论文标题:表皮生长因子对背根神经节神经元内向整流型钾离子通道调节的研究
Study on the Modulation of Kir Channel by EGF on DRG Neurons
论文作者 高霄飞
论文导师 路长林；张海林；何成；由振东；王雪琦,论文学位 博士,论文专业 神经生物学
论文单位 第二军医大学,点击次数 156,论文页数 100页File Size6960k
2005-05-01论文网 http://www.lw23.com/lunwen_7652077/ 表皮生长因子;内向整流型钾离子通道;背根神经节神经元;爪蟾卵母细胞;G蛋白偶联受体;酪氨酸蛋白激酶型受体
EGF; Kir channel; DRG neuron; XenoPus oocyte; GPCR; receptor tyrosine kinase
背根神经节(dorsal root ganglia,DRG)位于脊髓旁侧锥间孔内,主要由神经元/雪旺氏细胞和卫星细胞组成,传导痛觉/温觉/触觉和本体感觉。DRG神经元为假单极神经元,胞体在DRG内,轴突两个分支分别延伸到脊髓后角和周围感受器。DRG神经元的兴奋性影响着感觉信息的上传。 内向整流型钾离子通道(Inwardly rectifying potassium channel,Kirchannel)介导的电流具有内向整流特性,在偏离钾离子平衡电位相同距离的电压下,负向偏离下通道的内向电流要大于正向偏离下的外向电流,即使膜两边钾离子的浓度相等时也是如此。迄今为止,已发现了7个Kit通道亚家族成员,分别命名为Kit1.x-Kir7.x。其中,Kir2.x又称为IRK(inwardly rectifyingpotassium channel),Kir3.x又称为GIRK(G protein coupled inwardlyrectifving potassium channel)。已有文献报道Kir2.X和Kit6.x在DRG神经元上有表达,但尚不清楚DRG神经元上是否还存在其他类型Kir通道。 Kir通道具有维持静息电位、钾离子动态平衡、起博细胞活性、突触抑制以及调制神经沖动速度等多种细胞功能。其中,由于GIRK还受到G蛋白的调节,所以GIRK还参与G蛋白偶联受体(G protien coupled receptor,GPCR)对细胞兴奋性、递质分泌的调节作用。Kir通道的活性可以被许多因素调控,包括PIP_2、花生四烯酸、钠离子、镁离子、PH值,G蛋白异源三聚体(尤其是G_(βγ))以及ATP等。 表皮生长因子(epidermal growth factor,EGF)是具有53个氨基酸残基的单链多肽,分子量6kD。表皮生长因子受体(epidermal growth factorreceptor,EGFR)属酪氨酸蛋白激酶型受体,是一个170kD的膜蛋白,由130kD的单多肽链和N端相连的多糖组成,包括细胞外配体结合区、单跨膜区、细胞内
DRGs (dorsal root ganglias) are located at or beside Foramen in tervertebrale, and composed of neurons, Schwann cells and satellitic cells etc. DRGs transmit sense of pain, heat, touch, ect. DRG neuron is pseudounipolar neuron. Its cell body settled in DRG, but two branches of axon are extended to Cornu dorsale or periphery sensors each. Excitability of DRG neurons influences the sensor messages upward transduction.Kir channels (Inwardly rectifying potassium channels) can conduct much larger inward currents at membrane voltages negative to the K+ equilibrium potential than outward currents at voltages positive to it, even when K+ concentrations on both sides of the membrane are made equal. So far, seven members of Kir channel subfamilies have been defined, named Kirl. x-Kir7. x. Kir2. x is also called IRK (inwardly rectifying potassium channel), and Kir3.x GIRK (G protein coupled inwardly rectifying potassium channel)0 It has been reported that Kir2 and Kir6 exist on DRG neurons. It is not clear whether the other members of Kir channels are expressed in DRG neurons.Inward-rectifier K+ channels are a group of membrane proteins that enact important physiological tasks such as controlling the resting membrane potential, regulating cardiac and neuronal electrical activity, coupling insulin secretion to blood glucose levels, and maintaining electrolyte balance. GIRKs play a role in controlling cell excitability and transmitter release, as coupled with GPCR (G protien coupled receptor). Kir channels are regulated by many intracellular ligands, including the G_(βγ) subunits of heterotrimeric GTP-binding proteins, the membranephospholopid phosphatidylinositol-4, 5-bisphos-phate (PIP2), ATP and protons etc.EGF (epidermal growth factor) is a single chain polypeptide composed of 53 amino acid residues, MW 6kD. EGFR (epidermal growth factor receptor) is a 170 kD membrane-spanning protein composed of a 130 kD single polypeptide chain and N-linked oligosaccharides. The EGFR, a receptor tyrosine kinase, is activated through diraerization and then auto-phosphorylated on its tyrosine residues, following EGF or TGF a binding. Through four downstream singling pathways, namely PLC-y, PI3K, MAPK and Jak-Stat pathway, EGFR are involved in gene expression, cell proliferation, cell survive, etc. Many growth factors and their receptors, eg. EGF and EGFR, are expressed in DRGs. It is not clear whether EGF contributes to neuronal Kir channel modulation.In this study, electrophysiological, molecularbiological and morphological methods were used to investigate the mechanism underlying EGF mediated modulation of Kir channel current on DRG neurons.The main results are listed as following:1. Whole-cell patch voltage clamp model used, inwardly rectifing currents are observed on hyperpolarized DRG neuron. Reversal potential of the current changed following the potassium concentration change outside of the neuron. Its regular pattern agreed with Nernst equation. Further more,300u M BaCl2 could block this current. These results suggested that the current was inwardly rectifying potassium current.2. The Kir current on DRG neurons could be inhibited by applying EGF(100ng/ml).3. GIRK1-4 cDNA were amplified by RT-PCR from DRG tissue, suggesting the transcriptions of GIRK1-4 subunits in DRG.4. GIRK1 and GIRK2 protein were detected on DRG neurons through
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