先天性肌肉強直症相關之突變對於人類肌肉第一型 氯離子通道蛋白質生合成之影響

Abstract

先天性肌肉強直症是一種會影響骨骼肌舒張的神經肌肉遺傳疾病，這是由於負責轉錄骨骼肌上人類肌肉第一型氯離子通道的CLCN1基因突變造成的結果。人類肌肉第一型氯離子通道（簡稱為hClC-1），其功能主要為調節骨骼肌細胞膜的興奮性。 前人的研究指出一些與先天性肌肉強直症相關的突變會改變hClC-1通道的電位依賴性，然而除了藉由影響離子通道的門閥開關比例之外，其他由於基因突變造成hClC-1通道的缺失也可能為先天性肌肉強直症的致病機轉的成因。本篇研究想探討的主題即是這些與先天性肌肉強直症相關的突變，是否影響了hClC-1通道蛋白之生成以及於細胞中的分布型態。 首先，我們將正常型及突變型的hClC-1通道位於細胞內的N端接上可供特定抗體辨認的Flag epitope，轉殖後的HEK293T細胞利用穿膜式免疫螢光染色以及共軛焦雷射螢光顯微鏡技術分別觀察並比較其分布型態。由觀察結果發現，正常型的hClC-1通道主要分布於細胞膜上，而Y261C、R338Q以及P480T三種突變型則明顯滯留於細胞質中。在進一步的實驗中，我們直接在hClC-1上位於細胞膜外的D12-D13 linker區段上插入了小片段的Myc epitope，並且利用非穿膜式免疫螢光染色方式來加以證實hClC1位於細胞膜上的分布。另外為了釐清這些突變是否會影響hClC-1通道蛋白質的生成量或者藉由影響轉譯後修飾作用影響蛋白質成熟程度，我們也利用西方墨點法做進一步的分析比較。由實驗結果發現， Y150C, P480T 與A531V這三組突變型的蛋白質表現總量相對於正成型hClC-1有明顯偏低的現象。研究中同時發現A531V突變型hClC-1其蛋白質成熟程度比正常型hClC-1低。 本研究中證實與先天性肌肉強直症相關之突變會影響hClC-1蛋白質的表現總量與蛋白質分布型態，為先天性肌肉強直症的致病機轉提供一個新的方向。

Myotonia congenita is a neuromuscular disorder that affects skeletal muscle relaxation. The disorder is caused by mutations in the gene CLCN1 which encodes the human ClC-1 chloride channels (hClC-1) . hClC-1 channels regulate the membrane excitability of skeletal muscles. Previous studies indicate that several mutations alter the voltage dependence of hClC-1 channels. Besides changing the gating property of the Cl- channel, other defects in mutant hClC1 channels may also contribute to the mechanism of myotonia congenita. In this study, we want to investigate whether these myotonia congenita-related mutations affect the biogenesis and the subcellular localization of hClC-1 channels. By using immunofluorescence staining and confocal microscopy, we first compared the membrane localization patterns of Flag-tagged wild-type hClC-1 channels with those for mutants in HEK293T cells. While wild-type hClC-1 channels displayed surface localization, Y261C, R338Q and P480T showed a significant cytoplasmic retention pattern. To further confirm the proteins surface expression pattern, we also applied extracellular c-myc-tagging at D12-D13 linker for surface labeling under non-permeabilized configuration. Furthermore, we utilized Western blotting analysis to determine if these myotonia congenita related mutants affect the protein expression amount or the maturation level. In our results, we discovered that Y150C, P480T and A531V cause a decrease of total protein expression level. We also found out that the ratio of protein maturation level of A531V is significant lower than wild type. In this study, we show that myotonia congenita-related mutations influence the biogenesis and the subcellular localization of hClC-1 channels.