Charybdotoxin

Impairment of brain mitochondrial charybdotoxin- and ATP-insensitive BK channel activities in diabetes.[Pubmed:25344764]

Neuromolecular Med. 2014 Dec;16(4):862-71.

Existing evidence indicates an impairment of mitochondrial functions and alterations in potassium channel activities in diabetes. Because mitochondrial potassium channels have been involved in several mitochondrial functions including cytoprotection, apoptosis and calcium homeostasis, a study was carried out to consider whether the gating behavior of the mitochondrial ATP- and ChTx-insensitive Ca(2+)-activated potassium channel (mitoBKCa) is altered in a streptozotocin (STZ) model of diabetes. Using ion channel incorporation of brain mitochondrial inner membrane into the bilayer lipid membrane, we provide in this work evidence for modifications of the mitoBKCa ion permeation properties with channels from vesicles preparations coming from diabetic rats characterized by a significant decrease in conductance. More importantly, the open probability of channels from diabetic rats was reduced 1.5-2.5 fold compared to control, the most significant decrease being observed at depolarizing potentials. Because BKCa beta4 subunit has been documented to left shift the BKCa channel voltage dependence curve in high Ca(2+) conditions, a Western blot analysis was undertaken where the expression of mitoBKCa alpha and beta4 subunits was estimated using of anti-alpha and beta4 subunit antibodies. Our results indicated a significant decrease in mitoBKCa beta4 subunit expression coupled to a decrease in the expression of alpha subunit, an observation compatible with the observed decrease in Ca(2+) sensitivity. Our results thus demonstrate a modification in the mitoBKCa channel gating properties in membrane preparations coming from STZ model of diabetic rats, an effect potentially linked to a change in mitoBKCa beta4 and alpha subunits expression and/or to an increase in reactive oxygen species production in high glucose conditions.

In silico analysis of potential inhibitors of Ca(2+) activated K(+) channel blocker, Charybdotoxin-C from Leiurus quinquestriatus hebraeus through molecular docking and dynamics studies.[Pubmed:26069365]

Indian J Pharmacol. 2015 May-Jun;47(3):280-4.

OBJECTIVE: Charybdotoxin-C (ChTx-C), from the scorpion Leiurus, quinquestriatus hebraeus blocks the calcium-activated potassium channels and causes hyper excitability of the nervous system. Detailed understanding the structure of ChTx-C, conformational stability, and intermolecular interactions are required to select the potential inhibitors of the toxin. MATERIALS AND METHODS: The structure of ChTx-C was modeled using Modeller 9v7. The amino acid residues lining the binding site were predicted and used for toxin-ligand docking studies, further, selected toxin-inhibitor complexes were studied using molecular dynamics (MD) simulations. RESULTS: The predicted structure has 91.7% of amino acids in the core and allowed regions of Ramachandran plot. A total of 133 analog compounds of existing drugs for scorpion bites were used for docking. As a result of docking, a list of compounds was shown good inhibiting properties with target protein. By analyzing the interactions, Ser 15, Lys 32 had significant interactions with selected ligand molecules and Val5, which may have hydrophobic interaction with the cyclic group of the ligand. MD simulation studies revealed that the conformation and intermolecular interactions of all selected toxin-inhibitor complexes were stable. CONCLUSION: The interactions of the ligand and active site amino acids were found out for the best-docked poses in turn helpful in designing potential antitoxins which may further be exploited in toxin based therapies.

Triclosan, an environmental pollutant from health care products, evokes charybdotoxin-sensitive hyperpolarization in rat thymocytes.[Pubmed:22004961]

Environ Toxicol Pharmacol. 2011 Nov;32(3):417-22.

The effects of triclosan, an environmental pollutant from household items and health care products, on membrane potential and intracellular Ca(2+) concentrations of rat thymocytes were examined by a flow cytometry with fluorescent probes, di-BA-C(4) and fluo-3-AM, because triclosan is often found in humans and wild animals. Triclosan at a concentration of 3 muM decreased the intensity of di-BA-C(4) fluorescence, indicating the triclosan-induced hyperpolarization. The application of Charybdotoxin, a specific inhibitor of Ca(2+)-dependent K(+) channels, and the removal of external Ca(2+) eliminated the triclosan-attenuation of di-BA-C(4) fluorescence. Furthermore, triclosan augmented the fluo-3 fluorescence under normal Ca(2+) condition, indicating that triclosan increased intracellular Ca(2+) concentration. These results suggest that triclosan induces membrane hyperpolarization by increasing intracellular Ca(2+) concentration that activates Ca(2+)-dependent K(+) channels. Since the change in membrane potential of lymphocytes influence cellular immune functions, triclosan may exert adverse actions on immune system in human and wild animals.

Charybdotoxin and margatoxin acting on the human voltage-gated potassium channel hKv1.3 and its H399N mutant: an experimental and computational comparison.[Pubmed:22490327]

J Phys Chem B. 2012 May 3;116(17):5132-40.

