Proton signalling in Drosophila photoreceptors

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.

Drosophila phototransduction is mediated by PLC-mediated hydrolysis of PIP2 leading to the opening of TRP and TRPL channels by a mechanism that has long been enigmatic. We recently found that the channels can be activated by a combination of PIP2 depletion and protons released by PLC. Another potential proton target is the INAD scaffolding protein, which assembles TRP, PKC and PLC into "signalplexes". Recently we found that this undergoes a pH dependent conformational change. These results introduce proton release by PLC as a new paradigm in cellular signalling.

A) TRP channel activation. We aim to identify sites on TRP and TRPL involved in gating by mutagenising protonatable residues close to the membrane interface. We will also make measurements of light-induced global and local pH changes using indicator dyes and genetically encoded pH sensitive GFP probes. One way that PIP2 depletion could contribute to activation is by a physical change in the lipid bilayer. Remarkably, we found that photoreceptors contract in response to light. We will test whether this is a direct effect of PIP2 depletion. Another hypothesis is that depletion of PIP rather than PIP2 is key. We will explore this by tracking PIP2 and PIP dynamics with GFP based probes, and by depleting PIP2 with a voltage-sensitive phosphatase that generates PIP from PIP2.

B) INAD. Acidification switches INAD from a reduced state with high affinity for TRP and PLC to an oxidized state with reduced affinity. We will assess the significance of this using mutants that lock INAD in one or other state. We will also test the effects of small-peptide antagonists that compete with TRP and PLC binding to INAD. To assay the conformational state of the complex in vivo we will design GFP probes that bind to only the reduced state. Finally, the role of INAD phosphorylation by PKC will be tested by generating and testing mutants of putative PKC phosphorylation sites.

Staff and students: Staff and students engaged in the research will receive multidisciplinary training in neuroscience, electrophysiology, molecular biology, genetics, imaging and basic lab skills. Previous post-docs/students have become successful not only in academia, but also in a broad range of employment (incl., government, education and hi-tech industry in both UK and abroad). As well as post-docs and PhD students, we engage medical and basic science undergraduate students in lab-based projects, for whom the experience can be inspirational. Apart from lab skills required for the project, staff and students are encouraged and/or required to acquire a variety of further generic skills via courses or teaching experience. I also routinely welcome short-term (up to 3 months) visitors (e.g. from Russia, USA, India and several European countries) to learn specific skills and, by invitation have taught courses on several international graduate schools.

Health practitioners and pharmaceutical industry: TRP channels are the second largest ion channel family in our genome with 28 mammalian members distributed amongst 6 subfamilies. They are ubiquitous, of vital physiological importance, and increasingly implicated in a broad spectrum of hereditary and non-inherited disease. Within the TRPC subfamily alone, they have been implicated in cardiovascular disease (eg hypertension, cardiac hypertrophy); pulmonary disease, cancer, renal disease, rheumatoid arthritis, and neurological disorders such as cerebellar ataxia, bipolar disorder, and myasthenia gravia. TRP channels are hence widely regarded as promising novel therapeutic targets under intensive investigation by several pharmaceutical companies, with clinical trials underway for e.g. TRPV1, TRPM8 and TRPV3. As such my (BBSRC funded) seminal discovery of the first TRP channel has had, and continues to have a huge worldwide impact on clinically oriented research in both academia and the pharmaceutical industry. I was also the first to show that TRP channels could be lipid-regulated - now recognised to be true for many if not most TRP channels. The novel mechanism under study in this proposal -combinatorial activation by lipid and protons - may also prove to be a fundamental, widely conserved mechanism, which may also impact on future product-oriented research in the pharmaceutical industry.

General Public:
We expect the general public to be interested in several aspects of our work:
i) The sensory capabilities of insects, which are very different from our own (eg flies can detect movement up to 10x faster than can we can and can see UV and polarised light)
ii) Related TRP channels in humans mediate many specific sensory experiences - particularly sensations of hot and cold, pain and many tastes (chilli, menthol, horseradish, garlic, oregano, thyme) and play many other vital roles in the body
iii) The contributions Drosophila, as a model genetic organism, can make to our understanding of human biology and disease.

At a more advanced level, our research is widely considered as the definitive account of phototransduction in microvillar photoreceptors: it is taught at both 2nd and 3rd year level at our University and is widely taught in relevant University courses internationally.