布魯格達氏症候群相關的基因突變KCNT1 R1106Q改變FPD和細胞質中的鈣離子恆定

Abstract

有文獻指出，近期發現的KCNT1基因，又稱Slack，為一種鉀離子通道，其胺基酸序列1106位置的突變與罕見的遺傳性心臟離子通道病變-布魯格達氏症候群有關，此疾病會造成心律不整及突發性的猝死，而目前對KCNT1 R1106Q在心臟功能上的影響還尚未有相關研究。 先前研究指出，和布魯格達氏症候群相關的心肌離子通到的突變，造成心肌細胞流入的鈉、鈣離子及流出的鉀離子之間不平衡(內向正電流下降或外向正電流增加)導致此症候群心電圖典型的ST間段上升，表現出心律不整，是廣泛被接受的電位復極化(repolarization)理論。而KCNT1 R1106Q 是否會影響鉀離子的通透性，如使大量的鉀流出，增加電位復極化的速度，又或者，如此致命性的心臟疾病除了造成鉀離子在細胞內的變化外，對於維持心臟正常功能及心肌細胞電性活動皆很重要的鈣離子，是否也會有影響，也是我們很想了解的。本篇研究的目的即是想探討KCNT1 R1106Q突變是如何造成布魯格達氏症候群，其機制為何。 本篇研究使用可自發性放電的HL-1 細胞，大量表現KCNT1野生型和R1106Q突變型，監測其胞外電生理訊號。結果發現，KCNT1野生型的HL-1細胞測到比突變型還長的field potential duration (FPD)，而突變型的FPD跟控制組比則是無太大差異的。而從HEK293T 細胞大量表現KCNT1野生型和R1106Q 突變型，並利用Fura-2 A.M.來觀察細胞質內鈣離子的變化的實驗中，發現KCNT1野生型的組別，細胞質內鈣離子濃度比突變型組高，且使細胞模擬產生SOCE (store-operated calcium entry)的實驗結果也發現，突變型組SOCE的能力跟野生型比有顯著性的降低，且此SOCE現象在兩組中皆可被BTP2 (SOCE抑制劑)抑制。 從上述實驗結果得知，KCNT1 R1106Q除了影響鉀離子的通透性外，在細胞中對鈣離子的恆定也扮演重要的角色。且KCNT1 R1106Q造成細胞質鈣離子濃度偏低，及影響 SOCE的能力，可能是造成布魯格達氏症候群的原因之一，為了驗證此臆測，還需要更多的實驗去證明。

The KCNT1 gene (also known as SLACK, SLO2.2) encodes a sodium-activated potassium (KNa) channel. KCNT1 R1106Q mutation has been previously linked to Brugada syndrome (BrS), a rare cardiac channelopathy associated with sudden cardiac death (SCD). The mechanism by which KCNT1 R1106Q causes Brugada syndrome is still unknown. Previous studies have shown that in the reported channel mutations linked to BrS, the imbalance between inward and outward currents leads to the development of a characteristic notch and loss of the action potential dome mediated by an increase (relative or absolute) of the outward potassium currents. The alteration of repolarization produces the characteristic ST-segment elevation in the ECG of BrS. We want to find out if the KCNT1 R1106Q mutation can also change potassium permeability, increasing the outward current that causes fast repolarization. Furthermore, we want to know if it could also affect calcium permeability in such a lethal cardiac disease, since calcium is an important ion that maintains normal heart function and electric activity. The aim of the present study is to understand whether and how KCNT1 mutation is related to Brugada syndrome by determining the functional consequences of this mutation and the mechanisms. We used spontaneous beating HL-1 cells overexpressing KCNT1 wild-type (WT) and R1106Q to detect its extracellular field potential duration and found that KCNT1 WT has longer field potential duration (FPD) than in mutants. The mutant’s FPD was similar to control groups. Furthermore, we used HEK293T cells overexpressing KCNT1 wild-type (WT) and R1106Q to observe calcium homeostasis by using calcium indicator Fura-2 A.M. Interestingly, HEK293T overexpressed with the KCNT1 WT had a higher baseline calcium level than the mutant baseline level. Later on, we induced SOCE effect by depleting ER calcium, which showed that mutants can diminish SOCE effect. The SOCE inhibitor BTP2 could block this Ca2+ influx. Together with these results, we hypothesized that KCNT1 may have a significant role in both controlling potassium permeability as well as maintaining cardiac Ca2+ homeostasis. Furthermore, the disruption of Ca2+ signaling by mutant KCNT1 may contribute to Brugada syndrome. To verify such speculations, further experiments still need to be conducted.