Expression Of Protein Kinase C Isforms In Chinese Hamster Ovary Cell And Xenopus Oocyte

Inwardly rectifying K channels (Kir channels) are expressed in many tissues. The physiological function of the Kir channels includes maintaining resting potential and mediating K⁺ transmember transport. Since 1996, Henry[1] and his colleagues reported that protein kinase C (PKC) inhibits inward rectifier potassium channel (Kir) currents in Xenopus oocyte, study of mechanisms of PKC-dependent inhibition of Kir currents has became one of hot research field. In our laboratory, previous studies have shown that PMA inhibited Kir2.3 currents through a PKC-mediated mechanism in Xenopus oocyte whereas PMA did not inhibit Kir2.3 currents expressed in CHO cell. However, we did not know what attributed to the difference. One of the possible mechanisms underlying PKC-dependent inhibition of Kir currents may due to differences of PKC isforms expressed in CHO cell and Xenopus oocyte. PKC is a key factor in cell signal transduction, and is a down stream product of PIP₂ hydrolysis. PIP2 is the substrate for PLC, which hydrolyzes PIP₂ into inositol triphosphate (IP₃) and diacylglycerol (DAG), two ubiquitous signaling molecules. They regulate diverse cellular physiological functions through increasing intracellular Ca²⁺ concentration and activating PKC, respectively. Now, there are at least 12 isoforms of PKC that have been detected. Each of PKC isoforms has its own characteristics of biology and enzymology. They are expressed in different tissues and possess different functions in cell signal transduction. In recent years, studying of PKC as a whole gradually was replaced by the investigation of each of the PKC isoforms. In the current proposal, we seek to study the expression of PKC isforms in CHO cell and Xenopus oocyte, aim to identify mechanisms of PKC-dependent inhibition of Kir currents including Kir2.3. Aim: To study expression of PKC isforms in CHO cell and Xenopus oocyte. Method: 1 CHO cell culture and Preparation of Xenopus oocytes. 2 Primer design: The PKC isoform-specific primer pairs were derived from GeneBank sequence information based on similar species sequence such as Rattus norvegicus, Rat, mouse, Mus musculus, M.musculus, H.sapiens musculus, rabbit, human, Homo sapiens, Bos taurus. Using EMBL Data Library clustalw tool, the highest homologues sequences were screened among the different species for a particular PKC isoform, and were used to design the primers. The primers were designed by using Generunner program. 3 PKC isoform was profiled by the RT-PCR technique: Total RNA was extracted from CHO cell; cDNA was subsequently made from the mRNA, using AMV Reverse Transcriptase according to the manufacturer's instruction. PCR Products were sent for DNA sequencing. 4PCR products were ligated to the pGEM-T plasmid vector. Transformants were taken from E.coli DH5α. Blue/white screening for recombinants in indicator plate. The plasmid constructs were digested with restriction enzyme and were analyzed with gel electrophoresis. The products of restriction digestion with the sizes consistent with the predicated values were sent for DNA sequencing. 5 Western blot was used to analyze the PKC isoforms expressed in CHO cell and Xenopus oocyte. Result: The presence of mRNA for the PKC isoforms expressed in CHO cell was determined using 12 designed primer pairs by RT-PCR. The result revealed amplified cDNA fragments that corresponded to the predicted sizes of PKC α, δ, λ, μ, ι. PCR Products of PKC α, δ, λ, ιwere confirmed by DNA sequencing. The PCR product of PKC μwas ligated to the pGEM-T plasmid vector and was transformed to E.coli DH5α. After screened with blue/white and analyzed with restriction enzyme, the plasmids with a insert consistent with the predicated values was confirmed to be PKC μby DNA sequencing. PKC isoforms were not detected in Xenopus oocyte by using the primers we designed for CHO cells. In GeneBank, no cDNA sequences of Xenopus PKC isoforms could be found. So specific primers designed based on the species other than amphibians nay not suit for Xenopus oocyte. 2 Using Western blot analysis, the PKC isoforms of PKC α, β1, δ, ζwere found in CHO cell whereas PKC α, β1, β2, ζ, δ, γ, εwere found in...