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Title: Single photon responses of Drosophila photoreceptors
Author: Henderson, S.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2000
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The Drosophila phototransduction cascade is an example of a highly optimised G-protein signalling system, but with a unique potential for genetic dissection. Photoreceptors are also unique in their ability to produce a response (a quantum bump) to the stimulation of a single receptor (rhodopsin), with remarkable rapidity, and fidelity. The study represents a detailed analysis of quantum bumps using the whole cell patch clamp technique upon dissociated photoreceptors. Ch.1. There is a signal class of quantum consistent with a single transduction pathway, and each bump almost certainly corresponds to one and only one photon. Under control conditions bumps in wild type photoreceptors have a mean amplitude of ca. 10 pA corresponding to ca. 35 simultaneously open channels. The macroscopic flash response is accurately modelled by the linear summation of bumps. No correlation was found between latency and bump amplitude suggesting different underlying molecular mechanisms. Ch2. As extracellular [Ca2+] is decreased the bump shape broadens (ca. 20-fold), and the latency dispersion increases. A positive feedback dependent upon Ca2+ influx is suggested by the discovery of a slow ramping of the bump rising phase at [Ca2+]e levels intermediate between physiological and Ca2+ free. Ch3. Intracellular buffering of Ca2+ influx by BAPTA but not EGTA mimics the "creep" bump response. Influx of Ca2+ not steady state mediates this effect, and suggests that the targets of Ca2+ influx are closely apposed to the light-sensitive channels. Ch4. The quantum bumps of the light-sensitive channel mutants trp and trpl are examined. trpl bumps are very similar to wild-type, but have a slightly prolonged decay. This is the first reported difference between trpl and wild-type under physiological conditions. By contrast the quantal responses of trp are far smaller than those of wild type (2.6 pA c.f. 10 pA). There is however a lesser sub-set of larger quantal responses closer to the size of wild-type bumps. The quantum efficiency of both mutants is close to that of wild-type (trp slightly less). Ch5. The PKC mutant inaC and scaffolding protein mutant InaD have prolonged response termination to bright flashes of light. Contrary to previous reports the quantum bump of InaD is shown to be similarly prolonged but to have similar latency to that of wild-type. The range of phenotypic severity in InaD, and the similarity of some bumps in both mutants suggest a structural but perhaps not direct role for the InaD protein in coupling the inaC protein to modulation via Ca2+ influx, and/or the light sensitive channels.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available