Calcium and lipid signalling in Drosophila photoreceptors

眼睛中的光感受器通过将光转换成电信号来对光做出反应。这个“光转导”过程涉及一系列生化步骤，以嵌入细胞周围膜中的称为“离子通道”的专门蛋白质的开放结束。一旦打开，离子通道允许带电离子（如钠和钙）进入细胞，从而产生电信号，这些电信号沿着沿着神经传递到大脑。这一过程可以在果蝇中得到很好的研究，原因有几个。首先，我们现在知道了果蝇的整个遗传密码，并且可以操纵它的基因，这样单个基因（以及蛋白质）就可以被改变、删除或引入果蝇。其次，我们可以分离苍蝇感光器，并使用一种称为“膜片钳”的技术极其精确地记录它们的电信号。果蝇中负责视觉的生化事件与在整个身体中发现的广泛转导级联基本相同。这种级联反应的特征在于一种酶（磷脂酶C），它将膜中的特定化学物质分解成两个小的“第二信使”分子。这导致被称为TRP通道的特定离子通道的开放。TRP通道的一个重要特征是它们允许钙离子（Ca 2+）进入细胞。Ca 2+本身也是细胞中重要的第二信使-例如在光感受器中，它根据光照水平调节敏感度，使我们能够在夜间和白天看到-这一过程称为适应。在蝇眼中，TRP通道产生负责视觉的电信号和适应所需的Ca 2+内流。TRP通道实际上是最早发现的，因为没有这些通道的突变果蝇是盲目的。由于脊椎动物的大多数基因与苍蝇的基因相似，这导致了在人体中发现了类似的通道，几乎在身体的每一个组织中都发现了类似的通道。它们在广泛的过程中很重要，包括激素反应，血压调节，癌症，味觉和听觉，疼痛和冷热感觉。由于这些通道是最近才发现的，因此它们的确切功能以及它们的活性如何调节仍然不清楚。然而，这是理解所有这些不同过程的核心，不仅科学家，而且许多制药公司都有兴趣尽可能多地了解它们。在我们的研究中，我们用果蝇光感受器的“膜片钳”记录来测量这些通道的活性，通常使用基因工程果蝇，改变通道本身或怀疑对其功能重要的成分（酶等）。我们的研究有几个目标：其中包括。1)使用基因工程来计算出通道的“孔”的分子结构，并找出是什么使其对Ca 2+具有渗透性2）直接测量通道允许进入细胞的Ca 2+的量，然后通过重新排列孔的分子结构来改变果蝇，以测试Ca 2+的重要性，例如对于适应。3)研究Ca 2+如何通过特异性改变单个蛋白质来控制级联反应的其他组分，使Ca 2+不再影响它们。4)确定调节通道的第二信使小分子;不同的酶负责产生不同的信使物质，我们将产生候选酶缺陷的突变果蝇，以测试哪种酶是重要的。我们从这些研究中获得的知识不仅会让我们对光感受器如何看到的分子有详细的了解，而且因为基本的潜在生化机制被广泛发现，将为身体中许多其他临床重要过程提供新的见解。

Photoreceptors in the eye respond to light by converting it into electrical signals. This process of 'phototransduction' involves a sequence of biochemical steps, ending with the opening of specialised proteins known as 'ion channels', embedded in the membrane surrounding the cell. Once opened, ion channels allow charged ions, such as sodium and calcium, into the cell, thereby generating electrical signals that are transmitted along nerves to the brain. This process can be particularly well studied in the fruitfly Drosophila for several reasons. Firstly, we now know the entire genetic code of the fruitfly, and can manipulate its genes so that individual genes (and hence proteins) can be altered, deleted or introduced into the fly. Secondly, we can isolate fly photoreceptors and record their electrical signals with extreme precision using a technique known as 'patch clamp'. The biochemical events responsible for vision in the fly are essentially the same as in a widespread transduction cascade found throughout the body. This cascade is characterised by an enzyme (phospholipase C) that splits a specific chemical in the membrane into two small '2nd messenger' molecules. This results in the opening of specific ion channels known as TRP channels. An important feature of TRP channels is that they allow calcium ions (Ca2+) to enter the cell. Ca2+ itself is also an important 2nd messenger in cells - e.g. in photoreceptors it adjusts sensitivity according to light levels, allowing us to see both at night and during the day - a process known as adaptation. In fly eyes, TRP channels generate the electrical signals responsible for vision and the Ca2+ influx required for adaptation. TRP channels were in fact first discovered, because mutant flies without these channels were blind. Because most genes in vertebrates are similar to those in flies, this led to the discovery of similar channels in humans, found in virtually every tissue of the body. They are important in a wide range of processes including hormonal responses, regulation of blood pressure, cancer, taste and hearing, pain and sensations of hot and cold. Because these channels have only been recently discovered, exactly how they function and how their activity is regulated is still not well known. However, it is central to understanding of all these different processes, and not only scientists, but also many drug companies are interested in finding out as much as possible about them. In our research, we measure the activity of these channels with 'patch clamp' recordings from fly photoreceptors, often using genetically engineered flies with alterations to the channels themselves or components (enzymes, etc) suspected of being important for their function. Our study has several aims: which include. 1) Use genetic engineering to work out the molecular structure of the 'pore' of the channel and to find out what makes it permeable to Ca2+ 2) Directly measure the amount of Ca2+ that the channel allows into the cell and then generate flies in which this is altered by rearranging the molecular structure of the pore, to test the importance of Ca2+ , e.g. for adaptation. 3) Study how Ca2+ controls other components of the cascade by specifically altering individual proteins so that Ca2+ can no longer affect them. 4) Identify the small 2nd messenger molecules that regulate the channels; different enzymes are responsible for generating different messenger substances, and we will generate mutant flies defective in candidate enzymes to test which is important. The knowledge we gain from these studies will not only give us a detailed molecular 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.