The Study Of Interaction Between Gα Subunits And GIRK

G protein coupled inwardly rectifier potassium channels (GIRK) are part of a superfamily of inwardly rectifier K+ channels, GIRK are composed of five subunits (GIRK1-5). GIRK channels are involved in the regulation of the excitability of neurons and heart. They may contribute to the resting and inhibitory postsynaptic potential. Their molecular counterparts have been identified and the channel has been shown to be members of the Kir3.x family of K+ channels. As the name implies, GIRK channels are modulated by G-proteins; they are also modulated by phosphatidylinositol-4,5-bisphosphate, intracellular sodium, ethanol and mechanical stretch. 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 hyperpolarization 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 has been shown that the Gαsubunit is the key determinant of specificity of channel activation for receptors coupling predominantly to Gi/o as against those that couple to Gq and Gs. In native atrial myocytes, only receptors coupled to PTX-sensitive G proteins (Gi/Go) were believed to couple to K(ACh) (GIRK) channels, such as M2 muscarinic, A1 purinergic, and a sphingolipid receptor belonging to the EDG family. In other hand, activation of receptors coupled to PTX-insensitive G proteins (Gs), such asβ-adrenergic, could not activate K(ACh) in atrial myocytes under normal conditions. It is now well established that Gβγthat is liberated from PTX-sensitive Gi specifically activates GIRK channels. To date, 5 kinds ofβ-subunits and 12 kinds ofγ-subunits have been cloned and identified, but Gβγdoes not seems to be a plausible explanation for this specificity, since GIRKs are similarly activated by many different combinations of Gβand Gγsubtypes. Our previous work has shown that Gαsubunit may play a key role in determining the specificity of receptor-mediated modulation of GIRK. In this study, using the method of co-immunoprecipitation, we investigated if there is a direct interaction between Gαsubunit and GIRK channel expressed in Xenopus oocytes. We established that endogenous Gαi/o subunits of oocyte membrane co-immunoprecipitated with GIRK channel, whileas endogenous Gαq or Gαs subunits did not. However, this specificity was altered when Gαq or Gαs subunits were over-expressed. We present evidence to suggest a key role for Gαsubunit in determining the specificity of coupling between receptors and the channel.PartⅠConstruction of FLAG tagged GIRK4 channel, its expression and functional studyAim: FLAG tag will be inserted into the upstream of GIRK4-pGEMHE DNA sequence. FLAG-GIRK4 will be expressed in the Xenopus oocytes. The function of the FLAG tagged GIRK4 channel will be studied through the two-electrode voltage clamp (TEVC) technique. Western Blot will be applied to confirm that the FLAG tagged GIRK4 has expressed on the membrane of the Xenopus oocytes.Methods: (1) Construction of recombinant plasmid DNA: The FLAG tagged GIRK4 segment about 1,300 bases was produced by PCR. Then the FLAG-GIRK4 DNA segment and the pGEMHE plasmid DNA were both digested by double restriction enzymes SmaⅠand EcoRⅠ. After the products were purified, the ligation was carried out with the molar ratio of 10 to 1 between the inserts (FLAG-GIRK4 DNA segment) and vectors (pGEMHE plasmid DNA). The transfected Top 10 competent cells were spread along the LB agar plate with anti-Amp+ property and then the plasmid DNA of the clones were extrated following assessed by Sma Ⅰand EcoRⅠdouble restriction enzyme digestion. The correct clones were chosen and sent for sequencing. Finally we identified a clone with a completely correct sequence.(2) Recombinant plasmid DNA expression in the Xenopus oocytes: cRNA of FLAG-GIRK4, GIRK4 and M2 receptor were produced by in vitro transcription using RibomaxTM Large Scale RNA Production Systems-T7 Kit and were injected in the range of 1-2ng/oocyte depending on the functional expression level. After injection, oocytes were incubated in ND96 supplemented with 2.5mM sodium pyruvate at 19-20℃. Two-microelectrode voltage clamp (TEVC) was used to detect the channel expression. Acetylcholine (ACh) was added to the perfusate to activate GIRK4 further on top of the basal currents of expressed channels.