致病性 ClC-1 氯離子通道突變導致電壓依賴門控特性改變

Abstract

ClC-1是電壓依賴性氯離子通道，為homodimer的雙槍管結構，每個單體成獨立孔道，氯離子在通道中有三結合位點，分別是外側、中央、內側，並且有兩種門控機制，protopore gate主要由孔道內的glutamate gate與氯離子競爭外側結合位，而common gate則涉及蛋白質構型變化。ClC-1會在骨骼肌中大量表現，若突變會導致先天性肌強直症，患者會因肌肉膜電位無法迅速恢復，出現肌肉僵直與動作困難等症狀。 本研究旨在探討ClC-1突變型的門控變化與疾病的關聯。我們選定多個ClC-1突變位點（如A218、A313、A525、A531、A529、A535、A566），進行點突變。並利用非洲爪蟾（Xenopus laevis）卵母細胞進行異源表現模式，進行電生理(TEVC)實驗分析。我們發現突變後的ClC-1通道，其一類會嚴重影響common gate的電壓依賴性，使其開啟速率變慢，關閉速率變快 (A218T、A218V、A313T、A313V、A535V)，另一類會使通道表現量下降很多 (A529V、A531V、A566T、A566V)，兩類皆會導致先天性肌強直症發生。此外，藉由共同表現野生型與突變型通道，可以模擬異型合子患者的生理狀態，進一步分析dominant negative效應與臨床遺傳模式之關聯性。 有研究指出common gating可能是透過 E232 與中央陰離子結合位點中的高度保留的tyrosine (Y578) 之間的交互作用來關閉通道孔。因此在本研究中，我們置換細胞外溶液，改成不同濃度比例之碘離子，結果發現細胞外碘離子會影響通道的門控機制，使ClC-1傾向開啟的狀態，相較於WT，A313T、A313V通道在deactivation的過程中對點離子的親和力下降，而A566T則類似於WT，由此推論，細胞外碘離子對於WT以及突變型通道A313T、A313V有著不同的作用，可能也顯示了A313T、A313V會影響E232 與Y578的互動，進而改變ClC-1的common gating。

ClC-1 is a voltage-dependent chloride channel with a homodimeric double-barrel structure, where each monomer forms an independent pore. Within the pore, chloride ions interact with three binding sites: external, central, and internal. The channel exhibits two types of gating mechanisms: the protopore gate, primarily governed by a glutamate residue that competes with chloride ions at the external binding site, and the common gate, which involves larger conformational changes in the protein. ClC-1 is highly expressed in skeletal muscle, and mutations in this channel can lead to myotonia congenita, characterized by muscle stiffness and impaired relaxation due to delayed membrane repolarization. This study aims to investigate the gating alterations of ClC-1 mutations and their association with disease. Several ClC-1 mutants (e.g., A218, A313, A525, A529, A531, A535, A566) were generated via site-directed mutagenesis and expressed in Xenopus laevis oocytes for two-electrode voltage clamp (TEVC) recordings. We found that one group of mutations (A218T, A218V, A313T, A313V, A535V) severely disrupted the voltage dependence of the common gate, leading to slower activation and faster deactivation. Another group (A529V, A531V, A566T, A566V) significantly reduced the overall channel expression. Both types of mutations can cause congenital myotonia. Furthermore, co-expression of wild-type and mutant channels was used to mimic the heterozygous condition in patients, allowing us to assess dominant negative effects and their implications for the clinical inheritance pattern. Previous studies suggest that common gating may involve an interaction between E232 and a highly conserved tyrosine residue (Y578) at the central anion-binding site. In this study, we substituted extracellular chloride with varying concentrations of iodide and observed that iodide promoted channel opening. Compared to WT, the A313T and A313V mutants showed reduced affinity for iodide during deactivation, while A566T behaved similarly to WT. These findings suggest that extracellular iodide affects the gating of both WT and mutant channels differently and may indicate that A313T and A313V mutations disrupt the E232–Y578 interaction, thereby altering ClC-1 common gating.