Structure-guided transformation of charybdotoxin yields an analog that selectively targets Ca2+-activated over voltage-gated K+ channels

We have used a structure-based design strategy to transform the polypeptide toxin charybdotoxin, which blocks several voltage-gated and Ca2+-activated KC channels, into a selective inhibitor. As a model system, we chose two c hannels in T-lymphocytes, the voltage-gated channel Kv1.3 and the Ca2+-acti vated channel IKCa1, Homology models of both channels were generated based on the crystal structure of the bacterial channel KcsA. Initial docking of charybdotoxin was undertaken with both models, and the accuracy of these do cking configurations was tested by mutant cycle analyses, establishing that charybdotoxin has a similar docking configuration in the external vestibul es of IKCa1 and Kv1.3. Comparison of the refined models revealed a unique c luster of negatively charged residues in the turret of Kv1.3, not present i n IKCa1, To exploit this difference, three novel charybdotoxin analogs were designed by introducing negatively charged residues in place of charybdoto xin Lys(32), which lies in close proximity to this cluster. These analogs b lock IKCa1 with similar to 20-fold higher affinity than Kv1.3, The other ch arybdotoxin-sensitive Kv channels, Kv1.2 and Rv1.6, contain the negative cl uster and are predictably insensitive to the charybdotoxin position 32 anal ogs, whereas the maxi-K-Ca, channel, hSlo, lacking the cluster, is sensitiv e to the analogs. This provides strong evidence for topological similarity of the external vestibules of diverse K+ channels and demonstrates the feas ibility of using structure-based strategies to design selective inhibitors for mammalian K+ channels. The availability of potent and selective inhibit ors of IKCa1 will help to elucidate the role of this channel in T-lymphocyt es during the immune response as well as in erythrocytes and colonic epithe lia.