Proton signalling in Drosophila photoreceptors

光感受器通过一系列生化步骤将光转化为电信号，每个步骤都涉及特定的蛋白质分子（例如视觉色素和酶）。这种“光导级联”的最终结果是激活细胞周围脂质膜上被称为“离子通道”的蛋白质。一旦激活，离子通道打开，允许带电离子（如钠和钙）进入细胞，从而产生电信号并传输到大脑。我们可以很好地研究果蝇的光传导，因为我们可以很容易地操纵特定的基因（以及蛋白质），因为我们可以使用一种被称为“片钳”的技术高精度地记录它们的光感受器的电信号。参与光传导的分子并不是苍蝇光感受器所特有的，在我们身体的细胞中也发现了与之密切相关的分子。其中一个分子就是所谓的TRP通道。在果蝇中，这是由光导激活的通道；在哺乳动物中，色氨酸通道对于许多重要过程都是必不可少的，比如激素反应、血压调节、味觉、嗅觉、痛觉、冷热感觉。TRP通道如何被激活仍然是一个谜，尽管人们早就知道一种被称为磷脂酶C （PLC）的酶经常参与其中。PLC将细胞膜中特定的小脂质分子（PIP2）分裂成两种产物（称为DAG和InsP3）。此外，该反应还产生一个质子（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，以前从未认为它具有重要的功能。我们最近发现，TRP通道可以通过酸化和膜中PIP2浓度降低的结合而激活。我们还发现光导级联中的另一个关键分子在酸化时其结构发生了变化。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；都涉及PLC释放的质子。我们打算找出质子如何与TRP通道蛋白相互作用和控制，以及PIP2的减少如何控制质子激活通道的能力。通过设计定制的遗传编码荧光探针，我们还计划直接成像INAD分子在活体动物中的构象变化。这将允许一系列的实验来确定这种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看到的理解，而且由于基本的潜在生化机制被广泛发现，将为许多其他的，通常是临床上重要的身体过程提供新的见解。

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.