【作者】 赵志英；

【导师】 张海林；

【作者基本信息】 河北医科大学 ， 药理学， 2005， 硕士

【摘要】 内向整流性钾离子通道(Inwardly Rectifying K Channel, Kir)是在各种组织中广泛分布的一种钾离子通道,因其电压电流关系中所具有的内向整流特性而得名,即钾离子通过Kir 由细胞外向细胞内流的过程要比由细胞内向细胞外流的过程容易的多~[1]。Kir2.0 钾通道的特征就是具有很强的内向整流特性,这种通道的整流作用有利于维持细胞的静息电位使之接近钾离子的平衡电位并参与复极化过程~[2]。到目前为止发现Kir2.0 家族钾通道在心脏、神经、骨骼肌、肾脏和肺等组织中均有表达~[3],Kir2.3 是Kir2.0 家族中的重要一员,它具有内向整流性钾离子通道的普遍特征:(1)钾离子向内流较向外流容易,在正常生理状态下其反转电位在钾离子的平衡电位-90mV 左右。(2)内向整流性钾离子通道的功能均依赖于一种膜磷脂—PIP2~[4]。(3)内向整流性钾离子通道可以被多种因素调节,各种因素对不同的Kir 通道调节情况不同。(4)Kir 具有共有的基本结构,即:N 末端起始于细胞内,经两次跨膜折叠后,C 末端结束于细胞内~[5, 6]。现已知蛋白激酶C(PKC)的激活可以对Kir 通道的功能(如Kir2.3、Kir3.x 和Kir6.x)产生抑制作用~[7]。PKC 是依赖钙、磷脂(phospholipid, PL)和二酰基甘油(diacylglycerol, DAG)激活的丝氨酸/苏氨酸蛋白激酶的多基因超家族,为信号转导中的关键部分,PKC 通过催化多种蛋白质上Ser/Thr更多还原

【Abstract】 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更多还原