【作者】 李勃兴；

【导师】 高天明；

【作者基本信息】 第一军医大学 ， 神经生物学， 2006， 硕士

【摘要】 大电导钙激活钾通道(BK_Ca通道)作为一种由细胞去极化和细胞内钙离子浓度升高而激活的钾通道，广泛分布于神经细胞、肌细胞、腺细胞、淋巴细胞、成骨细胞等多种细胞类型。它不仅在调节动作电位的复极和发放频率、细胞膜兴奋性、平滑肌细胞收缩性、神经递质释放、学习记忆过程等中起重要作用，而且还参与了免疫调节和细胞凋亡过程，但目前对于BK_Ca通道下游的信号转导通路仍不了解。蛋白酪氨酸磷酸化是细胞重要的信号转导过程，它在细胞增殖、分化及凋亡等过程中都起到了重要的作用。细胞内蛋白酪氨酸磷酸化水平由蛋白酪氨酸激酶（PTKs）和蛋白酪氨酸磷酸酶（PTPases）二者共同调控。PTKs活性增加则细胞内蛋白酪氨酸磷酸化水平增加；PTPases活性增加则细胞内蛋白酪氨酸磷酸化水平降低。当生长因子、细胞因子、神经递质等与细胞上相应受体结合后，PTKs的活性将显著增加，导致细胞内蛋白酪氨酸磷酸化水平升高。同时也有研究表明，细胞膜去极化和钙离子内流同样能够影响细胞内PTKs的活性及蛋白酪氨酸磷酸化水平。本实验探讨了异源表达的BK_Ca通道及通道激动剂、阻断剂对细胞内蛋白酪氨酸磷酸化水平的调节，试图寻找BK_Ca通道下游可能的信号转导通路。1．BK_Ca通道的表达降低过钒酸盐处理后细胞蛋白酪氨酸磷酸化水平HEK293细胞不表达内源性BK_Ca通道，因此可作为研究BK_Ca通道异源表达的良好模型。生理状态下，HEK293细胞内PTPases的活性高于PTKs，因此细胞蛋白酪氨酸磷酸化水平相对较低。PTPases特异性抑制剂过钒酸盐能够时间依赖性地增加细胞内蛋白酪氨酸磷酸化水平。为检测BK_Ca通道对细胞内蛋白酪氨酸磷酸化水平的影响，我们比较了转染BK_Ca通道α亚基或对照质粒后HEK293细胞蛋白酪氨酸磷酸化水平。用过钒酸盐处理转染了BK_Ca通道或对照质粒的细胞后发现，NK_Ca通道的表达能够显著降低细胞的酪氨酸磷酸化水平（p＜0.05，独立样本t检验），同时，β-actin对照结果显示，BK_Ca通道的表达并不影响细胞总蛋白量。2．BK_Ca通道的表达降低过表达v-Src细胞的蛋白酪氨酸磷酸化水平为检测BK_Ca通道能否降低PTKs激活造成的蛋白酪氨酸磷酸化水平升高，我们比较了共转染BK_Ca通道和非受体型酪氨酸激酶v-Src或其无活性突变体R385A Src后细胞酪氨酸磷酸化水平的变化。v-Src是细胞内重要的非受体型酪氨酸激酶，转染细胞后可以显著提高细胞蛋白酪氨酸磷酸化水平。但共转染BK_Ca通道则使过表达v-Src细胞的蛋白酪氨酸磷酸化水平降低（p＜0.05，独立样本t检验），而共转染对照质粒则无此作用。无活性突变体R385A Src不影响细胞酪氨酸磷酸化水平，但共转染BK_Ca通道仍能使表达R385A Src细胞的蛋白酪氨酸磷酸化水平降低（p＜0.05，独立样本t检验）。3．BK_Ca通道特异性激动剂及阻断剂对细胞蛋白酪氨酸磷酸化水平的影响为确定BK_Ca通道的功能活动能否影响细胞蛋白酪氨酸磷酸化水平，我们使用BK_Ca通道特异性激动剂NS1619及特异性阻断剂paxilline，观察二者对细胞蛋白酪氨酸磷酸化水平的影响。HEK293细胞本身不表达BK_Ca通道，单独使用NS1619或paxilline对细胞蛋白酪氨酸磷酸化水平无影响（p＞0.05）。转染BK_Ca通道后，NS1619能进一步降低细胞蛋白酪氨酸磷酸化水平（p＜0.05，One-way ANOVA），而paxilline则部分逆转BK_Ca通道的作用（p＜0.05，One-way ANOVA）。海马神经元表达内源性BK_Ca通道，NS1619降低海马神经元蛋白酪氨酸磷酸化水平，paxilline则使蛋白酪氨酸磷酸化水平升高（p＜0.05，One-way ANOVA）。综合以上结果，BK_Ca通道的功能活动可以调控细胞蛋白酪氨酸磷酸化水平。更多还原

