This study reports the identification of an endogenous inhibitor of the G protein–gated (KACh) channel and its effect on the KACh channel kinetics. In the presence of acetylcholine in the pipette, KACh channels in inside-out atrial patches were activated by applying GTP to the cytoplasmic side of the membrane. In these patches, addition of physiological concentration of intracellular ATP (4 mM) upregulated KACh channel activity approximately fivefold and induced long-lived openings. However, such ATP-dependent gating is normally not observed in cell-attached patches, indicating that an endogenous substance that inhibits the ATP effect is present in the cell. We searched for such an inhibitor in the cell. ATP-dependent gating of the KACh channel was inhibited by the addition of the cytosolic fraction of rat atrial or brain tissues. The lipid component of the cytosolic fraction was found to contain the inhibitory activity. To identify the lipid inhibitor, we tested the effect of ∼40 different lipid molecules. Among the lipids tested, only unsaturated free fatty acids such as oleic, linoleic, and arachidonic acids (0.2–2 μM) reversibly inhibited the ATP-dependent gating of native KACh channels in atrial cells and hippocampal neurons, and of recombinant KACh channels (GIRK1/4 and GIRK1/2) expressed in oocytes. Unsaturated free fatty acids also inhibited phosphatidylinositol-4,5-bisphosphate (PIP2)-induced changes in KACh channel kinetics but were ineffective against ATP-activated background K1 channels and PIP2-activated KATP channels. These results show that during agonist-induced activation, unsaturated free fatty acids in the cytoplasm help to keep the cardiac and neuronal KACh channels downregulated by antagonizing their ATP-dependent gating. The opposing effects of ATP and free fatty acids represent a novel regulatory mechanism for the G protein–gated K+ channel.

Rapid desensitization of the muscarinic K+ current (KACh current) is observed in cell-attached patches with 10 microM acetylcholine in the pipette. When inside-out patches were formed within approximately 1 s after formation of cell-attached patches and GTP was applied to the cytoplasmic side of the membrane, desensitization was not observed, indicating that a cytosolic factor mediated the desensitization. Applying the atrial cytosolic extract directly to the cytoplasmic side of such inside-out patches elicited a rapid desensitization of the KACh current. ATP (1-4 mM) reversed this effect of the cytosol and reverted the KACh channel to the undesensitized state. These effects of ATP and cytosol on the KACh channel could occur in the absence of GTP or in the presence of 100 microM guanosine 5'-O-(3-thiotriphosphate), indicating that G protein was not involved. Treatment of the cytosol with proteases (trypsin, chymotrypsin, bacterial protease) or heat denaturation abolished the effect of the cytosol on the KACh channel kinetics, indicating that the cytosolic factor was a protein. Functional assay of the fractions collected from gel filtration column indicated that the molecular mass of the native protein was 95-130 kDa. We conclude that a large cytosolic protein mediates the rapid desensitization of the KACh channel current via a G protein-independent pathway.

Arachidonic acid has been shown to activate K(+)-selective, mechanosensitive ion channels in cardiac, neuronal and smooth muscle cells. Since the cardiac G protein (GK)-gated, muscarinic K+ (KACh) channel can also be activated by arachidonic acid, we investigated whether the KACh channel was also sensitive to membrane stretch. In the absence of acetylcholine (ACh), KACh channels were not active, and negative pressure failed to activate these channels. With ACh (10 microM) in the pipette, applying negative pressure (0 to -80 mm Hg) to the membrane caused a reversible, pressure-dependent increase in channel activity in cell-attached and inside-out patches (100 microM GTP in bath). Membrane stretch did not alter the sensitivity of the KACh channel to GTP. When GK was maximally activated with 100 microM GTP gamma S in inside-out patches, the KACh channel activity could be further increased by negative pressure. Trypsin (0.5 mg/ ml) applied to the membrane caused activation of the KACh channel in the absence of ACh and GTP; KACh channel activity was further increased by stretch. These results indicate that the atrial muscarinic K+ channels are modulated by stretch independently of receptor/G protein, probably via a direct effect on the channel protein/ lipid bilayer.

In atrial cells, the open probability of G protein-activated ACh-sensitive K+(KACh) channels can be increased approximately fivefold by intracellular ATP (ATPi). Using inside-out patches, we examined how proteases, changes in intracellular pH, and different anions affect G protein-mediated activation and ATP-induced stimulation of the KACh channel. Treatment with trypsin (0.5 mg/ml) removed the GTP dependence of the KACh channel and abolished the ATP-induced stimulation. Intracellular GTP activated KACh channels at all intracellular pH values tested (6.0–8.0), with the concentration at which half-maximal activation ( K ½) occurred ranging from 0.3 (pH 8.0) to 6.7 (pH 6.0) μM. However, the ATPi-induced increase in KACh channel activity was inhibited at pH 8.0 ( K ½ = pH 7.4). All anions tested except sulfate, phosphate, fluoride, and iodide supported GTP-induced activation. Of the anions that supported GTP-induced activation, only citrate blocked the ATP-induced stimulation of the KACh channel. These results indicate that the GTP- and ATP-mediated effects on the KACh channel use separate signaling pathways. The ATP-mediated effect involves a trypsin- and pH-sensitive mechanism.

Extracellular ATP (ATPo) and adenosine activate G protein-gated inwardly rectifying K+ currents in atrial cells. Earlier studies have suggested that the two agonists may use separate pathways to activate the K+ current. Therefore, we examined whether the K+ channels activated by the two agonists have different properties under identical ionic conditions. In cell-attached patches, K+ channels activated by 100 microM ATP in the pipette had a single-channel conductance and mean open time of 32.0 +/- 0.2 pS and 0.5 +/- 0.1 ms, respectively, compared with 31.3 +/- 0.3 pS and 0.9 +/- 0.1 ms for the K+ channels activated by adenosine (140 mM KCl). With ATPo as the agonist, the K+ channel activity in cell-attached patches was approximately threefold lower than that in inside-out patches with 100 microM GTP in the bath. Applying ATP to the cytoplasmic side of the membrane (ATPi) produced a biphasic concentration-dependent effect on channel activity: an increase at low [mean affinity constant (K0.5) = 190 microM] and a decrease at high (K0.5 = 1.3 mM) concentrations. In contrast, with adenosine as the agonist, K+ channel activity in cell-attached patches was approximately fourfold greater than that in inside-out patches with 100 microM GTP in the bath. In inside-out patches, ATPi only augmented the K+ channel activity (K0.5 = 32 microM). These results show that although both ATPo and adenosine activate kinetically similar K+ channels in atrial cells, the channels are regulated differently by intracellular nucleotides.