Binding of Correolide to Kv1 Family Potassium Channels

Correolide, a novel nortriterpene natural product, potently inhibits the voltage-gated potassium channel, Kv1.3, and [3H]dihydrocorreolide (diTC) binds with high affinity (Kd ∼ 10 nm) to membranes from Chinese hamster ovary cells that express Kv1.3 (Felix, J. P., Bugianesi, R. M., Schmalhofer, W. A., Borris, R., Goetz, M. A., Hensens, O. D., Bao, J.-M., Kayser, F., Parsons, W. H., Rupprecht, K., Garcia, M. L., Kaczorowski, G. J., and Slaughter, R. S. (1999) Biochemistry 38, 4922–4930). Mutagenesis studies were used to localize the diTC binding site and to design a high affinity receptor in the diTC-insensitive channel, Kv3.2. Transferring the pore from Kv1.3 to Kv3.2 produces a chimera that binds peptidyl inhibitors of Kv1.3 with high affinity, but not diTC. Transfer of the S5 region of Kv1.3 to Kv3.2 reconstitutes diTC binding at 4-fold lower affinity as compared with Kv1.3, whereas transfer of the entire S5-S6 domain results in a normal Kv1.3 phenotype. Substitutions in S5-S6 of Kv1.3 with nonconserved residues from Kv3.2 has identified two positions in S5 and one in S6 that cause significant alterations in diTC binding. High affinity diTC binding can be conferred to Kv3.2 after substitution of these three residues with the corresponding amino acids found in Kv1.3. These results suggest that lack of sensitivity of Kv3.2 to diTC is a consequence of the presence of Phe382 and Ile387 in S5, and Met458 in S6. Inspection of Kv1.1–1.6 channels indicates that they all possess identical S5 and S6domains. As expected, diTC binds with high affinity (Kd values 7–21 nm) to each of these homotetrameric channels. However, the kinetics of binding are fastest with Kv1.3 and Kv1.4, suggesting that conformations associated with C-type inactivation will facilitate entry and exit of diTC at its binding site. Taken together, these findings identify Kv1 channel regions necessary for high affinity diTC binding, as well as, reveal a channel conformation that markedly influences the rate of binding of this ligand.