"利用HEK293細胞株表現人類SCN1A,SCN1B及SCN2B蛋白質探討SCN1A變異蛋白質的功能"

Abstract

鈉離子通道的突變與遺傳性癲癇疾病極為相關，且臨床病患症狀嚴重度變異性高，其中最相關的基因為SCN1A。約一半SMEI病患帶有無義突變SCN1A基因，此類病患在癲癇疾病中症狀最為嚴重，且對於臨床藥物多具抗藥性。鑒於此類疾病的不良預後，帶有突變型SCN1A的SMEI症候群符合未滿足醫療迫切需求。過去研究團隊以抑制無義突變抑制治療對由無義突變引起的疾病進行藥物開發，概念為利用藥物在提前終止密碼子上插入任意胺基酸，使產生錯義突變的全長通讀蛋白質。最近有團隊利用酵母菌系統以慶大黴素 (gentamicin) 誘導通讀時，發現在提前終止密碼點會插入特定幾種胺基酸，此研究結果使得科學家可預測通讀蛋白種類及功能。基於上述，利用無義突變抑制治療策略對SCN1A無義突變的病患具有發展潛力。為了研究由通讀誘導出的SCN1A蛋白質是否具有正常的生理功能，本論文建構野生型及4種突變型SCN1A基因的載體pCMV-EGFP-hSCN1A (SCN1A蛋白N端以EGFP標記)，並轉染表現於穩定表現SCN1B及SCN2B蛋白的HEK293細胞株，以全細胞紀錄方式分析電生理特性。實驗結果顯示，HEK293穩定細胞株可測得SCN1B及SCN2B蛋白質表現，並成功地被2A胜肽切斷。全細胞紀錄發現SCN1A-E1099X完全無法產生電流，而SCN1A-E1099Q可回復表現與SCN1AWT相同電流量及通道開關動力學，顯示SCN1A-E1099Q異義突變蛋白能回復鈉離子通道的開關功能。本論文研究成果不僅提供以2A胜肽技術在HEK293細胞上同時穩定表現SCN1B及SCN2B的新方法，也提供以通讀誘導藥物治療SCN1A無義突變的未來發展潛力。-

Mutations in sodium channels are one of clinically relevant genetic epilepsies with a various range of severity. Especially, the most predominant target of mutation is the NaV1.1 channel encoded by the SCN1A gene. Truncation mutations in the SCN1A gene account for nearly 50% of SMEI patients, and most of them have intractable epilepsy after current epilepsy treatment. Due to the poor prognosis, patient with SCN1A nonsense mutation address an unmet medical need. Nonsense suppression therapy is a newly developed therapeutic approach for nonsense mutation diseases, which incorporates an amino acid at premature termination codons (PTCs). This process may lead to a missense mutation in a functional or non-functional readthrough protein. Recently, a study in S. cerevisiae identified that some specific sets of amino acids are inserted at PTCs after gentamicin treatment, which narrows the possible sets of proteins and its functionality after readthrough. Based on the above, the potential of nonsense suppression therapy for patients with SCN1A nonsense mutation is worthy of investigation. To investigate the electrophysiological properties of readthrough-induced SCN1A proteins in vitro, we generated chimeric constructs pCMV-EGFP-hSCN1A (N-terminal tagging of SCN1A) harboring wild-type (SCN1A-WT), nonsense mutation (SCN1A-E1099X) or three possible readthrough-induced proteins (SCN1A-E1099Q, SCN1A-E1099K, SCN1A-E1099Y). Meanwhile, we generated HEK293 cells stably expressing SCN1B and SCN2B proteins for modulating the kinetics of SCN1A protein. Our results showed that SCN1B and SCN2B proteins in stable cell lines were successfully cleaved by 2A peptides. By whole cell recordings, we characterized the electrophysiological properties of SCN1A-WT, SCN1AE1099X and SCN1AE1099Q. SCN1AE1099X predominantly lost the capacity of generating currents predominantly, decreasing Na+ current to <100 pA. SCN1AE1099Q retained the same Na+ currents and biophysical functions of wild-type channels, which indicated SCN1AE1099Q can restore the expression of Na+ channel with the normal function. Our analyses not only provide a new way to establish multi-protein expression of SCN1B and SCN2B proteins in HEK293 cell , but also validate the functionality of readthrough SCN1A protein.