kaliotoxin and noxiustoxin

Microglial cells were cultured from murine neonatal brain. Ramification of isolated microglia could be induced by the application of astrocyte-conditioned medium (ACM). Voltage-gated outward potassium currents (IK) were measured in ramified microglial cells 12-24 h after their treatment with ACM. The effects of the specific K+ channel blockers charybdotoxin (CTX), noxiustoxin (NTX) and kaliotoxin (KTX) on IK of ramified microglia were studied. All these peptide toxins blocked IK in a concentration-dependent manner, while showing a high sensitivity for IK. A half-maximal effective concentration (IC50) of CTX was estimated to be 1.13 nM, while IC50 values of 1.24 nM and of 0.81 nM were calculated for KTX and for NTX, respectively. In contrast, dendrotoxin (DTX) did not show any effect on IK. It is suggested that ramified microglial cells express outward K+ currents exhibiting pharmacological properties similar to that of outward K+ currents in cytokine-activated ameboid microglia.

Topics: Animals; Dose-Response Relationship, Drug; Mice; Mice, Inbred Strains; Microglia; Potassium Channels; Scorpion Venoms

The architecture of the pore-region of a voltage-gated K+ channel, Kv1.3, was probed using four high affinity scorpion toxins as molecular calipers. We established the structural relatedness of these toxins by solving the structures of kaliotoxin and margatoxin and comparing them with the published structure of charybdotoxin; a homology model of noxiustoxin was then developed. Complementary mutagenesis of Kv1.3 and these toxins, combined with electrostatic compliance and thermodynamic mutant cycle analyses, allowed us to identify multiple toxin-channel interactions. Our analyses reveal the existence of a shallow vestibule at the external entrance to the pore. This vestibule is approximately 28-32 A wide at its outer margin, approximately 28-34 A wide at its base, and approximately 4-8 A deep. The pore is 9-14 A wide at its external entrance and tapers to a width of 4-5 A at a depth of approximately 5-7 A from the vestibule. This structural information should directly aid in developing topological models of the pores of related ion channels and facilitate therapeutic drug design.

Topics: Amino Acid Sequence; Binding Sites; Charybdotoxin; Electric Conductivity; Electrochemistry; Ion Channel Gating; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Mutagenesis; Neurotoxins; Potassium Channels; Protein Structure, Tertiary; Scorpion Venoms; Solutions; Thermodynamics

The 3D structure of noxiustoxin, the first identified scorpion toxin acting on K+ channels, has been elucidated by NMR and molecular modeling. Thirty-nine solution structures were calculated using 572 distance and 42 dihedral restraints. The average atomic rms deviation between the refined structures and the mean structure is 0.75 A for the backbone atoms. Noxiustoxin adopts a alpha/beta scaffold constituted of a three-stranded beta-sheet (residues 2-3, 25-30, 33-38) linked to a helix (residues 10-20) through two disulfide bridges. A comparison between the 3D structure of noxiustoxin and those of other structurally and functionally related scorpion toxins (charybdotoxin, PO5-NH2, kaliotoxin) revealed a bending capacity of the helix and a variability in the relative orientations between the helix and the beta-sheet. These two features highlight the plasticity of the alpha/beta scaffold and offer a structural explanation for the capacity of the fold to accommodate an additional alanine residue in the Gly-x-Cys pattern of a previously proposed consensus sequence [Bontems et al. (1991) Science 254, 1521-1523]. Our structural data also emphasize the possibility that the beta-sheet of NTX is implicated in the capacity of NTX to recognize voltage-dependent K+ channels.

Topics: Amino Acid Sequence; Animals; Charybdotoxin; Disulfides; Electrochemistry; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Molecular Structure; Potassium Channels; Protein Structure, Secondary; Scorpion Venoms; Scorpions; Sequence Homology, Amino Acid; Thermodynamics

We have analyzed the biophysical and pharmacological properties of five cloned K+ (Kv) channels (Kv1.1, Kv1.2, Kv1.3, Kv1.5, and Kv3.1) stably expressed in mammalian cell lines. Kv1.1 is biophysically similar to a K+ channel in C6 glioma cells and astrocytes, Kv1.3 and Kv3.1 have electrophysiological properties identical to those of the types n and l K+ channels in T cells, respectively, and Kv1.5 closely resembles a rapidly activating delayed rectifier in the heart. Each of these native channels may be formed from the homomultimeric association of the corresponding Kv subunits, and pharmacological compounds that selectively modulate them may be useful for the treatment of neurological, immune, and cardiac disorders. The cell lines described in this report could be used to identify such drugs and we have therefore embarked on a pharmacological characterization of the five cloned channels. The compounds tested in this study include 4-aminopyridine, capsaicin, charybdotoxin, cromakalim, dendrotoxin, diltiazem, D-sotalol, flecainide, kaliotoxin, mast cell degranulating peptide, nifedipine, noxiustoxin, resiniferatoxin, and tetraethylammonium.

Topics: 3T3 Cells; Animals; Base Sequence; Benzopyrans; Capsaicin; Cell Line; Cells, Cultured; Charybdotoxin; Cloning, Molecular; Cromakalim; Diltiazem; Diterpenes; Elapid Venoms; Flecainide; Ion Channel Gating; Mice; Molecular Sequence Data; Nifedipine; Oligodeoxyribonucleotides; Peptides; Potassium Channels; Pyrroles; Scorpion Venoms; Sotalol