(3) Verification of the FLAG tagged GIRK4 channel protein:The membrane proteins from Xenopus oocytes expressing recombinant FLAG-GIRK4 channel were extracted and loaded for SDS-PAGE, western blot using anti-FLAG antibody was applied to identify the fusion of the FLAG tag and GIRK4 channel.Results: (1) The construction of recombinant plasmid DNA was successful. Confirmed by sequencing, the FLAG tag had been inserted into the upstream of the DNA base sequence of GIRK4-pGEMHE. So we successfully constructed the recombinant plasmid DNA of FLAG-GIRK4-pGEMHE. Sequence alignment in NCBI gene bank using Blast program shows that no base mutation was found and the recombinant plasmid DNA sequence was identical with the template sequence.(2) The recombinant FLAG-GIRK4 was successfully expressed in the Xenopus oocytes. Channel currents were recorded by TEVC. The level of its expression was the same with that of GIRK4. The increased activation of currents of FLAG-GIRK4 and GIRK4 channles by ACh was similar, and no significant difference was found.(3) Western Blot experiments showed that the FLAG tag was successfully fused with GIRK4 channel protein and they were expressed in the oocytes.Conclusions: The recombinant plasmid of FLAG-GIRK4-pGEMHE was successfully constructed by means of PCR technique. Moreover, the FLAG tagged protein had stably expressed and GIRK4 channel function was not affected. These results laid a solid basis for future study.PartⅡThe study of interaction between Gαsubunits and GIRKAim: (1) To observe quantitively endogenously expressed Gαsubunits and their interaction with Gβγor GIRK in Xenopus oocytes. (2) To study the effects of over-expressing Gαsubunits on interaction between Gαsubunits and Gβγor GIRK.Methods:(1) For Xenopus oocytes studies, cRNA transcription in vitro was made from linearized cDNA of FLAG-GIRK4, Gαq, Gαs, M1 muscarinic receptor (M1R),β2-adrenergic receptor (β2AR). The quality of cRNA was estimated using an ethidium bromide stained agarose gel and the concentration measured by UV spectrophotometry. Oocytes were injected with a 46nl solution containing cRNA for the G protein Gαq subunits, M1R and FLAG-GIRK4 channel or Gαs subunits,β2AR and FLAG-GIRK4 channel. Oocytes were incubated in ND96 supplemented with 2.5mM sodium pyruvate at 19-20℃. Recordings in Xenopus oocytes were performed 2-4 days following cRNA injection.(2) Western Blot: Oocytes were homogenized using a homogenizer on ice. Homogenates were centrifuged at 5000g at 4℃for 5min. Supernatants were centrifuged at 200,000g at 4℃for 1h and the pellets solubilized in homogenate buffer. SDS-polyacrylamide gel electrophoresis and transfer of membrane proteins from oocytes endogenous or overexpressing Gαsubunits to polyvinylidene difluoride membrane was performed using a LiuYi electrophoresis (Beijing) system according to the manufacturer's instructions. The homogenate was lysed with an equal volume of 2×gel loading buffer and probed with a primary polyclonal rabbit antibody to Gαi/o/t/z, Gαq/11, Gαs/olf (Santa Cruz) or GAPDH (Chemicon) and the appropriate secondary antibody before visualization of bands using the DAB Kit.(3) Co-immunoprecipitation: Anti-Gαi/o/t/z (Santa Cruz), anti-Gαq/11 (Santa Cruz), anti-Gαs/olf (Santa Cruz), or anti-FLAG (Sigma) 2-4μg was added to each respective preparation, and incubated overnight at 4℃. 20μl Protein A sepharose beads (Santa Cruz) was added and incubated for another 2-5h. The lysate was centrifuged at 1,000g for 3min at 4℃. The beads was rinsed 3-5 times with 500μl homogenate buffer; proteins were eluted with 20μl loading buffer containedβ-mercaptoethanol and then heat denatured at 99℃for 5 min before loading into 10% polyacrylamide gel. Proteins were transferred to polyvinylidene difluoride membranes (Millipore), blocked with 3% BSA for 1h, and probed with rabbit anti-Gβ(Santa Cruz), anti-Gαi/o/t/z (Santa Cruz), anti-Gαq/11 (Santa Cruz), anti-Gαs/olf (Santa Cruz), or mouse anti-FLAG (Sigma) overnight at 4℃. Secondary antibodies goat anti-rabbit or goat anti-mouse (Santa