Activation Of ε Protein Kinase C Isoform By Phorbol Ester Is Involved In Downregulation Of The Kir2.3 Currents

Inwardly rectifier K+ channels (Kir) play an important role in controlling membrane excitability. As a member of Kir, Kir2.3 is expressed in several tissues including the central nervous, heart, and kidney. Kir2.3 is known to be modulated by several intra-and extra-cellar signal molecules including Mg²⁺, polyamines, protons and protein kinase C (PKC). The modulation of channel activity by PKC is remarkable because the signal transduction pathway of PKC is so common that a large number of extracellular messengers can act on this K⁺ channel through the activation of PKC. To dissect the role of α,βandεprotein kinase c isozymes in the regulation of the currents of Kir2.3, which was expressed in Xenopus oocytes, peptide PKC isomyze-specific inhibitors were used. Whole-cell Kir2.3 was recorded using two-electrode voltage clamp technique (TEVC). We demanstrate the partial reversal of PMA induced inhibition of Kir2.3 currents by the hightly selected inhibitorεPKC peptide inhibitor, but not byαPKC peptide inhibitor and βPKC peptide inhibitor. Part 1 The expression of Kir2.3 channel in Xenopus oocyte and the properities of Kir2.3 channel Aim: To express Kir2.3 channel in the Xenopus oocytes and record the currents of the Kir channels through two-electrodes voltage clamp technique. Methods: â‘ Transcription of Kir2.3 channel in vitro: Kir2.3 cDNA construct was subcloned into the pGEMHE plasmid vector and the sequence was confirmed by sequencing. cRNA was transcribed using Ribomax^TM Large scale RNA production T7 Kit after linearizing the DNA construct with appropritate restriction endonuclease. â‘¡Preparation of oocytes: Xenopus oocytes were surgically extracted, dissociated, defolliculated by collagenase (2mg/ml) for 1.5～2 hours, then washed with ND96 solution. â‘¢Microinjection of oocytes: Each oocyte was injected with 50nl of water containing the desired cRNA. cRNA of the Kir2.3 channel was injected in the range of 0.5～1ng/oocyte. â‘£Currents recording: Whole cell currents was recorded under two-microelectrodes voltage clamp using Gene clamp 500B amplifier. Results: â‘ Analysis of linearized plasmid DNA by argarose electrophoresis; â‘¡The analysis of in vitro transcribed cRNA; â‘¢Electrophysiological experiment: Kir2.3 channel currents can be recorded using two-electrodes voltage clamp method at 2～4 days after mocroinjection of oocytes. With a voltage protocol that changed from –80mV to +80mV gradually, The current-voltage relationship (I-V) of Kir2.3 channel currents showed signicant inward rectifier property (the potassium ion can flow into the cell more easily than flow outthrough the channel). At the high K⁺ solution, the reversal potential of Kir currents was around 0mV. The reversal potential of Kir2.3 channel currents is consistent with the potassium equilibrium potential according the Nest equation. Ba²⁺ blocked the currents effectively. Conclusion: Kir2.3 channel can be expressed by microinjiecting the in vitro transcribed RNA into Xenopus oocytes. We can observe the characteristics of Kir2.3 channel currents by two electrodes voltage clamp 2～3 days after the cRNA microinjection. â‘ Kir2.3 channel currents showed a characteristic of inwardly rectifying. â‘¡The reveral potenrial of the Kir2.3 channel currents is equivalent to the potassium equilibrium potential. â‘¢Ba2+ can block Kir2.3 currents effectively. Part 2 Activation of protein kinase C by phorbol ester induces downregulation of the cloned Kir2.3 Aim: To investigate whether PKC is involved in the PMA inhibition of Kir2.3 currents. Methods: Kir2.3 channel was expressed in the Xenopus oocytes. Two-electrodes voltage clamp was used to record the Kir2.3 currents in Xenopus oocytes perfused in PMA-ND96K or in PMA-BISI-ND96K solutions. Results: 0.5μM PMA significantly inhibited Kir2.3 currents in Xenopus oocytes. After 30 min treatment of 0.5μM PMA, the Kir2.3 currents at –80mV in high K⁺ was inhibited by 52.36±9.09% (n=7). The PKC blocker,10μM BISI almostabolished the PMA effect; the inhibition is only 0.14 ±0.05%(n=5). This result suggested that PMA inhibited the Kir2.3 function through activating PKC. Conclusion: PMA, through actvation of PKC, inhibited the Kir2.3 currents expressed in Xenopus oocytes. Part 3 Activation ofεprotein kinase c isoforms is involved in phorbol ester-induced downregulation of the Kir2.3 Aim: To dissect the role of α,βandεprotein kinase C isozymes in the regulation of the Kir2.3 currents expressed in Xenopus oocytes. Methods: â‘´Kir2.3 channel was expressed in the Xenopus oocytes. ⑵Xenopus oocytes were divided into four groups, which were respectively injected with: â‘ H₂O; ②αPKC peptide inhibitor; ③β-PKC peptide inhibitor; ④εPKC peptide inhibitor. 10～15min later, two-electrodes voltage clamp was used to record the Kir2.3 currents in Xenopus oocytes perfused with PMA-ND96K+ solution. ⑶The maturation experiment of Xenopus oocytes: Xenopus oocytes were divided into four groups , which were respectively incubated with: â‘ PMA; â‘¡PMA and BISI; â‘¢PMA; â‘£PMA. The third group was injected with H2O and the fourth group was injected withβPKC peptide inhibitor, 10～15 min before incubation. Results: PMA significantly inhibited Kir2.3 currents in Xenopus oocyte groups which had been injected with H₂O, αPKC peptide inhibitor and βPKC peptide inhibitor. The Kir2.3 currents from the three groups mentioned above were inhibited...