Abstract
We previously showed that activation of the human endothelin A receptor (HETAR) by endothelin-1 (Et-1) selectively inhibits the response to mu opioid receptor (MOR) activation of the G-protein-gated inwardly rectifying potassium channel (Kir3). The Et-1 effect resulted from PLA2 production of an eicosanoid that inhibited Kir3. In this study, we show that Kir3 inhibition by eicosanoids is channel subunit-specific, and we identify the site within the channel required for arachidonic acid sensitivity. Activation of the G-protein-coupled MOR by the selective opioid agonist D-Ala(2)Glyol, enkephalin, released Gbetagamma that activated Kir3. The response to MOR activation was significantly inhibited by Et-1 activation of HETAR in homomeric channels composed of either Kir3.2 or Kir3.4. In contrast, homomeric channels of Kir3.1 were substantially less sensitive. Domain deletion and channel chimera studies suggested that the sites within the channel required for Et-1-induced inhibition were within the region responsible for channel gating. Mutation of a single amino acid in the homomeric Kir3.1 to produce Kir3.1(F137S)(N217D) dramatically increased the channel sensitivity to arachidonic acid and Et-1 treatment. Complementary mutation of the equivalent amino acid in Kir3.4 to produce Kir3.4(S143T)(D223N) significantly reduced the sensitivity of the channel to arachidonic acid- and Et-1-induced inhibition. The critical aspartate residue required for eicosanoid sensitivity is the same residue required for Na(+) regulation of PIP(2) gating. The results suggest a model of Kir3 gating that incorporates a series of regulatory steps, including Gbetagamma, PIP(2), Na(+), and arachidonic acid binding to the channel gating domain.
Highlights
We previously showed that activation of the human endothelin A receptor (HETAR) by endothelin-1 (Et-1) selectively inhibits the response to mu opioid receptor (MOR) activation of the G-protein-gated inwardly rectifying potassium channel (Kir3)
Effects of Endothelin-1 on Kir3 Activation by MOR—Heterologous expression of MOR and Kir3 in Xenopus oocytes generated a malleable system for the study of channel activation (Fig. 2)
The point mutation of Kir3.2(S146T) served as a control as this mutation in the pore was similar to Kir3.1(F137S) and Kir3.4(S143T)
Summary
G-protein-gated inwardly rectifying potassium channel; PIP2, phosphatidylinositol 4,5-bisphosphate; HETAR, human endothelin A receptor; MOR, mu opioid receptor; DAMGO, D-Ala2Glyol, enkephalin; Et-1, endothelin-1; G2, -2 subunit of the G-protein; PLC, phospholipase C. Huang and colleagues proposed that G␥ activates Kir by stabilizing interactions between PIP2 and the potassium channel They showed that PIP2 depletion blocks activation of Kir by both G␥ and Naϩ [8]. Sui et al [10] show that Kir activity is ATP-dependent and is mediated by PIP2; ATP hydrolysis enables both Naϩ and G␥ activation [10] These studies support the hypothesis that Kir gating components responsible for channel activation are interdependent. Arachidonic acid than heteromultimers consisting of Kir3.1 and Kir3.5 These results suggest that eicosanoids have direct effects on G-protein-gated inwardly rectifying potassium channels by modification of the channel conformation, but the molecular basis for eicosanoid inhibition of Kir was not defined. Using site-directed mutagenesis to identify the eicosanoid-sensitive regulatory site on the Kir channel, we provide evidence that eicosanoid-induced inhibition of Kir requires the Naϩ-dependent gating site
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