Binding of correolide to the K(v)1.3 potassium channel: Characterization of the binding domain by site-directed mutagenesis

Correolide is a novel immunosuppressant that inhibits the voltage-gated pot assium channel K(v)1.3 [Felix et al. (1999) Biochemistry 38, 4922-4930]. [H -3]Dihydrocorreolide (diTC) binds with high affinity to membranes expressin g homotetrameric K(v)1.3 channels, and high affinity diTC binding can be co nferred to the diTC-insensitive channel, K(v)3.2, after substitution of thr ee 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 iden tify those residues that contribute to high affinity binding of diTC. Bindi ng of monoiodotyrosine-HgTX(1)A19Y/Y37F ([I-125]HgTX(1)A19Y/Y37F) in the ex ternal vestibule of the channel was used to characterize each mutant for bo th tetrameric channel formation and levels of channel expression. Substitut ions 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 r egion of the protein that alters its conformation during channel gating. Mo lecular modeling of the S-5-S-6 region of K(v)1.3 using the X-ray coordinat es of the KcsA channel, and other experimental constraints, yield a templat e 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 c ontributed by the side chains of specific residues. High affinity binding i s predicted to be determined by the complementary shape between the bowl-sh ape of the cavity and the shape of the ligand. The conformational change th at occurs in this region of the protein during channel gating may explain t he state-dependent interaction of diTC with K(v)1.3.