探討人類第一型氯離子通道A531位點之功能重要性

Abstract

CLC-1是一種對細胞膜電位敏感(voltage-dependent)的氯離子通道。骨骼肌細胞膜(sarcolemma)含有大量的CLC-1通道蛋白，並貢獻了高達80%的靜止膜電導(rest membrane conductance)，使得骨骼肌的靜止膜電位得以被穩定。當骨骼肌第七對染色體上的CLCN1基因發生突變，可能會導致骨骼肌電壓敏感性氯離子通道CLC-1的功能改變，繼而引發先天性肌肉強直症(myotonia congenita)的產生。本實驗室先前的研究發現，與肌強直症相關的CLC-1突變型A531V其蛋白質無法順利的從內質網運輸至細胞膜上表現。此外，由於蛋白質的降解(degradation)增加，導致A531V在細胞質中的總表現量較正常型(wild-type, WT)顯著減少，而細胞膜上的氯離子流密度(Cl- current density)亦明顯下降。不過，A531V離子通道之電位調控門閘(voltage-dependent gating)性質卻與WT沒有明顯差異。本論文欲以電生理的角度來探討位於helix O上的A531位點在CLC-1離子通道電流的表現量與電位調控門閘性質上是否扮演重要的角色。 Alanine和valine都是非極性的胺基酸，在一般蛋白質內的體積大小分別約為92 Å3和142 Å3。我們在A531位點上額外製造了十三個不同的點突變，其胺基酸在蛋白質內的體積大小約由66 Å3至204 Å3不等。我們分別以cell-attached mode及whole-cell mode的方式記錄這十三個A531點突變之電流表現量。我們的實驗結果顯示，所有A531之點突變氯離子電流密度皆比WT顯著減少，而且電流大小減少的程度似乎與胺基酸體積大小成正比關係。在電位調控門閘方面，有部分的A531點突變出現明顯的變化，但其改變程度則與胺基酸體積大小不具有明顯的對應關係。 為了進一步了解A531位點是否為CLC-1在通道蛋白質表現與電位調控門閘性質之關鍵位點，我們還選擇與A531位點相當靠近之helix Q的A562位點，製造三個胺基酸大小介於106 Å3至168 Å3之間的點突變。我們也在羧基末端之A618位點，製造一個點突變，將其體積增加約至99 Å3。我們發現無論是A562或A618之點突變都不會顯著造成氯離子電流表現減少。以上研究結果顯示，A531位點或是helix O可能在CLC-1通道蛋白的合成過程中與電位調控機制扮演重要的功能。

The voltage-dependent chloride channel CLC-1 is essential for the stabilization of resting membrane potential in skeletal muscle. Mutations in the CLCN1 gene on the chromosome 7q, which encodes human CLC-1, and associated with the skeletal muscle disorder myotonia congenita. Previous studies from our lab have shown that the disease-causing CLC-1 mutant A531V displays enhanced protein degradation and defected membrane trafficking, resulting in significantly reduced Cl- current expression. Nevertheless, the voltage-dependent gating property of A531V does not appear to be notably different from that of the wild-type (WT) CLC-1 channel. The aim of this thesis is to apply electrophysiological techniques to determine whether the A531 residue on helix O may play an important role in the expression level and voltage-dependent gating. Both alanine and valine are non-polar amino acids with a volume of approximately 92 Å3 and 142 Å3 when buried in protein. We created 13 additional point mutations at A531, introducing amino acids with volumes ranging from 66 Å3 to 204 Å3. We examined the functional expression of these A531 point mutations using either cell-attached or whole-cell patch clamp recording. Our results demonstrate that the current density of all the A531 point mutations is significantly lower than WT, and the degree of current reduction seems to be proportional to the volume of the introduced amino acid. Moreover, some of the A531 point mutations show substantial changes in voltage-dependent gating property. The intent of alteration Cl- channel gating function, however, does not appear to correlated with the volume of the introduced amino acid. To further understand whether A531 is a key residue in CLC-1 protein expression and channel gating, we focus on the A562 residue on helix Q, which is very close to A531. We created three point mutations with volumes ranging from 106 Å3 to 168 Å3. We also created a point mutation at A618, which is located in the cytoplastic carboxyl terminal region, increasing its volume by approximately 99 Å3. We found that the point mutations at either A562 or A618 fail to significantly reduce CLC-1 current level. Overall, our data suggest that either A531 residue or helix O may play an important role in current expression and gating of CLC-1 channel.