Molecular Localization of the Inhibitory Arachidonic Acid Binding Site to the Pore of hIK1

We previously demonstrated that the endogenously expressed human intermediate conductance, Ca2+-activated K+ channel (hIK1) was inhibited by arachidonic acid (AA) (Devor, D. C., and Frizzell, R. A. (1998) Am. J. Physiol. 274, C138–C148). Here we demonstrate, using the excised, inside-out patch-clamp technique, that hIK1, heterologously expressed in HEK293 cells, is inhibited 82 ± 27 (n = 16) with 3 ॖm AA, being half-maximally inhibited (IC50) at 1.4 ± 0.7 ॖm. In contrast, AA does not inhibit the Ca2+-dependent, small conductance K+ channel, rSK2, another member of the KCNNgene family. Therefore, we utilized chimeric hIK1/rSK2 channels to define the AA binding domain on hIK1 to the S5-Pore-S6 region of the channel. Subsequent site-directed mutagenesis revealed that mutation of Thr250 to Ser (T250S) resulted in a channel with limited sensitivity to block by AA (8 ± 27, n = 8), demonstrating that Thr250 is a key molecular determinant for the inhibition of hIK1 by AA. Likewise, when Val275 in S6 was mutated to Ala (V275A) AA inhibited only 43 ± 117 (n = 9) of current flow. The double mutation T250S/V275A eliminated the AA sensitivity of hIK1. Introducing the complimentary single amino acid substitutions into rSK2 (S359T and A384V) conferred partial AA sensitivity to rSK2, 21 ± 37 and 31 ± 37, respectively. Further, introducing the double mutation S359T/A384V into rSK2 resulted in a 63 ± 87 (n= 9) inhibition by AA, thereby demonstrating the ability to introduce this inhibitory AA binding site into another member of theKCNN gene family. These results demonstrate that AA interacts with the pore-lining amino acids, Thr250 and Val275 in hIK1, conferring inhibition of hIK1 by AA and that AA and clotrimazole share similar, if not identical, molecular sites of interaction.