We previously demonstrated that the endogenously expressed human intermediate conductance, Ca 2؉ -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 ؎ 2% (n ‫؍‬ 16) with 3 M AA, being half-maximally inhibited (IC 50 ) at 1.4 ؎ 0.7 M. In contrast, AA does not

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... it the Ca 2؉ -dependent, small conductance K ؉ channel, rSK2, another member of the KCNN gene 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 Thr 250 to Ser (T250S) resulted in a channel with limited sensitivity to block by AA (8 ؎ 2%, n ‫؍‬ 8), demonstrating that Thr 250 is a key molecular determinant for the inhibition of hIK1 by AA. Likewise, when Val 275 in S6 was mutated to Ala (V275A) AA inhibited only 43 ؎ 11% (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 ؎ 3% and 31 ؎ 3%, respectively. Further, introducing the double mutation S359T/A384V into rSK2 resulted in a 63 ؎ 8% (n ‫؍‬ 9) inhibition by AA, thereby demonstrating the ability to introduce this inhibitory AA binding site into another member of the KCNN gene family. These results demonstrate that AA interacts with the pore-lining amino acids, Thr 250 and Val 275 in hIK1, conferring inhibition of hIK1 by AA and that AA and clotrimazole share similar, if not identical, molecular sites of interaction.