【Abstract】 Large conductance calcium-activated potassium channel (BK_Ca channel) is a special kind of potassium channel, which could be activated by cell membrane hypopolarization and the elevation of the intracellular calcium concentration. BK_Ca channels are widely expressed in many types of cells, such as neurons, muscle cells, gland cells, lymph cells, and osteoblasts. BK_Ca channels can not only regulate the repolarization and firing rate of action potential, cell membrane excitability, smooth muscle contraction, neurotransmitter release, learning and memory, but also participate in immunity regulation and cell apoptosis. But the downstream pathways of BK_Ca channels are still unknown.Protein tyrosine phosphorylation is an important process in cellular signal transduction. It plays an important role in cell proliferation, differentiation and apoptosis. The cellular protein tyrosine phosphorylation is regulated by the activities of protein tyrosine kinases （PTKs） and protein tyrosine phosphotases （PTPases）. PTKs increase the cellular protein tyrosine phosphorylation, while PTPases decrease it. When growth factors, cytokines, and neurotransmitters bind their receptors in the cell membrane, PTKs activities will increase dramatically, which will induce the elevation of the cellular protein tyrosine phosphorylation. It was also reported that hypopolarization of plasma membrane and calcium influx could influence the cellular PTKs activities and protein tyrosine phosphorylation.Our experiments discussed the effects of the expressed BK_Ca channels, the agonist and the antagonist of the channels on the cellular protein tyrosine phosphorylation and try to find the downstream signaling pathways of the channel.1. BK_Ca channel expression decreased endogenous protein tyrosine phosphorylation in pervanadate-treated HEK 293 cells. Cells were transfected with cDNA for either control vector or BK_Ca channels. 36 h after transfection, cells were treated with the membrane-permeant tyrosine phosphatase inhibitor pervanadate （250μM; 0-30 min）. Endogenous protein tyrosine phosphorylation in pervanadate-treated HEK 293 cells decreased after BK_Ca channels expression （p＜0.05, independent sample t test）.2. BK_Ca channel coexpression decreased v-Src-induced protein tyrosine phosphorylation. HEK 293 cells were cotransfected with vector together with v-Src, vector together with R385A Src, BK_Ca channels together with v-src, or BK_Ca channels together with R385A Src vectors. 36h after transfection, immunoblots were probed with anti-phosphotyrosine, v-Src expression increased the protein tyrosine phosphorylation and BK_Ca channel coexpression decreased v-Src-induced protein tyrosine phosphorylation （p＜0.05, independent sample t test）.3. BK_Ca channel agonist and antagonist affected protein tyrosine phosphorylation. HEK 293 cells were transfected with cDNA for either control vector or BK_Ca channels. We detected the effects of BK_Ca channels agonist NS1619 or antagonist paxilline on the protein phosphorylation. NS1619 and paxilline had no effects on the cellular protein tyrosine phosphorylation in the vector transfected 293 cells. But NS1619 could further decrease the protein tyrosine phosphorylation in BK_Ca channels expressing 293 cells; while paxilline could partly reverse the BK_Ca channels effects （p＜0.05, One-way ANOVA）. The cellular protein tyrosine phosphorylation of hippocampal neurons was decreased by NS 1619, but increased by paxilline （p＜0.05, One-way ANOVA）. The experiment was repeated 3 times and the results were reproducible.From the results above, we concluded that the functional activity of large conductance calcium-activated potassium channels could regulate cellular protein tyrosine phosphorylation.更多还原