分子伴護蛋白及泛素連接酶調控人類第一型氯離子通道之功能表現

Abstract

正常的骨骼肌細胞膜藉由表達大量的氯離子通道而維持細胞膜電位的穩定。過去的文獻報告指出，當骨骼肌細胞中第7對染色體上的CLCN1基因發生變異，可能會導致膜上電位依賴性氯離子通道CLC-1功能異常，繼而引發先天性肌強直症(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雖然在功能及閘道調控機制上與野生型(wild-type)並無明顯差異，但生成的CLC-1 A531V無法順利地由ER運送至細胞膜上表現，且由於蛋白酶體降解 (proteasome degradation) 增加導致蛋白質的總表現量減少，並因此造成細胞膜上的氯離子流密度 (Cl- current density) 明顯下降。若利用dominant negative (DN)-CUL4A/4B干擾CLC-1泛素蛋白酶體降解路徑中CRL4 E3 ligase complex的構築，或是藉由Ub-K0阻礙泛素鏈的聚合，則會促使細胞質中CLC-1的總表現量增加，顯示CLC-1可能是經由CRL4媒介泛素連結以及蛋白酶體降解路徑。此外，當Hsc70、FKBP8、Aha1及HOP大量表現後，亦會造成細胞質中CLC-1的總表現量增加，所以新生成的CLC-1可能藉由Hsp70-Hsp90伴護蛋白系統調控蛋白分子的折疊及穩定。然而，藉由干擾蛋白質降解路徑或促進Hsp70-Hsp90伴護蛋白系統所增加的CLC-1蛋白質並不一定能夠順利地被運送至細胞膜成為具有功能性的氯離子通道。因此，本論文以電生理的角度，探討影響CLC-1之Hsp70-Hsp90伴護蛋白系統及蛋白酶體降解途的參與因子，是否的確會影響細胞膜上CLC-1氯離子通道蛋白的功能表達。我們的實驗結果顯示，若增加Hsp70-Hsp90伴護蛋白系統中參與因子Aha1、FKBP8、HOP、Hsp90α/β及Hsc/Hsp70的表現量，得以促進CLC-1野生型和A531V氯離子通道的功能表現。此外，利用MLN4924藥物處理細胞，藉由DN-CUL4A/4B及DN-CRBN干擾CRL4的構築，或利用Ub-K0阻礙泛素鏈的聚合，亦皆能有效增加細胞膜上CLC-1通道之氯離子流。綜觀我們的研究結果足以推測，Hsp70-Hsp90伴護蛋白系統與CRL4蛋白酶體降解系統的確可以經由影響CLC-1蛋白質生成而調控細胞膜上的CLC-1氯離子通道之功能表現。

The expression of chloride channels is essential for the stabilization of membrane potential in skeletal muscle. Myotonia congenita is a skeletal muscle disorder associated with mutations in the CLCN1 gene (chromosome 7q), which encodes the voltage-gated chloride channel, CLC-1. Previous studies from our lab have demonstrated that despite the presence of a diminished whole-cell current density, the myotonia-related CLC-1 mutant A531V displays no detectable gating defects. Yet the A531V mutant is associated to a defective endoplasmic reticulum export and a reduction in the total protein expression that can be attributed to enhanced proteasomal degradation. Co-expression with Ub-K0 or dominant negative (DN)-CUL4A/4B increased the protein expression level of CLC-1, suggesting that the CRL4 E3 ligase system may mediate the ubiquitination and thus the proteasome degradation of CLC-1 proteins. Furthermore, over-expression of Hsc70, FKBP8, Aha1, or HOP also promoted CLC-1 expression, implying that the Hsp70-Hsp90 system may contribute to CLC-1 protein folding and stability. Elevated expression level of CLC-1 proteins, however, does not prove that the recued CLC-1 proteins are trafficked to cell surface and display proper chloride channel function. Hence, in this study, we aim to apply electrophysiological techniques to determine whether the Hsp70-Hsp90 and the CRL4 proteasome degradation systems modulate functional CLC-1 expression. Our results show that Aha1, FKBP8, HOP, Hsp90α/β, or Hsc/Hsp70 enhanced the functional expression WT and A531V CLC-1 channels. CLC-1 currents were also increased upon suppression of CRL4 function (by MLN4924 or DN-CUL4A/4B) or ubiquitin chain formation (by Ub-K0). Together, our data suggest that the Hsp70-Hsp90 system and the CRL4 proteasome degradation systems can indeed modulate the functional expression CLC-1 channels through the regulation of the biosynthesis of the channel protein.