Study On The Modulation Of Kir Channel By EGF On DRG Neurons

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...