G蛋白门控内向整流钾离子通道4基因突变与原发性醛固酮增多症相关性的研究

G-protein coupled potassium(K+) channel 4 (GIRK4) gene mutations are closely associated with primary aldosteronism. Institute of Hypertension Research, People's Hospital of Xinjiang Uygur Autonomous Region found 100% new mutations of X2 loci of GIRK4 gene in 46 cases with adrenal adenoma, and introduced the mutated genes into 293-T cells by stable transfection. Patch clamp detection was used to detect K+, Na+ and Ca2+ ion characteristics of GIRK4 to comprehend effects of X2 mutant loci on GIRK4 ion channel. In order to explain pathogenesis of GIRK4 in primary aldosteronism, we should understand the general electrical properties and dynamic characteristics of ion channel; understand the function of ion channel regulation using potassium channel blockers BaCl2, and tertiapin-Q (TPN-Q); determine calmodulin level by RT-PCR; detect intracellular Ca2+ concentration bycalcium determination and imaging technology; and evaluate CYP11B2 gene using RT-PCR to understand relationship between the second messenger and the cell proliferation. And in order to reveal pathogenesis of GIRK4 in primary aldosteronism, we should understand changes of electrophysiological characteristics caused by X2 mutation.

G蛋白偶联的内向整流型钾离子通道4 （GIRK4）基因突变与原发性醛固酮增多症密切相关。新疆高血压研究所发现GIRK4基因新的突变位点X2在46例肾上腺腺瘤中100%突变，制备点突变转染细胞，检测细胞的增殖效应，检测K+、Na+和Ca2+离子单通道的特性来了解X2位点突变对GIRK4通道的影响。了解其离子通道一般电学性质和动力学特性，并利用钾通道阻滞剂BaCl2、tertiapin-Q(TPN-Q)了解离子通道的调控关系；采用RT-PCR法对细胞钙调蛋白进行转录水平的测定；利用钙离子测定与成像技术对细胞内Ca2+浓度进行实时监控；采用RT-PCR法对细胞内CYP11B2基因水平进行测定，了解细胞合成分泌醛固酮的能力，与细胞增殖之间的关系，通过了解X2位点突变带来的电生理特性的改变来了解GIRK4的功能学改变，从而揭示GIRK4在原醛症中的发病机制。

KCNJ5基因编码G蛋白偶联的内向整流型钾离子通道亚型4（GIRK4），研究已证实其突变可导致醛固酮分泌瘤的发生，然而突变引起的通道功能的改变及其参与调控肾上腺球状带细胞增殖的机制尚未阐明。本课题通过制备KCNJ5 L102Q突变的293T细胞株，利用MTT法、EdU法观察细胞增殖情况，同时应用不同种类的通道阻断剂观察其对细胞增殖的影响，最后运用western blot 技术检测三种细胞中Akt、pAkt （Thr308和Ser473），其下游靶蛋白GSK-3β、pGSK-3β的表达, 从而推测突变所引起的通道功能改变。我们发现（1）经过12小时、24小时孵育后KCNJ5突变型细胞的增殖率明显高于野生型和空白质粒型细胞，进一步EDU实验也证实转染KCNJ5突变可使细胞增殖率显著增加；（2）细胞外利用不同浓度的氯化钡、布比卡因和维拉帕米等离子通道阻断剂，进行24小时孵育后，布比卡因可显著抑制KCNJ5突变型细胞的增殖，这可能与突变引起的GIRK4通道离子选择性缺失，允许Na+通过的通道改变有关。（3）与在野生型和空载质粒细胞相比，pAkt （Thr308和Ser473）、pGSK-3β等蛋白在KCNJ5 突变型细胞中的表达未见明显差异，提示该突变独立于PI3K/Akt通路的磷酸化参与醛固酮瘤的发病。本课题从增殖的角度进一步验证该突变的功能学意义，推测突变所引起的通道功能改变，并为今后的药物治疗提供理论基础。