Binding of Correolide to the Kv1.3 Potassium Channel: Characterization of the Binding Domain by Site-Directed Mutagenesis

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• 作者：Markus Hanner ; Brian Green ; Ying-Duo Gao ; William A. Schmalhofer ; Mary Matyskiela ; Daniel J. Durand ; John P. Felix ; Ana-Rosa Linde ; Carmen Bordallo ; Gregory J. Kaczorowski ; Martin Kohler ; and Maria L.
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：October 2, 2001
• 年：2001
• 卷：40
• 期：39
• 页码：11687 - 11697
• 全文大小：691K
• 年卷期：v.40,no.39(October 2, 2001)
• ISSN：1520-4995

文摘

Correolide is a novel immunosuppressant that inhibits the voltage-gated potassium channelK_v1.3 [Felix et al. (1999) Biochemistry 38, 4922-4930]. [³H]Dihydrocorreolide (diTC) binds with highaffinity to membranes expressing homotetrameric K_v1.3 channels, and high affinity diTC binding can beconferred to the diTC-insensitive channel, K_v3.2, after substitution of three nonconserved residues in S₅and S₆ with the corresponding amino acids present in K_v1.3 [Hanner et al. (1999) J. Biol. Chem. 274,25237-25244]. Site-directed mutagenesis along S₅ and S₆ of K_v1.3 was employed to identify those residuesthat contribute to high affinity binding of diTC. Binding of monoiodotyrosine-HgTX₁A19Y/Y37F([¹²⁵I]HgTX₁A19Y/Y37F) in the external vestibule of the channel was used to characterize each mutantfor both tetrameric channel formation and levels of channel expression. Substitutions at Leu³⁴⁶ and Leu³⁵³in S₅, and Ala⁴¹³, Val⁴¹⁷, Ala⁴²¹, Pro⁴²³, and Val⁴²⁴ in S₆, cause the most dramatic effect on diTC bindingto K_v1.3. Some of the critical residues in S₆ appear to be present in a region of the protein that alters itsconformation during channel gating. Molecular modeling of the S₅-S₆ region of K_v1.3 using the X-raycoordinates of the KcsA channel, and other experimental constraints, yield a template that can be used todock diTC in the channel. DiTC appears to bind in the water-filled cavity below the selectivity filter toa hydrophobic pocket contributed by the side chains of specific residues. High affinity binding is predictedto be determined by the complementary shape between the bowl-shape of the cavity and the shape of theligand. The conformational change that occurs in this region of the protein during channel gating mayexplain the state-dependent interaction of diTC with K_v1.3.