Targeted Mutagenesis with a Homology Model Identifies Critical Residues for Arachidonic Acid Inhibition of Kv4 Channels

Abstract

Polyunsaturated fatty acids such as arachidonic acid (AA) demonstrate inhibitory modulation of Kv4 potassium channels. Molecular docking approaches using a new Kv4.2 homology model predicted a membrane-embedded binding pocket for AA comprised of the S4-S5 linker on one subunit and several nearby hydrophobic residues within the internal side of S5 and S6 from an adjacent subunit. We tested the hypothesis that modulatory effects of AA on Kv4.2/KChIP channels require access to this site. Targeted mutation of a lysine residue and a nonpolar residue within the S4-S5 linker as well as a nonpolar residue in S3 significantly impaired the effects of AA on K+ currents in Xenopus oocytes. These residues may be important in stabilizing or regulating access to the negatively charged carboxylate moiety on the fatty acid. The structural specificity of this interaction was supported by the lack of disruption of AA effects observed with charge neutralizing mutations at residues located near but not within the predicted binding pocket. Furthermore, we found that the crystal structure of the Kv1.2/2.1 channel chimera lacks an AA docking site with the structural features present in the proposed hydrophobic pocket of Kv4.2 and the chimera was likewise unaffected by AA. We simulated the mutagenic substitutions of critical residues identified in our Kv4.2 model to provide a structural interpretation of the disruption of the AA binding pocket. We conclude that AA inhibits Kv4 channel currents and facilitates inactivation by interacting with a hydrophobic binding pocket in which a lysine residue within the S4-S5 linker is important for AA interaction.