All SAK-I toxins contain two a-helices and no P-sheet. They block Kv1 channels by plugging the channel pore. 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). For BgK, other residues such as F6 and N19 are also involved in the binding and selectivity to Kv1 channels (Racape et al. 2002). Molecular models of complexes between sea anemone toxins and Kv1 channels have been developed using the structure of KcsA (Doyle et al. 1998; Lanigan et al. 2002). They provide a molecular description for the interaction of toxin residues with the pore of the channel (Fig. 2c) (Doyle et al. 1998; Gilquin et al. 2002; Kalman et al. 1998; Lanigan et al. 2002; Norton et al. 2004). 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). The toxin diad makes electrostatic interactions with carbonyl oxygen atoms of the conserved tyrosine residue (motif GYGD) in the channel selectivity filter. The functional diad K22-Y23 of ShK interacts with the Y in the GYGD motif of the Kv1.3 channel pore domain (Gasparini et al. 2004; Kalman et al. 1998; Lanigan et al. 2002; Norton et al. 2004). This key step is completed by hydrophobic interactions between residues F6 and Y26 in BgK, (Gilquin et al. 2002), Y23 in ShK, (Lanigan et al. 2002) (Fig. 2c) and the hydrophobic Y379 residue in the S5-S6 region of the Kv1.1 channel. Mutations of the Y379 residue in Kv1.1 modify the affinity and the selectivity of several scorpion and sea anemone toxins such as BgK and ShK (Gilquin et al. 2005).
All these toxin-channel interaction studies have assisted in the design of new types of toxins, such as ShK-Dap22 a mutant peptide where K22 has been replaced by a diaminopropionic acid. Binding and electrophysiological studies have shown that ShK-Dap22 is a highly potent and selective blocker of the Kv1.3 channel with a 100-fold decreased affinity for Kv1.1, Kv1.4 and Kv1.6 channels (Kalman et al. 1998). NMR studies have shown that the overall structures of ShK and ShK-Dap22 are quite similar, but there are differences in the side chains involved in Kv1.3 binding (Kalman et al. 1998; Norton et al. 2004). A high expression level of Kv1.3 is considered as a marker for activated effector memory T cells (TEM cells), which are involved in the pathogenesis of autoimmune diseases. Therefore, the selective suppression of autoreactive TEM cells with Kv1.3 blockers might constitute a novel approach for the treatment of multiple sclerosis (MS) and other autoimmune diseases such as type-1 diabetes mellitus or psoriasis. Both ShK and ShK-Dap22 were proven to prevent and treat rat autoimmune encephalomyelitis (Beeton et al. 2001; Norton et al. 2004). In addition, ShK-F6CA, a fluorescein-labeled analogue of ShK, has been reported to have potential applications in the diagnostic of autoimmune diseases (Beeton et al. 2003; Norton et al. 2004). This peptide, containing an additional negatively-charged moiety at the N-terminus, has a higher affinity and selectivity than ShK for Kv1.3 channels, allowing a specific detection of activated TEM cells implicated in multiple sclerosis.
The kalicludines (AsKC1-3), which belong to the SAK-II family of toxins, also block Kv1.2 channels, although with less affinity (Schweitz et al. 1995); they do not contain the functional dyad "K-Y" conserved in SAK-I.
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