摘要
目的研究致遗传性远端肾小管酸中毒(dRTA)的囊泡型H^+-ATPaseB1亚基(ATP6V181)的点突变对大鼠内髓集合管(IMCD)细胞H^+-ATPase结构和泵氢功能的影响。方法模拟致人类遗传性dRTA的B1亚基点突变构建野生型(WT)和7种突变型(M)质粒,转染大鼠IMCD细胞并筛选稳定表达绿色荧光蛋白(CFP)-B1 M和GFP—B1 WT的IMCD细胞系。应用免疫荧光、免疫蛋白印迹法、ATP。NADH耦合实验和快速酸负荷后不依赖钠的细胞内pH的变化,来观察GFP—B1 M和GFP—B1 WT在细胞内的分布,及其与H^+-ATPase其他(E、H和c)亚基结合能力对ATP酶活性和H^+-ATPase泵氢功能的影响。结果GFP.B1wT在转染细胞中呈囊泡样分布,与H^+-ATPase的分布一致;而GFP—B1 M则为弥散分布。免疫沉淀结果显示只有GFP—B1 WT融合蛋白能和其他的H^+-ATPase亚基(E、H和c)结合形成复合物,而GFP-B1 M融合蛋白无此作用。ATP酶活性只有在GFP—B1 WT转染细胞株的免疫沉淀产物中存在,在GFP—B1 M转染细胞株的免疫沉淀产物中不存在。在GFP—B1 M转染的IMCD细胞快速酸负荷后H^+-ATPase介导的钠不依赖pHi的恢复受到显著抑制[pHi的恢复率(pHU/min)在L81P、R124W、M174R、P275R、G316E、P346R、G364S GFP—B1M转染的IMCD细胞分别为0.007±0.002、0.004±0.002、0.002±0.002、0.003±0.002、0.006±0.004、0.009±O.004、0.015±0.006,P〈0.05,n=51。而GFP—B1 WT转染的IMCD细胞pHi的恢复率与未转染IMCD细胞相似[(0.040±0.006)pH U/min],且能被1μmol/L巴弗洛霉素(H^+-ATPase特异性抑制剂)所抑制。结论遗传性dRTA囊泡型H^+-ATPaseB1亚基点突变影响GFP—B1融合蛋白与其他亚基正常结合组装形成完整的H^+-ATPase,并抑制H^+-ATPase的泌酸功能。
Objective To test the hypothesis that point mutations in the B1 subunit of vacuolar H +-ATPase, which cause inherited type 1 (or distal) renal tubular acidosis (dRTA), interfere with assembly and proton secretion function of the H^+-ATPase. Methods Eight constructs that mimic seven known point mutations in inherited dRTA (M) or wild type (WT) B1 were transfected into a rat inner medullary collecting duct (IMCD) cell line to express GFP-B1 WT or GFP-B1 M fusion proteins. Distributions of GFP-B1 WT or M, and its assembly ability with other subunits (E, H and c), as well as its effect on the ATP hydrolysis activity and proton secretion function of H^+-ATPase were tested by immunofluorescent methods, immunoprecipitates, ATP/NADH coupled assay or Na^+-independent pHi recovery following acute acid load respectively. Results Immunofluorescence revealed that GFP-B1 WT displayed the same typical vesicular distribution pattern as H^+-ATPase, but all GFP-B1 M exhibited more diffused cytoplasm pattern. In co-immunoprecipitation studies, GFP-B1 WT formed complexes with other H^-ATPase subunits (c, H and E) whereas GFP-B1 M did not. Proteins immunoprecipitated with anti-GFP antibody from GFP-B1 WT cells had ATPase activity whereas proteins from GFP-B1 M cells did not. Proton pump-mediated pHi transport was significantly inhibited in GFP-B1 M transfected cells [pHi recovery rate (pH U/min) was 0.007±0.002, 0.004±0.002, 0.002±0.002, 0.003±0.002, 0.006± 0.004, 0.009±0.004, 0.015±0.006 in L81P, R124W, M174R, P275R, G316E, P346R, G364S B1 M-transfected IMCD cells, P〈0.05, n=5]. However, pHi recovery rates in both GFP-B1 WT ceils and the untransfected IMCD cells were similar[(0.040±0.006) pH U/mini, and were abolished by 1 μmol/L bafilomycin (specific H^+-ATPase inhibitor). Conclusion B1 point mutations that produce dRTA prevent normal assembly of the H^+-ATPase B1 and affect the proton secretion of H^+- ATPase in IMCD cells.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2009年第1期24-30,共7页
Chinese Journal of Nephrology
基金
广东省自然科学基金(7001636,20001380)
留学归国人员基金