Abstract
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 microm AA, being half-maximally inhibited (IC(50)) at 1.4 +/- 0.7 microm. In contrast, AA does not inhibit 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.
Highlights
Intermediate conductance, Ca2ϩ-activated Kϩ channels play crucial roles in a wide array of physiological processes, including agonist-mediated transepithelial ClϪ secretion across airway and intestinal epithelia [1,2,3,4,5,6]
We previously demonstrated that inhibition of cytosolic PLA2 resulted in a potentiated ClϪ secretory response to the Ca2ϩ-mediated agonist, carbachol in T84 cells and that arachidonic acid (AA) was a potent negative modulator of the intermediate conductance, Ca2ϩ-dependent Kϩ channel in these cells [2]
Arachidonic Acid Inhibits Heterologously Expressed hIK1—We previously demonstrated that AA inhibits endogenously expressed hIK1 in T84 cells with high affinity [2]
Summary
Intermediate conductance, Ca2ϩ-activated Kϩ channels play crucial roles in a wide array of physiological processes, including agonist-mediated transepithelial ClϪ secretion across airway and intestinal epithelia [1,2,3,4,5,6]. Ca2ϩ-mediated agonists are known to increase AA levels in a wide range of tissues where hIK1 is expressed, including colon and lung (14, 16 –18). This can occur in several ways [19], including: 1) Ca2ϩ directly activating PLA2, 2) either diacylglycerol itself or protein kinase C activating PLA2, or 3) diacylglycerol lipase directly generating AA from diacylglycerol. We previously demonstrated that inhibition of cytosolic PLA2 resulted in a potentiated ClϪ secretory response to the Ca2ϩ-mediated agonist, carbachol in T84 cells and that AA was a potent negative modulator of the intermediate conductance, Ca2ϩ-dependent Kϩ channel in these cells [2]. We demonstrate that AA directly inhibits heterologously expressed hIK1, whereas rSK2 is insensitive
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