ABSTRACT
G protein-activated inwardly rectifying K+ channel subunits GIRK1 (Kir 3.1), GIRK2 (Kir 3.2), and CIR (Kir 3.4) were expressed individually or in combination in Xenopus oocytes and CHO cells. GIRK1 coexpressed with CIR or GIRK2, produced currents up to 10-fold larger than any of the subunits expressed alone. No such clear synergistic effects were observed upon coexpression of CIR/GIRK2 under the same conditions. Coexpression of G protein beta gamma (G beta 1 gamma 2) increased the current through GIRK1/GIRK2 and GIRK2 channels. G beta gamma subunits purified from bovine brain, increased channel activity 50-1000-fold in patches from cells expressing GIRK1/GIRK2 or GIRK2 alone. The single GIRK1/GIRK2 channels resembled previously described neuronal G protein-gated K+ channels. In contrast, single GIRK2 channels were short-lived and unlike any previously described neuronal K+ channel. We propose that some neuronal G protein-activated inward rectifier K+ channels may be formed by a GIRK1/GIRK2 heteromultimer and that G beta gamma activation may involve both subunits.
Subject(s)
GTP-Binding Proteins/metabolism , Neurons/metabolism , Potassium Channels, Inwardly Rectifying , Potassium Channels/chemistry , Animals , Brain/metabolism , CHO Cells , Cattle , Cricetinae , Electrophysiology , Female , G Protein-Coupled Inwardly-Rectifying Potassium Channels , In Vitro Techniques , Ion Channel Gating , Oocytes/metabolism , Potassium Channels/genetics , Potassium Channels/metabolism , Protein Conformation , RNA, Messenger/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , XenopusABSTRACT
In locus coeruleus neurons, substance P (SP) suppresses an inwardly rectifying K+ current via a pertussis toxin-insensitive guanine nucleotide binding protein (G protein; GnonPTX), whereas somatostatin (SOM) or [Met]enkephalin (MENK) enhances it via a pertussis toxin-sensitive G protein (GPTX). The interaction of the SP and the SOM (or MENK) effects was studied in cultured locus coeruleus neurons. In neurons loaded with guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), application of SOM (or MENK) evoked a persistent increase in the inward rectifier K+ conductance. A subsequent application of SP suppressed this conductance to a level less than that before the SOM (or MENK) application; the final conductance level was independent of the magnitude of the SOM (or MENK) response. This suppression by SP was persistent, and a subsequent SOM (or MENK) application did not reverse it. When SP was applied to GTP[gamma S]-loaded cells first, subsequent SOM elicited only a small response. In GTP-loaded neurons, application of SP temporarily suppressed the subsequent SOM- (or MENK)-induced conductance increase. These results suggest that the same inward rectifier molecule that responds to an opening signal from GPTX also responds to a closing signal from GnonPTX. The closing signal is stronger than the opening signal.
Subject(s)
Enkephalin, Methionine/pharmacology , GTP-Binding Proteins/metabolism , Locus Coeruleus/physiology , Potassium Channels/physiology , Somatostatin/pharmacology , Substance P/pharmacology , Animals , Atrial Function , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , In Vitro Techniques , Ion Channel Gating , Neurons/physiology , RatsABSTRACT
Somatostatin enhances an inward rectifier K conductance in cultured locus coeruleus neurons, while substance P reduces an inward rectifier K conductance in cultured nucleus basalis and locus coeruleus neurons. The role of arachidonic acid metabolites in these responses was studied. The somatostatin-induced response was reduced by phospholipase A2 inhibitors, non-specific lipoxygenase inhibitors and specific 5-lipoxygenase inhibitors. A cyclooxygenase inhibitor and a 12-lipoxygenase inhibitor had no effect. 5(S)-HPETE occasionally increased the K conductance, but failed to occlude the somatostatin response. The substance P response was suppressed by a 5-lipoxygenase inhibitor but not by a 12-lipoxygenase inhibitor. These results suggest that the 5-lipoxygenase pathway is not a specific messenger of either one of these responses, but that it plays a more general role in maintaining or enhancing the effectiveness of both somatostatin and substance P responses.
Subject(s)
Arachidonic Acid/metabolism , Brain/physiology , Neurons/physiology , Potassium/physiology , Somatostatin/pharmacology , Substance P/pharmacology , Animals , Arachidonic Acid/antagonists & inhibitors , Brain/cytology , Electric Conductivity , Leukotrienes/pharmacology , Locus Coeruleus/cytology , Locus Coeruleus/physiology , Prosencephalon/cytology , Prosencephalon/physiology , Rats , Rats, Inbred StrainsABSTRACT
Effects of substance P on cultured neurons of the locus coeruleus of the rat were studied using the whole-cell patch clamp technique. In some cells substance P produced a decrease in a K conductance which showed an inwardly rectifying property. In other cells substance P produced an initial inward current which was accompanied by a conductance increase. The rest of the cells showed responses which were mixtures of the above two responses. The measurement of the reversal potential of the initial inward current after suppressing the voltage-gated Ca and K conductances suggests that it is caused by an increase in a non-selective ionic conductance. In cells loaded with 260 microM GTP gamma S, application of substance P produced an irreversible reduction of the K conductance, while the initial inward current could still be recorded, suggesting that the former is mediated by a G protein, whereas the latter may be activated by a different signal transduction mechanism. The initial inward current was not eliminated by external application of high concentrations of tetrodotoxin, d-tubocurarine or amiloride. Nor was it affected by the intracellular application of cyclic GMP or cyclic AMP.