The effect of the pore-blocking peptides Charybdotoxin and margatoxin, both scorpion toxins, on currents through human voltage-gated hK(v)1.3 wild-type and hK(v)1.3_H399N mutant potassium channels was characterized by the whole-cell patch clamp technique. In the mutant channels, both toxins hardly blocked current through the channels, although they did prevent C-type inactivation by slowing down the current decay during depolarization. Molecular dynamics simulations suggested that the fast current decay in the mutant channel was a consequence of amino acid reorientations behind the selectivity filter and indicated that the rigidity-flexibility in that region played a key role in its interactions with scorpion toxins. A channel with a slightly more flexible selectivity filter region exhibits distinct interactions with scorpion toxins. Our studies suggest that the toxin-channel interactions might partially restore rigidity in the selectivity filter and thereby prevent the structural rearrangements associated with C-type inactivation.

Charybdotoxin-sensitive K+ channels regulate the myogenic tone in the resting state of arteries from spontaneously hypertensive rats.[Pubmed:7679030]

Br J Pharmacol. 1993 Jan;108(1):214-22.

1. To determine the possible role of Ca(2+)-activated K+ (KCa) channels in the regulation of resting tone of arteries from spontaneously hypertensive rats (SHR), the effects of agents which interact with these channels on tension and 86Rb efflux were compared in endothelium-denuded strips of carotid, femoral and mesenteric arteries from SHR and normotensive Wistar-Kyoto rats (WKY). 2. Strips of carotid, femoral and mesenteric arteries from SHR exhibited a myogenic tone; that is, the resting tone decreased when either the Krebs solution was changed to a 0-Ca2+ solution or 10(-7) M nifedipine was added. 3. The addition of Charybdotoxin (ChTX, 10(-9)-10(-7) M), a blocker of large conductance KCa channels, to the resting strips of these arteries produced a concentration-dependent contraction, which was significantly greater in SHR than in WKY. Relatively low concentrations of tetraethylammonium (0.05-5 mM) produced a concentration-dependent contraction which was similar to the ChTX-induced contraction in these strips. 4. The ChTX-induced contractions in SHR were greatly attenuated by 10(-7) M nifedipine and by 3 x 10(-6) M cromakalim, a K+ channel opener. Cromakalim alone abolished the myogenic tone in SHR. 5. The addition of apamin (a blocker of small conductance KCa channels, up to 10(-6) M), or of glibenclamide (a blocker of ATP-sensitive K+ channels, up to 5 x 10(-6) M), to the resting strips failed to produce a contraction. 6. In resting strips of carotid, femoral and mesenteric arteries preloaded with 86Rb, the basal 86Rb efflux rate constants were significantly greater in SHR than in WKY. The addition of 10-7 M nifedipine to the resting strips decreased the basal 86Rb efflux rate constants only in SHR.7. The cellular Ca2+ uptake in the resting state of carotid and femoral arteries from SHR was significantly increased when compared to WKY, and this increase in SHR was significantly reduced by 10-7M nifedipine.8. These results suggest that the ChTX-sensitive KCa channels were highly activated to regulate the myogenic tone in the resting state of carotid, femoral and mesenteric arteries from SHR. The increased Kca channel functions in SHR arteries appeared to be secondary to the increased Ca2' influx via L-type voltage-dependent Ca2+ channels in the resting state of these arteries.

Purification, sequence, and model structure of charybdotoxin, a potent selective inhibitor of calcium-activated potassium channels.[Pubmed:2453055]

Proc Natl Acad Sci U S A. 1988 May;85(10):3329-33.

Charybdotoxin (ChTX), a protein present in the venom of the scorpion Leiurus quinquestriatus var. hebraeus, has been purified to homogeneity by a combination of ion-exchange and reversed-phase chromatography. Polyacrylamide gel electrophoresis, amino acid analysis, and complete amino acid sequence determination of the pure protein reveal that it consists of a single polypeptide chain of 4.3 kDa. Purified ChTX is a potent and selective inhibitor of the approximately 220-pS Ca2+-activated K+ channel present in GH3 anterior pituitary cells and primary bovine aortic smooth muscle cells. The toxin reversibly blocks channel activity by interacting at the external pore of the channel protein with an apparent Kd of 2.1 nM. The primary structure of ChTX is similar to a number of neurotoxins of diverse origin, which suggests that ChTX is a member of a superfamily of proteins that modify ion-channel activities. On the basis of this similarity, the three-dimensional structure of ChTX has been modeled from the known crystal structure of alpha-bungarotoxin. These studies indicate that ChTX is useful as a probe of Ca2+-activated K+-channel function and suggest that the proposed tertiary structure of ChTX may provide insight into the mechanism of channel block.

Charybdotoxin, a protein inhibitor of single Ca2+-activated K+ channels from mammalian skeletal muscle.[Pubmed:2578618]

Nature. 1985 Jan 24-30;313(6000):316-8.

The recent development of techniques for recording currents through single ionic channels has led to the identification of a K+-specific channel that is activated by cytoplasmic Ca2+. The channel has complex properties, being activated by depolarizing voltages and having a voltage-sensitivity that is modulated by cytoplasmic Ca2+ levels. The conduction behaviour of the channel is also unusual, its high ionic selectivity being displayed simultaneously with a very high unitary conductance. Very little is known about the biochemistry of this channel, largely due to the lack of a suitable ligand for use as a biochemical probe for the channel. We describe here a protein inhibitor of single Ca2+-activated K+ channels of mammalian skeletal muscle. This inhibitor, a minor component of the venom of the Israeli scorpion, Leiurus quinquestriatus, reversibly blocks the large Ca2+-activated K+ channel in a simple biomolecular reaction. We have partially purified the active component, a basic protein of relative molecular mass (Mr) approximately 7,000.