Binding of correolide to the Kv1.3 potassium channel:: Characterization of the binding domain by site-directed mutagenesis

Correolide is a novel immunosuppressant that inhibits the voltage-gated potassium channel K(v)1.3 [Felix et al. (1999) Biochemistry 38, 4922-4930]. [H-3]Dihydrocorreolide (diTC) binds with high affinity to membranes expressing homotetrameric K(v)1.3 channels, and high affinity diTC binding can be conferred to the diTC-insensitive channel, K(v)3.2, after substitution of three nonconserved residues in S-5 and S-6 with the corresponding amino acids present in K(v)1.3 [Harmer et al. (1999) J. Biol. Chem. 274, 25237-25244]. Site-directed mutagenesis along S-5 and S-6 of K(v)1.3 was employed to identify those residues that contribute to high affinity binding of diTC. Binding of monoiodotyrosine-HgTX(1)A19Y/Y37F ([I-125]HgTX(1)A19Y/Y37F) in the external vestibule of the channel was used to characterize each mutant for both tetrameric channel formation and levels of channel expression. Substitutions at Leu(346) and Leu(353) in S-5, and Ala(413), Val(417), Ala(421), Pro(423), and Val(424) in S-6, cause the most dramatic effect on diTC binding to K(v)1.3. Some of the critical residues in S6 appear to be present in a region of the protein that alters its conformation during channel gating. Molecular modeling of the S-5-S-6 region of K(v)1.3 using the X-ray coordinates of the KcsA channel, and other experimental constraints, yield a template that can be used to dock diTC in the channel. DiTC appears to bind in the water-filled cavity below the selectivity filter to a hydrophobic pocket contributed by the side chains of specific residues. High affinity binding is predicted to be determined by the complementary shape between the bowl-shape of the cavity and the shape of the ligand. The conformational change that occurs in this region of the protein during channel gating may explain the state-dependent interaction of diTC with K(v)1.3.