Delineation of the Functional Site of α-Dendrotoxin

Abstract

We identified the residues that are important for the binding of ␣-dendrotoxin (␣DTX) to Kv1 potassium channels on rat brain synaptosomal membranes, using a mutational approach based on site-directed mutagenesis and chemical synthesis. Twenty-six of its 59 residues were individually substituted by alanine. Substitutions of Lys 5 and Leu 9 decreased affinity more than 1000-fold, and substitutions of Arg 3 , Arg 4 , Leu 6 , and Ile 8 by 5-30-fold. Substitution of Lys 5 by norleucine or ornithine also greatly altered the binding properties of ␣DTX. All of these analogs displayed similar circular dichroism spectra as compared with the wild-type ␣DTX, indicating that none of these substitutions affect the overall conformation of the toxin. Substitutions of Ser 38 and Arg 46 also reduced the affinity of the toxin but, in addition, modified its dichroic properties, suggesting that these two residues play a structural role. The other residues were excluded from the recognition site because their substitutions caused no significant affinity change. Venomous animals from four distinct phyla produce small toxic proteins that block a variety of Kv1 voltage-gated potassium channels. These are the scorpions (1), sea anemones (2-4), marine cone snails (5), and snakes (6 -8), which are arthropods, cnidarians, molluscs, and chordates, respectively. At least four different folds, the sizes of which range from approximately 30 to 60 residues, are associated with these different potassium channel-blocking toxins. These are (i) the ␣/␤-toxin fold, which is found in scorpion toxins, such as charybdotoxin (9); (ii) the fold that comprises two short helices and is only adopted by toxins from sea anemone toxins such as ShK (10) and BgK (11); (iii) the -conotoxin fold, which has three ␤-sheet strands and is adopted by -conotoxin from cone snails (12, 13); and (iv) the BPTI 1 -type fold (14), composed of two short helices and a two-stranded ␤-sheet, which is adopted by the snake dendrotoxins (15-17) and probably by the sea anemone kalicludines (4).Although structurally unrelated, the Kv1 channel-blocking toxins produced by scorpions, snakes, sea anemones, and snails all are likely to bind to the peptide loop between the membranespanning segments S5 and S6 of Kv1 channels (18 -25, 11). Therefore, all of these toxins may possess a functional surface that is complementary to this loop, an observation that raises the question as to how similar these surfaces are from one toxin to another. The answer to such a question may not only shed light on the evolution of these toxins but should also help characterize the surface by which Kv1 channels interact with these toxins. Mutational analyses have finely delineated the functional sites of scorpion toxins (26) and sea anemone toxins (11,25). Although the sea anemone and scorpion toxins are not structurally related, their functional sites share some similarities. They are all flat surfaces of comparable size (ϳ700 Å 2 ) with five functionally important residues, including a similar critical function...

Mapping the Functional Anatomy of BgK on Kv1.1, Kv1.2, and Kv1.3

“…37-41), they are all likely to bind to the P-region of Kv1 channels (32,(42)(43)(44)(45)(46). Recent progress has characterized the sites by which these peptides bind to the vestibule of Kv1 channels (14, 16 -17, 27, 32-33, 36, 47-50), and comparison of these sites revealed that all of these blockers possess at least a common diad composed of two functionally important residues: a lysine and an hydrophobic residue (32,36). A model of the interaction of these blockers with Kv1 channels is now commonly accepted in which this critical lysine plugs the pore of the channel (14 -17, 27).…”

“…Indeed, it was previously proposed that in BgK, Lys-25 and Tyr-26 constitute the two elements of a critical diad formed by a lysine and a spatially close hydrophobic residue (aromatic or aliphatic) found in all Kv1 channels blockers from sea anemone, snakes, and scorpions (32,36). In order to evaluate the functional contribution of the diad, we synthesized the analog possessing both substitutions, K25A/Y26A.…”

Characterization of a Novel Radiolabeled Peptide Selective for a Subpopulation of Voltage-gated Potassium Channels in Mammalian Brain

“…DTX-K (26) was synthesized using the same procedure as for ␣DTX (25). Synthetic charybdotoxin (ChTX) was purchased from Latoxan (Valence, France).…”

“…25. Protein concentration was determined according to Lowry, using bovine serum albumin as a standard.…”

“…Peptides-Native BgK, BgK analogs and ␣DTX were synthesized as described previously (24,25). DTX-K (26) was synthesized using the same procedure as for ␣DTX (25).…”