Altered Expression Of G Protein Coupled Inwardly Rectifer Potassium Channels Subunit 1 (GIRK1) And Subunit 2 (GIRK2) In Hippocampus Of Tremor Rat And SER

IntroductionThere is about 30 - 40 percentage of epilepsy etiological factors that are associated with genetic factors. The human genome project (HGP) is being completed and research methods are being improved gradually, the study about the relationship of heritage epilepsy and genovariation increase gradually. The study has revealed that the mutations of 18 different gene locus are related to epilepsy genesis.GIRK channel ( G protein coupled inwardly rectifier potassium channels) as a member of IRK (inwardly rectifier potassium channels) plays an important role in the regulation of nervous excitation. GIRK channel is composed of five subtypes, GIRK1 ~ GIRK5 orderly (Kir3.1 - Kir3.3). There are evidences that GIRK channel is constructed by homotetramer or hereotetramer, GIRK1 -GIRK4 subtype express in different locus of central nervous system (CNS) of animals. GIRK1/2 distribute mainly in CNS and it plays an important role in surstaining resting potential of postsynaptic neuron and nervous excitation . The investigation has confirmed that the point mutation of GIRK2 protein may relate with spontaneous seizure. Some one found Weavor rat's point mutation of GIRK2 may construct unselective ion channel, the mutated channel may influence resting potential of membrane to change the cells electrophysiology activity. Otherwise, Sigmorini and his assistants cultivated the mutated rat that is lack of the GIRK protein in 1997. Similar with Weavor rat is that they are more sensitive to spontaneous or drug induced seizure, they display that the GIRK1 and GIRK2 decreased significantly. These facts confirm that the deficiency of GIRK2 proteinmay cause spontaneous seizure. All the evidences demonstrate that the GIRK protein variations have close relationship with the disfunction of GIRK channel and seizure.This experiment applied RT - PCR method to test the expression of GIRK1 and GIRK2 in Tremor rat and SER hippocampus on the level of gene. By analyzing the relationship of the change of GIRK1 and GIRK2 mRNA expression and seizure , we approach its role in the genesis and development of epilepsy.Material and Method1. investigated obiect;Normal adult Wistar rat, tremor rat and SER five cases separately , pento-barbite sodium anesthesia rat, decapitation and taking brain, isolating hippocampus. Take the tissue of hippocampus to store in the - 70°C refrigerator. Wistar rat hippocampus tissue is the control group.2. ReagentsTrizol and reverse transcription kit were purchased from TaKaRa Inc. The primers of GIRK1 GIRK2 and (3 - actin were synthesized by Beijing AoKe biological Inc.3. Laboratory apparatus:PCR amplification:PTC - 100?(America) Hypothermia desk centrifuge;TC -480 type(Germany) Gel -image automatic analysis system: ID KODAK (Japan) DYY-31A electrophoresis bath (Beijing)4. Mehod;Semiquantitative Reverse transcription polymerase chain reaction ( RT -PCR)Statistical analysis was performed using SPSS for windows 10. 0 software. The data of the experiment was expressed as means. And comparisons of means were carried out by t test.Result1. Expression level of GIRK1 and GIRK2 in normal hippocampus tissue and the tremor rat hippocampus tissue were as follow;174. 24 ±15. 84,175.13 ±24. 80,183. 60 ±9. 84,189. 93 ± 13. 38O There is not significant difference between these two group (P<0.05 n=5).The expression level of GIRK1 and GIRK2 in SER hippocampus tissue was;239. 98 ±41. 14 , 94.28 ±56. 13 . The GIRK1 mRNA expression level was significantly higher than that in normal hippocampus tissue. But the GIRK2 mRNA expression level was significantly decreased than that in normal hippocampus tissue (P<0.05 n=5).DiscussionBecause of the increase of the potassium conductance, kalium ion influxes to the cell more easily than outfluxes from the cell, that results in intracellular kalium concentration increasing, cell membrane hyperpolarization, accordingly decreasing the amplitude and frequency of the action potential, sustaining the rest potential to regulate the neural excitation. GIRK agonist can reversibly degrade the glutamate which induced excitatory postsynaptic potential (EPSP) to 1/3 , its antagonist can decrease potassium conductance to increase EPSP, suggests that GIRK plays important role in excitation. SER hippocampus GIRK2 mRNA down regulation may contribute to neural excitation. Weaver mouse GIRK2 gene point mutation (G953 -A) changes amino acid sequence (glyl56 - ser) , as well as potassium channel highly conservative " fingerprint sequence" GYG to SYG, losses the selectivity of monovalent cation, generates spontaneous seizure, cerebellum and dopamine neuron death;gene knock - out GIRK - / - mouse nervous system development well, but from CA1 pass by CA3 to gate region, cell spontaneous discharge increase. Consequently, the reduction of GIRK2 will increase the neuron excitation, which will cause overexci-tation diffusing in the hippocampus .We apply RT - PGR method to find that GIRKl and GIRK2 mRNA express abundantly in rat hippocampus. Functional GIRK is composed of four subunits which can construct homotetramer or heterotetramer , the heterotetramer is constructed by two different subunit. GIRK2 and GIRK4 can be homotetramer or heterotetramer, but GIRKl must be with other subunit to construct the functional heterotetramer. The efficiency of the heterotetramer is obviously higher than homotetramer, GIRKl and other subunits coexpression can generate several order of magnitude current strength than expression alone . GIRKl ,2 distribute similarly in the whole hippocampus, the reason is that GIRK1/2 construct the main functional channel in the brain. Although GIRK4 distributes limited in the brain, but it mainly distributes in hippocampus CA3 region. The increase of GIRKl mRNA may result from GIRK1/4 expression increasing, which makes the neuron hyperpolarization , decreases the neuron excitation, inhibits generating seizure . This increase may be orgnism& ego protected mechanism.ConclusionThe low expression of GIRK2 in SER hippocampus tissue, results in high neuron excitation, and maybe the reason of seizure. GIRKl's high expression may be the adaptation reaction of the organism to neuron overexcitation.