Cruz), respectively, were applied for 1h at 37℃, and the membrane was developed with DAB Kit.Results: (1) Endogenous Gαq or Gαs subunits of oocyte had a higher expression level than that of Gαi subunits.One of the possible reseason for selective activation of GIRK by PTX-sensitive Gi/o coupled receptors over PTX-insensitive G protein coupled receptors is that the amount of Gβγdimers released from Gq or Gs proteins activation are far less than that released from Gi/o activation. Thus we first compared the quantity of endogenous Gαsubunits of Gi/o, Gq and Gs by using western blot. GAPDH was used as an internal control to normalize the measurement of Gαsubunits. Anti-Gαi/o/t/z, anti-Gαq/11, anti-Gαs/olf and anti-GAPDH were applied to determine the amount of each one of these Gαsubunits. The results showed that endogenous Gαq or Gαs subunits of oocyte had a higher expression level than that of Gαi subunits.(2) Endogenous Gαq or Gαs subunits from oocytes bound more Gβγsubunits than Gαi subunits did.The concentration of the membrane proteins was measured by BCA protein assay. 4μg of either anti-Gαi/o/t/z, or anti-Gαq/11, or anti-Gαs/olf antibody was membrane samples that contained same amount of proteins for co-immunoprecipitation. The amount of Gβγthat these Gαsubunits bound was analyzed using immunobloting using anti-Gβantibody. The relative intensities of the bands were analyzed by light spectrophotometry through Bio1D software. The results indicated that endogenous Gαq or Gαs subunits from oocytes bound more Gβγsubunits than Gαi subunits did.(3) Gαi/o, but not Gαq or Gαs subunits of Xenopus oocytes co-immunoprecipitated with GIRK4 channel.Co-immunoprecipitation was used to study the coupling between Gαsubunits and GIRK4 channel. Anti-Gαi/o/t/z, anti-Gαq/11 or anti-Gαs/olf antibody was added to co-immunoprecipitate the Gαsubunits and the channel, and the GIRK4 was recognized by anti-FLAG antibody. The results indicated Gαi/o, but not Gαq or Gαs subunits of Xenopus oocytes co-immunoprecipitated with GIRK4 channel. A reversed experiment, using anti-FLAG antibody to immunoprecipitate and anti-Gαi/o/t/z antibody for bloting, gave a same result.(4) Overexpression of Gαq or Gαs subunits confered coupling of Gαq or Gαs with GIRK4 channel.We first used anti-Gαq/11 or anti-Gαs/olf antibody to immunoprecipitate, and anti-FLAG antibody for immunobloting; and then, we used anti-FLAG antibody to immunoprecipitate and anti-Gαq/11 or anti-Gαs/olf antibody for immunobloting. Both of these experiments displayed that Gαq or Gαs subunits could couple with GIRK4 channel when over-expressed.We also compared the amount of Gβγsubunits that Gαq or Gαs bound either in the absence or presence of Gαq or Gαs overexpression. The results showed that overexprssion of Gαsubunits bound more Gβγthan endogenous Gαsubunits did. Conclusions: Endogenous Gαq and Gαs subunits of oocyte had a higher expression level than that of Gαi subunits. Endogenous Gαi/o, but not Gαq or Gαs subunits of Xenopus oocytes co-immunoprecipitated with GIRK4 channel. Overexpression of Gαq or Gαs subunits confered coupling of Gαq or Gαs with GIRK4 channel, and bound more Gβγthan endogenous Gαsubunits did.SUMMARY1 A recombinant plasmid DNA, FLAG-GIRK4-pGEMHE, was successfully constructed by means of PCR technique. The FLAG tagged GIRK4 expressed well in Xenopus oocytes and GIRK4 channel function was not affected. These results laid a solid basis for future study.2 Endogenous Gαq or Gαs subunits of Xenopus oocytes had a higher expression level than that of Gαi subunits.3 Endogenous Gαq or Gαs subunits from oocytes bound more Gβγsubunits than Gαi did.4 Endogenous Gαi/o, but not Gαq or Gαs subunits of Xenopus oocytes co-immunoprecipitated with GIRK4 channel.5 Overexpression of Gαq or Gαs subunits confered coupling of Gαq or Gαs with GIRK4 channel.6 Overexprssion of Gαq or Gαs subunits bound more Gβγ than endogenous Gαsubunits did.7 The mechanism underlying the altered specificity of G protein coupled receptor-mediated activation of GIRK in the condition of Gαq or Gαs overexpression is probably due to an increased unspecific coupling of these Gαsubunits with GIRK channel.