Study Of The Specificity Of GPCR Regulation Of GIRK Channels

The G protein-gated inward rectifier K+ channels (GIRK) are part of a superfamily of inward-rectifier K channels. They are expressed in cardiac myocytes and central neurons. GIRK channels are G-protein-activated and mediate the effects of certain G-protein-coupled receptors (GPCRs) on electrical activity in cardiac, neuronal and neurosecretory cells. GIRK1 and GIRK4 are specifically localized in the atrial and sinoatrial node cells of the heart and fulfill important physiological roles. In the heart, activation of GIRK by acetylcholine via the muscarinic m2 receptor and a pertussis toxin (PTX)-sensitive G protein induces hyperpolarizaton and slows of cardiac frequency. GIRK2 channels mainly exist in neuronal cells. In the brain, GIRKs are activated via PTX-sensitive G proteins by a number of neurotransmitters: GABA, dopamine, serotonin, opioids, etc. and the modulation of such channels regulate neuronal excitability. It is currently widely accepted Gβγt?hat is liberated from PTX-sensitive Gi specifically actives GIRK channels. Whereas PTX-sensitive G proteins activate the channel under normal physiological conditions, PTX-insensitive G ?protein such as Gq and Gs can't activate GIRK channels. So G protein can specifically regulate GIRK channels. To date, 5 kinds of β?subunits and 12 kinds of γ?subunits have been cloned and identified, but Gβγd?oes not seems to be a plausible explanation for this specificity, since GIRKs are similarly activated by many different combinations of Gβand Gγsubtypes. In this study, we expressed GIRK4* channels and G protein coupled receptors in Xenopus oocytes to try to understand the underlying mechanism of specificity in GIRK activation by G protein coupled receptors. Then by overexpressing Gα, such as Gαq,Gαs, in Xenopus oocytes, we investigated the effects of overexpressing Gαon specific regulation of G protein on GIRK4* channels . Ⅰ. The specificity of regulation GIRK by G proteins Aim: To observe the electrophysiological properties of the regulation of GIRK channels by different GPCRs and to investigate the specificity of these regulation by coexpressing GIRK channels and differentGPCRs in Xenopus oocytes. Methods: (1) cRNA Transcription in vitro(:1.1)channels: In our study we utilized a point mutant of GIRK4 channel, GIRK4 (S143T), designated as GIRK4*. (1.2)GPCR: m2 and m1 muscarinic receptors, β2 adrenergic receptors and Bradykinin receptors (BK2R). All cDNA constructs were subcloned into the pGEMHE plasmid vector and the sequences of all constructs were confirmed by sequencing. All cRNAs were transcribed in vitro.(2)Microinjection of oocytes: cRNA of the various channels and receptors were injected in the range of 1-2 ng/ oocyte depending on the functional expression level.After injection, oocytes were incubated in ND96 supplemented with 2.5 mM sodium pyruvate at 19-20 ℃. ( 3 ) The Two-Electrode Voltage Clamp (TEVC) technique was used to record GIRK currents and characters of regulation GIRK by G protein coupled recptor in oocytes Oocytes. Results: 1 GIRK currents were increased with m2 receptor activation. In our study we utilized a point mutant of GIRK4 channel, GIRK4 (S143T), designated as GIRK4* that is highly active when expressed as homomers. m2 receptor is known to be coupled with Gi protein. m2 receptor and GIRK4﹡were co-expressed in oocytes, Currents were recorded using the TEVC technique. Current amplitudes were measured at +80 mV and –80 mV. In high potassium (HK) solution, Xenopus oocytes exhibited large agonist-independent inwardly rectifying potassium currents that were called basal currents. Upon application of ACh, GIRK currents were further increased from 13±2.38μA to 39.25±3.9μA. 2 GIRK currents were inhibited with m1 receptor activation. m1 receptor is known to be coupled with Gq protein. m1 receptor and GIRK4﹡were coexpressed in oocytes. ACh application led to GIRK current inhibition from 23.75±2.39μA to 11±1.35μA (P<0.01), and at the mean time, transient calcium-activated chloride currents were activated. 3 GIRK currents were inhibi...