Eicosanoids Inhibit the G-protein-gated Inwardly Rectifying Potassium Channel (Kir3) at the Na+/PIP2 Gating Site

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 G␤␥ 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 G␤␥, PIP 2 , Na ؉ , and arachidonic acid binding to the channel gating domain.

Molecular Determinants for Activation of G-protein-coupled Inward Rectifier K+ (GIRK) Channels by Extracellular Acidosis

“…However, previous studies have demonstrated that the mutation of a single amino acid residue in the pore-forming sequence (P) allows both GIRK1 and GIRK4 to be expressed homomerically (35). This homomeric expression technique has been conducive to elucidating the modulation of GIRK channels by several modulators (11,35,37,38). Therefore, we constructed the GIRK1 with Phe-137 mutated to serine (GIRK1-F137S) and GIRK4 with Ser-143 mutated to threonine (GIRK4-S143T) as shown previously by Vivaudou et al (35).…”

M1Muscarinic Receptors Inhibit L-type Ca2+Current and M-Current by Divergent Signal Transduction Cascades

“…This model does not require a loss of PIP 2 bound to channels. In a second scenario, AA could occupy a critical position around the channel, competing with PIP 2 for the same site of interaction (Liu et al, 2004), as has been proposed for some K ϩ channels (Rogalski and Chavkin, 2001). A third possibility is that AA acts directly on the channel at a site distinct from PIP 2 .…”

A Role for the Middle C Terminus of G-protein-activated Inward Rectifier Potassium Channels in Regulating Gating

“…The Glu-224 residue in Kir2.1 and a histidine at a similar position in Kir6.2 (His-216) were also shown to affect polyamine block by electrostatic interactions, suggesting that they face the permeation pathway (19 -21). Positively charged residues within the proximal C terminus are also known to be important for binding phosphoinositides, and an aspartate within this region of GIRK2 and GIRK4 mediates the activation of GIRK channels by Na ϩ (7) and inhibition by eicosanoids (22).The role of the highly conserved middle C-terminal region in Kir channel function is not well understood. It is also involved in binding phosphoinositides (6,23), and determinants of G ␤␥ binding have been identified within this region and within the proximal N and C termini (24).…”

“…The Glu-224 residue in Kir2.1 and a histidine at a similar position in Kir6.2 (His-216) were also shown to affect polyamine block by electrostatic interactions, suggesting that they face the permeation pathway (19 -21). Positively charged residues within the proximal C terminus are also known to be important for binding phosphoinositides, and an aspartate within this region of GIRK2 and GIRK4 mediates the activation of GIRK channels by Na ϩ (7) and inhibition by eicosanoids (22).…”