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

Abstract

BgK is a peptide from the sea anemone Bunodosoma granulifera, which blocks Kv1.1, Kv1.2, and Kv1.3 potassium channels. Using 25 analogs substituted at a single position by an alanine residue, we performed the complete mapping of the BgK binding sites for the three Kv1 channels. These binding sites included three common residues (Ser-23, Lys-25, and Tyr-26) and a variable set of additional residues depending on the particular channel. Shortening the side chain of Lys-25 by taking out the four methylene groups dramatically decreased the BgK affinity to all Kv1 channels tested. However, the analog K25Orn displayed increased potency on Kv1.2, which makes this peptide a selective blocker for Kv1.2 (K D 50-and 300-fold lower than for Kv1.1 and Kv1.3, respectively). BgK analogs with enhanced selectivity could also be made by substituting residues that are differentially involved in the binding to some of the three Kv1 channels. For example, the analog F6A was found to be >500-fold more potent for Kv1.1 than for Kv1.2 and Kv1.3. These results provide new information about the mechanisms by which a channel blocker distinguishes individual channels among closely related isoforms and give clues for designing analogs with enhanced selectivity.

Developmental Biology of Neoplastic Growth

“…Three residues (S20, K22, and Y23 in ShK; S23, K25 and Y26 in BgK) are strictly conserved in all SAK-I toxin sequences, and represent a common core of hot spot residues for pharmacological activity ( Fig. 2a) (Alessandri-Haber et al 1999;Castaneda et al 1995;Gasparini et al 2004;Gendeh et al 1997b;Minagawa et al 1998;Pennington et al 1996a, b;Schweitz et al 1995). Alanine mutation analyses have shown that other residues (I7, R11, H19, R24 and F27 in ShK), which are clustered around the three conserved residues, are also important for the binding activity of ShK to Kv1.2 channels in brain membranes, and to Kv1.3 channels in T lymphocytes (Pennington et al 1996a, b;Rauer et al 1999).…”

“…This is why, to allow a more extensive use of these very useful pharmacological tools, especially for in vivo studies which use large amounts of material, a number of the sea anemone K + channel inhibitors like ShK or BgK, have been chemically synthesized. Solid-phase synthesis allows the obtaining of "mg" of linear peptides which then require to be folded to their biologically active forms (Alessandri-Haber et al 1999;Cotton et al 1997;Pennington et al 1995). The synthetic peptides display the same activity on K + channels as the native toxins.…”

“…2c) (Doyle et al 1998;Gilquin et al 2002;Kalman et al 1998;Lanigan et al 2002;). Since the different Kv1 subtypes are highly homologous (83% identity) in the P region, it is not really surprising that a single type of toxin can bind to several members of the Kv1 channel family (Alessandri-Haber et al 1999). Double-mutant cycles experiments have shown that a diad, composed of an aromatic hydrophobic amino acid and a lysine, constitutes a conserved functional core and acts as a common anchor in different models of toxin-channel interaction (Dauplais et al 1997;Gasparini et al 2004;Gilquin et al 2002).…”

“…Although they are minor constituents of sea anemone extracts, their highly basic character and thermal stability (even at 100°C) facilitated their isolation and subsequent sequence determinations. They were readily synthesized by solid-phase methods (Pennington et al 1995); this allowed detailed structure-activity analyses, including so-called "alanine" scans, to identify residues critical for ion channel binding (Pennington et al 1996a, b;Alessandri-Haber et al 1999). The solution structure of synthetic ShK ( Fig.…”

Allurin: Exploring the Activity of a Frog Sperm Chemoattractant in Mammals

“…Last is the ion channel regulatory (ICR) domain at the C-terminal (magenta), named for its ability to block or modulate a variety of voltage-dependent potassium and calcium channels as well as ryanodine receptors (Gibbs et al 2006 ). The ICR domain is similar in tertiary structure to ion channelblocking peptides from sea anenomes (Pennington et al 1999 ;Alessandri-Haber et al 1999 ;Cotton et al 1997 ).…”