Calcium and lipid signalling in Drosophila photoreceptors

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.

Activation of PLC In Drosophila photoreceptors leads to the opening of two light sensitive channels, TRP and TRPL. The mechanism of permeation, activation and regulation of these channels, as well as of their vertebrate homologues (TRPC's),is complex and often controversial. To address a range of outstanding issues, we will combine Drosophila's genetic potential with high-resolution patch clamp recordings, making particular use of novel experimental approaches, we have pioneered. These include genetically targeted electrophysiological biosensors and exploitation of the Na+/Ca2+ exchanger, to control cytosolic Ca2+ levels. Our program includes the following main objectives. 1) Drosophila TRP has the highest reported Ca2+ selectivity of any TRPC channel and a voltage dependent divalent ion block, but the molecular determinants of these pore properties are unknown. Guided by sequence alignment, we will identify residues responsible for TRP pore properties. Since TRP channels are not reliably heterologously expressed, we will generate TRPL channels with single or multiple residues replaced with equivalent residues from the TRP pore. These will be tested both in vivo and in cell lines. Similarly we will mutate equivalent residues in TRP and express these channels in vivo. This strategy will allow us to define residues critical for permeation, to explore the functional significance of high Ca2+ permeability and to formally establish the identity of TRP as the native light-sensitive conductance. 2) The ionic selectivity of TRP and TRPL has been defined by reversal potential measurements but the fraction of the current carried by Ca2+ is unknown. Since this is a critical parameter for Ca2+ dynamics and homeostasis, we will estimate the fractional Ca2+ conductance of both TRP and TRPL channels in vivo, by exploiting the electrogenic Na/Ca exchanger. Since this extrudes Ca2+ with a known stoichiometry, the ratio of the charge integral of influx (TRP/TRPL) currents and efflux (Na/Ca exchange) currents should provide an empirical estimate of fractional Ca2+ conductance. 3) Both the TRP channels and PLC are inhibited by Ca2+, but over different concentration ranges; in addition, PLC (but not TRP) is inhibited by a PKC dependent mechanism. We will investigate the molecular mechanisms of this Ca2+ dependent feedback, by a)testing whether Ca2+ and PKC dependent phosphorylation of the INAD scaffolding molecule is required to inhibit PLC (monitored with electrophysiological PIP2 biosensors); and b) using a combination of genetic and pharmacolgical approaches, test whether Ca2+ dependent inactivation of TRP and/or TRPL is mediated by calmodulin. 4) The only ligands that reliably activate TRP and TRPL channels are poly-unsaturated fatty acids (PUFAs). In principle these could be released from DAG by a DAG lipase, but no evidence exists for this enzyme in Drosophila photoreceptors. We will explore the requirement of PUFAs for phototransduction in vivo by generating and characterising mutants of a novel putative DAG lipase gene. We will also express an electrophysiological DAG biosensor(the DAG sensitive TRPC6 channel) to monitor the fate of DAG during transduction 5) The rapid kinetics and amplification of transduction in Drosophila rely on a powerful Ca2+ dependent facilitation, but the mechanism is unknown. We will test two hypotheses: i) does Ca2+ increase the effective affinity of the channels for the activating ligand? This will be tested by measuring the dose response function for PUFAs whilst systematically varying Cai using the Na/Ca exchange equilibrium. ii) Is PLC activity enhanced by Ca2+ influx? This will be tested using PIP2 biosensors to monitor PLC activity in vivo as a function of [Ca2+].