. Protein C and activated protein C resistance can be measured by means of RVV and Protac, a fast acting inhibitor from Southern copperhead snake venom and von Willebrand factor can be studied with Botrocetin from Bothrops jararaca venom. Finally, phospholipase A2 enzymes and the disintegrins, a family of Arg-Gly-Asp (RGD)-containing proteins found in snake venoms, show great potential for the study of haemostasis including, notably, platelet glycoprotein receptors GPIIb/IIIa and Ib.

Venomous Snakebite in Thailand I: Medically Important Snakes

Thailand has an abundance of venomous snakes. Among the neurotoxic family Elapidae, there are three species of the genus Naja (cobras), three of the genus Bungarus (kraits), and the king cobra of the genus Ophiophagus. Other Elapidae snakes in Thailand include sea snakes and Asian coral snakes of the genus Calliophis. They have potent venoms but rarely bite humans. Tissue and hemotoxic snakes are represented by family Viperidae, subfamilies Viperinae and Crotalinae. They remain an occupational hazard for farmers and rubber tappers, causing serious morbidity but only rare deaths, since competent treatment is now widely available throughout Thailand. Purified equine antivenin is manufactured locally for the monocled and Siamese spitting cobras (Naja kaouthia and N. siamensis), king cobra (Ophiophagus hannah), banded krait (Bungarus fasciatus), most green pit vipers (Trimeresurus sp.), Malayan pit viper (Calloselasma rhodostoma), and the Siamese Russell's viper (Daboia russelli siamensis).

Comparison of venom phospholipases A 2 from six Daboia russelli geographic samples revealed the existence of two types of the Asia viper

Comparison of venom phospholipases A 2 from six Daboia russelli geographic samples revealed the existence of two types of the Asia viper

Synaptosomal binding of 125I-labelled daboiatoxin, a new PLA 2 neurotoxin from the venom of Daboia russelli siamensis

Daboiatoxin (DbTx), the PLA 2 neurotoxin from Daboia russelli siamensis venom, was shown to bind specifically and saturably to rat cerebrocortical synaptosomes and synaptic membrane fragments. Two families of binding sites were detected by equilibrium binding analysis in the presence and absence of Ca 2+. Scatchard analysis of biphasic plateaus revealed K d1 5 nM and B max1, 6 pmoles/mg protein, and K d2 80 nM and B max2 20 pmoles/mg protein, respectively, for the high- and low-affinity binding sites. The binding of 125I-DbTx to synaptosomes did not show marked dependence on Ca 2+, Mg 2+, Co 2+ and Sr 2+. Native DbTx was the only strong competitor to 125I-DbTx synaptosomal binding ( ic 50 12.5 nM, K 1 5.5 nM). Two other crotalid PLA 2 neurotoxins, crotoxin CB and mojave toxin basic subunit, and non-toxic C. atrox PLA 2 enzyme, were relatively weaker inhibitors, while two viperid PLA 2 neurotoxins, ammodytoxin A and VRV PL V, were very weak inhibitors. Crotoxin CA was a poor inhibitor even at μM concentrations, whereas no inhibitory effect at all was observed with crotoxin CACB, ammodytoxin C, VRV PL VIII a, taipoxin, β-bungarotoxin, or with PLA 2 enzymes from N. naja venom, E. schistosa venom, bee venom and porcine pancreas. All other pharmacologically active ligands examined (epinephrine, norepinephrine, histamine, choline, dopamine, serotonin, GABA, naloxone, WB-4101, atropine, hexamethonium and α-bungarotoxin) also failed to interfere with 125I-DbTx binding. As those competitors that showed partial inhibition were effective only at μM concentration range compared to the K d (5 nM) of 125I-DbTx synaptosomal binding, DbTx could well recognize a different neuronal binding site. Rabbit anti-DbTx polyclonal antisera completely blocked the specific binding. When a range of Ca 2+ and K + channels modulators were examined, Ca 2+ channel blockers (ω-conotoxins GVIA and MVIIC, taicatoxin, calciseptine and nitrendipene) did not affect the binding even at high concentrations, while charybdotoxin was the only K + channel effector that could partially displace 125I-DbTx synaptosomal binding amongst the K + channel blockers tested (apamin, dendrotoxin-I, iberiotoxin, MCD-peptide, 4-aminopyridine and tetraethylammonium), suggesting that neither K + nor Ca 2+ channels are associated with DbTx binding sites.