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Proton signalling in Drosophila photoreceptors

果蝇光感受器中的质子信号传导

基本信息

批准号：
BB/J009253/1
负责人：
Roger Hardie
金额：
$ 85.73万
依托单位：
University of Cambridge
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2012
资助国家：
英国
起止时间：
2012 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FJ009253%2F1
关键词：
Proton signalling Drosophila photoreceptors

项目摘要

Photoreceptors transduce light into electrical signals by a series of biochemical steps, each involving specific protein molecules (e.g. visual pigments and enzymes). The end result of this "phototransduction cascade" is the activation of proteins known as "ion channels", in the lipid membrane surrounding the cell. Once activated, ion channels open to allow charged ions, such as sodium and calcium, into the cell, thereby generating electrical signals for transmission to the brain. Phototransduction can be particularly well studied in the fruitfly Drosophila because of the ease with which we can manipulate specific genes (and hence proteins) and because we can record the electrical signals of their photoreceptors with high precision using a technique known as "patch-clamp". The molecules involved in phototransduction are not unique to fly photoreceptors and closely related molecules are found in cells throughout our own bodies. One such molecule is the so-called TRP channel. In flies, this is the channel activated by phototransduction; in mammals, TRP channels are essential for a wide range of vital processes such as hormonal responses, regulation of blood pressure, taste, smell, and sensations of pain, hot and cold. How TRP channels are activated remains mysterious, although it has long been known that an enzyme, known as phospholipase C (PLC) is often involved. PLC splits a specific small lipid molecule (PIP2) in the cell membrane into two products (called DAG and InsP3). In addition, the reaction also yields a proton (a hydrogen ion), which results in acidification. This simple chemical fact has been largely ignored and never previously considered to be of functional significance. We have recently found that TRP channels can be activated by a combination of acidification and the reduction in concentration of PIP2 in the membrane. We also found that another key molecule in the phototransduction cascade undergoes a change in its structure upon acidification. This molecule, (INAD), is a so-called scaffolding molecule, which normally binds the TRP channel and the PLC enzyme together into a signalling complex; but releases them upon acidification. Our research builds on these findings, which suggest radical new mechanisms for TRP channel activation and its subsequent inactivation; both involving protons released by PLC. We intend to find out how protons interact with and control the TRP channel protein and how a reduction in PIP2 can control the ability of protons to activate the channel. By designing tailor-made genetically encoded fluorescent probes we also plan to directly image the conformational change in the INAD molecule in living animals. This will permit a range of experiments to determine the mechanism and function of this pH regulated molecular switch. The knowledge we gain from these studies will not only further our understanding of how photoreceptors see but, because the basic underlying biochemical mechanisms are so widely found, will provide new insight into many other, often clinically important processes in the body.

光感受器通过一系列生化步骤将光转换为电信号，每个生化步骤都涉及特定的蛋白质分子（例如视觉色素和酶）。这种“光转导级联”的最终结果是激活细胞周围脂质膜中称为“离子通道”的蛋白质。一旦激活，离子通道就会打开，允许带电离子（例如钠和钙）进入细胞，从而产生电信号传输到大脑。光转导可以在果蝇中得到特别好的研究，因为我们可以轻松地操纵特定基因（以及蛋白质），并且因为我们可以使用称为“膜片钳”的技术高精度记录其光感受器的电信号。参与光转导的分子并不是苍蝇光感受器所独有的，在我们体内的细胞中也发现了密切相关的分子。其中一种分子就是所谓的 TRP 通道。在果蝇中，这是由光转导激活的通道；在哺乳动物中，TRP 通道对于多种生命过程至关重要，例如激素反应、血压调节、味觉、嗅觉以及疼痛、冷热感觉。 TRP 通道的激活方式仍然是个谜，尽管人们早已知道其中经常涉及一种称为磷脂酶 C (PLC) 的酶。 PLC 将细胞膜中的特定小脂质分子 (PIP2) 分裂成两种产物（称为 DAG 和 InsP3）。此外，该反应还产生质子（氢离子），从而导致酸化。这个简单的化学事实在很大程度上被忽视了，以前从未被认为具有功能意义。我们最近发现 TRP 通道可以通过酸化和膜中 PIP2 浓度降低的组合来激活。我们还发现光转导级联中的另一个关键分子在酸化时其结构发生变化。这种分子 (INAD) 是一种所谓的支架分子，通常将 TRP 通道和 PLC 酶结合在一起形成信号复合物；但在酸化时会释放它们。我们的研究建立在这些发现的基础上，这些发现提出了 TRP 通道激活及其随后失活的全新机制；两者都涉及 PLC 释放的质子。我们打算找出质子如何与 TRP 通道蛋白相互作用并控制 TRP 通道蛋白，以及 PIP2 的减少如何控制质子激活通道的能力。通过设计定制的基因编码荧光探针，我们还计划直接对活体动物中 INAD 分子的构象变化进行成像。这将允许进行一系列实验来确定这种 pH 调节分子开关的机制和功能。我们从这些研究中获得的知识不仅将进一步加深我们对光感受器如何观察的理解，而且由于基本的生化机制已被广泛发现，还将为体内许多其他通常具有临床意义的过程提供新的见解。