EA2相關突變種之顯性抑制作用對P/Q型鈣離子通道在細胞合成作用之影響

P/Q型鈣離子通道是由形成離子孔道 (pore-forming) 的α1A subunit與其他α2δ、β等auxiliary subunits所組成，其神經生理功能主要是在調控突觸傳遞作用和神經細胞的興奮性。陣發性不協調第二型(episodic ataxia type 2，EA2)是一種與小腦運動功能失常有關的遺傳性自體顯性疾病。根據分子遺傳學的研究結果顯示，EA2是由於P/Q型鈣離子通道之α1A subunit突變是所導致。生物物理研究顯示，與正常型鈣離子通道相比，EA2相關的突變種鈣離子通道，幾乎完全失去離子通道功能。如何由α1A subunit突變進而導致EA-2諸多症狀的機制，至今仍不完全清楚。我們實驗室先前的電生理研究結果顯示數種與EA2相關之鈣離子通道縮短突變(truncation mutants)對於正常型鈣離子通道有明顯的顯性抑制作用(dominant-negative effect)。因此，我們進一步以細胞生物學的方法，探討EA2相關突變種之顯性抑制作用對P/Q型鈣離子通道在細胞內運送之影響，以對EA2突變種造成之顯性抑制作用之分子機制有更深一層的了解。我們的研究方法是將帶有GFP-tag的正常型與突變種鈣離子通道α1 subunit (R1281X 、F1406C 、R1549X、R1669X、E1761K 、△681;783X 、Ins1004;1070X、△1177;1189X)表現在HEK293T細胞，結合一些細胞胞器的螢光標記，以共軛焦顯微鏡觀察，從次細胞層次探討突變種鈣離子通道以及正常型混合突變種鈣離子通道在細胞內的biogenesis。Missense mutation F1406C和正常型鈣離子通道最相似，在細胞膜都有很高的分佈比例。相較於正常型，E1761K、R1549X、R1669X則是細胞膜的分佈比例皆有下降的現象。R1281X和△681;783X都是只有兩個domain的縮短突變，卻能夠使大部分的鈣離子通道滯留在細胞質中。利用內質網標記，顯示R1281X、△681;783X突變蛋白存在於內質網中。混合表達正常型與突變種鈣離子通道，發現正常型蛋白較容易滯留在細胞質中，進一步利用內質網標記確認正常型蛋白質亦滯留於內質網中。利用低溫處理，能夠促進混合表達之正常型鈣離子通道運送至細胞膜的能力，亦為一強烈的間接證明滯留的胞器確為內質網。因此，顯性抑制作用的分子機制之一可能是正常型鈣離子通道蛋白質受到EA2相關突變種蛋白質的影響而滯留在內質網。此外，我們還發現4種在loop domain II-III以及domain III S1 segment區域發生truncation mutation之EA2突變種的細胞質滯留現象特別明顯，而且這四個突變種有隨著胺基酸序列越短，滯留在細胞質的情形越為顯著的現象。這個結果暗示了loop domain II-III對於鈣離子通道蛋白的膜運送過程的重要性。

關鍵字

鈣離子通道；陣發性小腦運動失調

並列摘要

P/Q-type calcium channel are composed of pore-forming α1A subunit and auxiliary α2δ, β and γ subunits. The neurophysiological role of P/Q-type calcium channels is to modulate synaptic and neuronal excitability. Episodic ataxia type-2 (EA2) is an inherited autosomal dominant disease related to cerebellar dysfunction. Molecular genetic studies have demonstrated that EA2 is associated with mutations in human α1A subunit. Biophysical studies have shown that most EA2 related mutations result in significant loss of function of P/Q-type Ca2+ channels. The exact mechanism of how mutant P/Q-type Ca2+ channels result in EA2 phenotypes is, however, not fully understood. Our previous functional studies in Xenopus oocytes support the idea that EA2 mutants may exert dominant-negative effects on wild-type P/Q-type Ca2+ channels. To further pursue the molecular mechanism underlying the dominant-negative effect of EA2 mutations, we test the effect of the EA2 mutations on the membrane trafficking of human P/Q-type Ca2+ channels. By expressing GFP-tagged wild type or mutants in HEK293T cells, we study the subcellular distribution of GFP signals using confocal microscopy. The missense mutation F1406C shows similar subcellular distribution pattern with that of wild-type, whereas E1761K, R1549X, R1669X mutants display less efficient membrane trafficking pattern. Furthermore, two 2-domain truncation mutants, R1281X and △681;783X, exhibit extensive cytoplasm retention pattern. When we co-express GFP-tagged WT with EA2 mutations, we notice a significant enhanced retention of GFP-WT signals in the cytoplasm. The cytoplasm retention pattern of WT Cav2.1, as well as those of R1281X and △681;783X, colocalize with an ER marker, suggesting that these proteins are trapped in the ER. Thus, the mechanism of the dominant-negative effect of EA2 mutations may involve ER-retention of wild-type Cav2.1 channels. In addition, four EA2 mutations involving premature stop codon within the protein region of loop domainⅡ-Ⅲand domain Ⅲ S1 segment display significant cytoplasm retention pattern in HEK293T cells. Interestingly, with these four mutations, the tendency for cytoplasm retention enhances as the extent of deletion increases, suggesting that the loop domainⅡ-Ⅲ region may be essential in determining the membrane trafficking process of human P/Q –type Ca2+ channels.

並列關鍵字

calcium channel ； episodic ataxia

參考文獻

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EA2相關突變種之顯性抑制作用對P/Q型鈣離子通道在細胞合成作用之影響

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