| Inwardly Rectifying K Channel (Kir) is a group of potassium channels extensively distributed in many kinds of tissues. It is known for its inwardly rectifying property. This property provides easier access for K+ influx from exterior of the cell than its efflux from the interior of the cell. Kir2.0 is characteristic for its strong inward rectifying property. This property can help to maintain the membrane resting potential of the cell by regulating it back to the K+ equilibrium potential. Kir 2.0 is also involved in the process of repolarization. Till now, the expression of Kir2.0 family group has been found in the heart, nerve, kidney, lung and many other types of organs and tissues. Kir2.3 is an important member of Kir2.0 family group. It shares the common characters of inward rectifying potassium channel: (1) K+ influx is easier than its efflux. Under normal physiological conditions, its reverse potential is around –90mV. (2) The channel function depends on a kind of membrane phospholipid—PIP2. (3) the channel is under regulation of many types of factors. (4) Kir channels all share the same basic structure. The N-terminus starts from the interior of the cell, after twice transmembrane folding, the C-terminus also ends in the interior of the cell. At present, it is known that PKC activation can lead to inhibition of Kir channel function such as Kir2.3,Kir3.x and Kir6.x. PKC is a Ser/Thr protein kinase multiple gene superfamily whose activation depends on calcium, phospholipid and diacylglycerol. It is a pivotal part of the signal transduction pathway. PKC regulates the cell metabolism, growth, proliferation and differentiation by phosphorylation of Ser/Thr in many kinds of proteins. PMA is an activator of PKC, or in another sense, PKC is an intracellular receptor of PMA. Generally, it is believed that PMA can lead to a translocation of PKC from the cytosol to the membrane, which temporarily affects PKC's activity both in the cytosol and on the membrane respectively. PMA has an inhibitory effect on Kir channels through PKC activation. However, the effects of PMA on different Kir channels or the same channel expressed in different cells are hardly the same. PMA can inhibit Kir2.3 channel, and this inhibitory effect can be blocked by applying staurosporine, which is a PKC inhibitor. Other types of PKC activators can also inhibit channel function. When the inactive form of PMA, such as PDBu, is applied, no inhibitory effect on channel function is observed. This shows that PMA affects the channel by PKC activation. However, Kir2.1 is not sensitive to PMA. Kir2.3 channel is very sensitive to the inhibitory effect by PMA when it is expressed in the Xenopus laevis oocytes. But no inhibition appears when Kir2.3 channel is expressed in CHO cells. Although there have been many researches on theinhibitory effect of PMA on Kir2.3 by PKC activation, the mechanism is full of suspicion. It has been shown by some experiments that PKC modulates the function of Kir including Kir2.3 by phosphorylating the channel protein directly. When the phosphoryation sites of Kir were mutated, the inhibitory effect disappeared. However, till now, there has no direct evidance of channel phosphorylation. The ultimate aim of this study is to look for the direct evidance of Kir 2.3 phosphorylation by PKC. We expressed Kir2.3 in the Xenopus laevie oocytes and tried to purify it for the study of direct channel phosphorylation by PKC. Aim: Flag tag will be inserted into the upstream of Kir2.3-pGEMHE DNA sequence. Flag-Kir2.3 will be expressed in the Xenopus laevie oocytes. Consequence of Flag tagging on the Kir2.3 channel protein function will be studied. Immunocytochemistry method will be applied to confirm that the Flag tagged Kir2.3 has expressed on the membrane of the Xenopus oocyte. Methods: (1) Construction of recombinant plasmid DNA: The DNA base sequence corresponding to the amino acid sequence of Flag peptide was inserted into the upstream of the DNA base sequence of Kir2.3 by PCR. Enzyme digestion site by EcoRI and protective bases were als...
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