Mechanisms of inactivation in Drosophila phototransduction

Photoreceptors respond to light by converting it into electrical signals. This process of 'phototransduction' involves a cascade of biochemical steps, each of which involves one or more specific protein molecules (e.g. visual pigments and catalytic enzymes). The end result is the activation of specialised proteins known as 'ion channels', embedded in the membrane surrounding the cell. Once activated, 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. Fly photoreceptors are remarkable in being able to generate responses 10-100 x more rapidly than equivalent photoreceptors in vertebrate eyes thereby representing the fastest biochemical signalling cascade of this sort in the animal kingdom (one of the main reasons why it is so hard to swat a fly!). Phototransduction can be particularly well studied in the fruitfly Drosophila for several reasons. Firstly, we now know its entire genetic code, 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'. Thirdly, our laboratory has developed specialized and sophisticated physiological tools that allow us to monitor the rates of individual molecular steps in phototransduction in living, responding cells. In order to respond quickly and reliably photoreceptors must not only activate in response to light, but must also be able to terminate their activity when the light goes off. Although equally important for photoreceptor performance, the molecular steps involved in inactivation are poorly understood. In this research programme we will combine our biochemical and physiological approaches with genetic manipulation of different molecular components of the phototransduction cascade to provide a detailed understanding of how these inactivation mechanisms are controlled and co-ordinated to generate the remarkable performance of these photoreceptors. The molecules involved in generating the fly's response to light are not unique to fly photoreceptors. Even in humans, molecules closely related to those we are studying are found throughout the body. They play important roles in a wide range of processes such as all manner of hormonal responses, regulation of blood pressure, taste and smell, and sensations of pain, hot and cold. The knowledge we gain from these studies will not only give us a detailed 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.

The fly phototransduction cascade represents the fastest known G-protein coupled signalling system. Its performance is remarkable, generating large quantum bumps in response to single photons with kinetics 10-100x faster than in vertebrate rods. High temporal resolution requires not only fast activation but also rapid inactivation. Inactivation steps include quenching of active metarhodopsin (M*) by arrestin, termination of G-protein and PLC activity, metabolism of second messenger (diacylglycerol), closure of the channels, and resynthesis of PIP2. However, the contribution of these steps to response kinetics remains largely unexplored as does the question of whether and how they are regulated (eg by Ca2+). Rapid response termination also requires myosin III (NINAC), but how NINAC functions in phototransduction has remained largely mysterious. Recently we have developed a method (instantaneous photoinactivation of M* by photoreisomerisation) which allows us to measure the response deactivation time course of different steps of the cascade. Our preliminary results using this technique showed that M* deactivation by arrestin is strongly Ca2+ dependent, and implicated NINAC in this process. Objectives: i) To determine the mechanism underlying NINAC's role in Ca2+ dependent M* deactivation. ii) To investigate the molecular basis of two further separable phenotypes of ninaC mutants, (the occurrence of spontaneous quantum bump-like events in the dark and an increase in quantum bump duration) iii) To determine the contribution of specific deactivation steps to overall response deactivation. kinetics iv) To investigate the Ca2+ dependence of specific inactivation steps by using the Na/Ca exchanger to quantitatively control cytosolic Ca2+ whilst measuring the response deactivation time-course of different steps of the cascade.