A new tool to sense pH changes at the neuromuscular junction synaptic cleft.
A new tool to sense pH changes at the neuromuscular junction synaptic cleft.
复制标题
DOI：
10.1038/s41598-020-77154-3
复制
发表时间：
2020-11-24
影响因子：
4.6
通讯作者：
Uchitel OD
中科院分区：
文献类型：
--
作者：
Blaustein M;Wirth S;Saldaña G;Piantanida AP;Bogetti ME;Martin ME;Colman-Lerner A;Uchitel OD
突触传递触发突触间隙的短暂酸化。最近，它已被证明，pH值影响突触后通道的开放，因此，允许研究这些行为的工具的生产应导致最重要的价值。我们将α-银环蛇毒素（一种来源于银环蛇的神经毒素，可与乙酰胆碱受体胞外区不可逆结合）与pH敏感的GFP超荧光蛋白融合，并在毕赤酵母中高效表达。该传感器允许测量pH值的突触变化，而无需将转基因掺入动物细胞中。在这里，我们表明，小鼠耳提肌与含有这种重组蛋白的溶液孵育足以荧光标记终板突触后膜。此外，我们可以生理上改变和测量突触后pH值通过评估结合到乙酰胆碱受体的荧光信号的变化。事实上，使用这种工具，我们能够检测到神经电刺激触发囊泡胞吐后乙酰胆碱受体附近的0.01至0.05 pH单位的下降。进一步的实验将允许了解突触激活期间和之后pH的精确变化。
Synaptic transmission triggers transient acidification of the synaptic cleft. Recently, it has been shown that pH affects the opening of postsynaptic channels and therefore the production of tools that allow to study these behaviors should result of paramount value. We fused α-bungarotoxin, a neurotoxin derived from the snake Bungarus multicinctus that binds irreversibly to the acetylcholine receptor extracellular domain, to the pH sensitive GFP Super Ecliptic pHluorin, and efficiently expressed it in Pichia pastoris. This sensor allows synaptic changes in pH to be measured without the need of incorporating transgenes into animal cells. Here, we show that incubation of the mouse levator auris muscle with a solution containing this recombinant protein is enough to fluorescently label the endplate post synaptic membrane. Furthermore, we could physiologically alter and measure post synaptic pH by evaluating changes in the fluorescent signal of pHluorin molecules bound to acetylcholine receptors. In fact, using this tool we were able to detect a drop in 0.01 to 0.05 pH units in the vicinity of the acetylcholine receptors following vesicle exocytosis triggered by nerve electrical stimulation. Further experiments will allow to learn the precise changes in pH during and after synaptic activation.
登录
查看更多内容
影响因子：
5.5
作者：
AXELSSON, J;THESLEFF, S
通讯作者：
THESLEFF, S
影响因子：
5.3
作者：
Gonzalez-Inchauspe, Carlota;Urbano, Francisco J.;Uchitel, Osvaldo D.
通讯作者：
Uchitel, Osvaldo D.
影响因子：
8.8
作者：
Vincent, Philippe F. Y.;Cho, Soyoun;Dulon, Didier
通讯作者：
Dulon, Didier
DOI：
10.1073/pnas.1407018111
发表时间：
2014-06-17
影响因子：
11.1
作者：
Du, Jianyang;Reznikov, Leah R.;Welsh, Michael J.
通讯作者：
Welsh, Michael J.
影响因子：
3.4
作者：
Sankaranarayanan, S;De Angelis, D;Ryan, TA
通讯作者：
Ryan, TA