kaliotoxin and agitoxin-2

BmKTX is a toxin recently purified from the venom of Buthus Martensi, which belongs to the kaliotoxin family. We have determined its solution structure by use of conventional two-dimensional NMR techniques followed by distance-geometry and energy minimization. The calculated structure is composed of a short alpha-helix (residues 14 to 20) connected by a tight turn to a two-stranded antiparallel beta-sheet (sequences 25-27 and 32-34). The beta-turn connecting these strands belongs to type I. The N-terminal segment (sequence 1 to 8) runs parallel to the beta-sheet although it cannot be considered as a third strand. Comparison of the conformation of BmKTX and toxins of the kaliotoxin family clearly demonstrates that they are highly related. Therefore, analysis of the residues belonging to the interacting surface of those toxins allows us to propose a functional map of BmKTX slightly different from the one of KTX and AgTX2, which may explain the variations in affinities of these toxins towards the Kv1.3 channels.

Topics: Amino Acid Sequence; Animals; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Potassium Channel Blockers; Protein Conformation; Scorpion Venoms; Scorpions; Sequence Homology, Amino Acid

Potent and selective peptidyl blockers of the Shaker-type (Kv1) voltage-gated potassium channels were used to determine the role of these channels in regulating the spontaneous motility of smooth muscle preparations. Margatoxin (MgTX), kaliotoxin, and agitoxin-2 at 1 to 10 nM and agitoxin-1 at 50 to 100 nM induce twitches in guinea pig ileum strips. These twitches are abolished by tetrodotoxin (TTX, 0.5 microM), atropine (1 microM), hexamethonium (10 microM), or nifedipine (0.1 microM). It is proposed that blockade of Kv1 channels by MgTX, kaliotoxin, or the agitoxins increases excitability of intramural nerve plexuses in the ileum, promoting release of acetylcholine from excitatory motor nerve terminals. This, in turn, leads to Ca2+-dependent action potentials and twitching of the muscle fibers. MgTX does not induce twitches in several other guinea pig and/or rat vascular, genitourinary, or gastrointestinal smooth muscles, although small increases in spontaneous myogenic activity may be seen in detrusor muscle exposed to >30 nM MgTX. This effect is not reversed by TTX or atropine. The TTX- and atropine-sensitive twitches of guinea pig ileum are also induced by nanomolar concentrations of alpha-dendrotoxin, a selective blocker of Shaker Kv1.1 and 1.2 subtypes, or stichodactylatoxin, a peptide isolated from sea anemone that displays high affinity for Kv1.1 and 1.3, but not by charybdotoxin, which blocks Kv1.2 and 1.3 but not 1.1. The data taken together suggest that high-affinity blockade of Kv1.1 underlies the ability of MgTX, kaliotoxin, agitoxin-1, agitoxin-2, alpha-dendrotoxin, and stichodactylatoxin to elicit TTX-sensitive twitches in guinea pig ileum.

Topics: Acetylcholine; Animals; Atropine; Enteric Nervous System; Female; Guinea Pigs; Hexamethonium; Ileum; In Vitro Techniques; Isometric Contraction; Kv1.1 Potassium Channel; Male; Muscle, Smooth; Muscle, Smooth, Vascular; Neurotoxins; Nifedipine; Peptides; Portal Vein; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Voltage-Gated; Rats; Rats, Wistar; Scorpion Venoms; Shaker Superfamily of Potassium Channels; Tetrodotoxin; Toxins, Biological; Urinary Bladder

A new generation of structural models were developed of the outer vestibule and ion-selective portion of the voltage-gated Shaker K+ channel. Some features of these models are similar to those that we have developed previously [Durrel S. R. and Guy H. R. (1992) Biophys. J. 62, 238-250; Guy H. R. (1990) In Monovalent Cations in Biological Systems (Pasternak C. A., Ed.), pp. 31-58, CRC Press, Boca Raton, FL; Guy H. R. and Durell S. R. (1994) In Molecular Evolution of Physiological processes (Fambrough D., Ed.), pp. 197-212, The Rockefeller University Press, NY; Guy H. R. and Durell S. R. (1995) In Ion Channels and Genetic Diseases (Dawson D., Ed.), pp. 1-16, The Rockefeller University Press, NY] and other features were modified to make the models more consistent with recent experimental findings. The first part of the P segment is postulated, as always, to form a short alpha helix that spans only the outer portion of the membrane. The helix is tilted so that its C-terminal is nearer the pore than its N-terminal. The latter part of the P segment, P2, is postulated to have a relatively elongated conformation that is positioned approximately parallel to the axis of the pore. Four of the P2 segments assemble to form an ion-selective region that has two narrow regions; one formed by the Y445 side-chains at the outer entrance of the pore and one formed by the backbone of the T442 residues near the innermost part of the P segments. The S6 segment is postulated to form two alpha helices. The first S6 helix packs next to the P segments in our models. The NMR structures of two scorpion toxins, charybdotoxin and agitoxin 2, have been docked into the models of the outer vestibules. The shape of the outer vestibule has been modeled so that specific toxin-channel residue-residue interactions correspond to those that have been identified experimentally.

Topics: 4-Aminopyridine; Charybdotoxin; Ion Channel Gating; Models, Molecular; Mutation; Potassium Channels; Protein Structure, Secondary; Scorpion Venoms; Shaker Superfamily of Potassium Channels; Tetraethylammonium; Tetraethylammonium Compounds; Toxins, Biological