鈣調素對鈉鈣交換蛋白之生理調控機制

Abstract

細胞內鈣離子濃度([Ca2 +] i)的變化是許多生理機制重要的訊息傳導途徑。鈉鈣交換蛋白（Na+/Ca2+ exchangers，NCX）根據胞內外鈉鈣離子的電化學梯度，可控制細胞膜內外鈣離子的進出以維持[Ca2 +]i動態平衡。鈣調素﹙Calmodulin，CaM﹚蛋白感知鈣離子濃度變化並藉與目標蛋白（如通道或是運輸蛋白）結合的方式調控鈣離子信號，但目前尚不清楚鈣調素與NCX1活性之間的相關性。在此篇研究，我們使用鈣調蛋白作為釣餌，與NCX1的兩種異構體的胞內片段（NCX1.1CL和NCX1.3CL）進行GST融合蛋白質免疫沉澱實驗﹙pull-down assay﹚。實驗結果顯示鈣調素在鈣離子存在時，可藉NCX1上的鈣調蛋白結合片段（calmodulin-binding segment，CaMS）與NCX結合。為了確定此結合使否調控NCX1活性，我們利用鈣離子顯影技術（calcium image）偵測[Ca2 +]i變化且同時在HEK 293T細胞內表現NCX1及鈣調素，或是同時表現不同的突變體來確定其中的調控機制。實驗結果顯示缺乏CaMS的NCX1.1及NCX1.3在細胞膜上的表現量及活性都顯著降低，此突變的NCX1.3若同時表現CaM1234則可回復此抑制效果。我們進一步依序點突變CaMS序列中4個重要胺基酸，顯示CaM對NCX1不同異構體有不同的交互機制。點突變第一、二個胺基酸位置會使NCX1.1活性大幅下降，而NCX1.3則是第二、三個位置抑制了NCX的活性。我們的研究結果發表了NCX1被CaM調控的直接證據，也提供了其結合的可能機制。

The change in the intracellular Ca2+ concentration ([Ca2+]i) is an important signal for various physiological activities. The Na+/Ca2+ exchangers (NCX) at the plasma membrane transport Ca2+ into or out of the cell according to the electrochemical gradients of Na+ and Ca2+ to modulate the [Ca2+]i homeostasis. Calmodulin senses [Ca2+]i changes and binds to proteins, such as channels and transporters, to relay Ca2+ signaling. However, it is not clear how calmodulin modulates NCX activity. Using calmodulin as bait, we pulled down the intracellular loops of alternative splicing isoforms of NCX1: NCX1.1 and NCX1.3. This interaction requires both Ca2+ and a putative calmodulin-binding segment (CaMS). To determine whether calmodulin modulates NCX activities, we co-expressed NCX1 isoforms with CaM or CaM1234 (a Ca2+-binding deficient mutant) in 293T cells and measured the increase in [Ca2+]i caused by the influx of Ca2+ through NCX. Deleting the CaMS from NCX1.1 and NCX1.3 attenuated exchange activities and decreased localization to the plasma membrane. Removing the mutually exclusive exons from NCX1.3 reduced exchange activity, which could be rescued by CaM1234 co-expression. Point-mutations at any of the 4 conservative a.a. residues in the CaMS had differential effects in NCX1.1 and NCX1.3. Mutating the first two conservative a.a. in NCX1.1 decreased the exchange activity. In contrast, mutating the 2nd and 3rd conservative a.a. in NCX1.3 decreased the exchange activity. Taken together, our results demonstrate that calmodulin senses changes in [Ca2+]i and binds to the cytoplasmic loop of NCX to regulate exchange activity and Ca2+ homeostasis.