THE IONIC MECHANISM OF THE PHOTORECEPTOR IN MUTANT AND WILD TYPE DROSOPHILA MELANOGASTER

MICHAEL JOHN WILCOX, Purdue University

Abstract

Available evidence suggests that the light coincident receptor potential (LCRP) and the prolonged depolarizing afterpotential (PDA) are generated by closely related mechanisms. However, in two classes of Drosophila mutants the PDA has been affected in ways that suggest that an uncoupling of the LCRP and PDA occurs. In ina mutant photoreceptors the LCRP appears normal but the PDA decays or repolarizes within a few seconds. In nina mutant photoreceptors the LCRP appears normal but a PDA is completely absent. Therefore in these two mutants uncoupling of the PDA from the LCRP appears to occur. Using intact flies, the responses of wild type and mutant photoreceptors to a PDA inducing stimulus were examined using intracellular recording to make sure that the phenotype was expressed in the mutant photoreceptor itself. Conductance properties during PDA induction in wild type and mutant photoreceptors were examined by injecting current into the photoreceptor using a constant current amplifier while recording intracellularly. The conductance increase in response to an intense blue stimulus returned to the dark resting conductance level after PDA induction in ina photoreceptors, while the conductance remained increased for wild type photoreceptors. The conductance increased in nina photoreceptors when stimulated by intense blue light but decreased to the dark resting level when the stimulus was withdrawn. The above results ruled out the possibility that the decline or absence of the PDA in the mutants might arise from a simultaneous increase in two or more opposing conductances. The amplitude of the LCRP in response to various intensities of 480 nm light (absorbance maximum of rhodopsin) was examined and no differences were apparent for wild type or mutant photoreceptors, indicating that the mutations did not affect the stimulus-response function. I applied a technique for examining the ionic mechanism of photoreceptors, which enabled me to perfuse isolated retinas in salines containing various ionic concentrations, while recording the LCRP intracellularly from a single photoreceptor. The ionic mechanism of wild type photoreceptors was examined by perfusing isolated retinas in salines containing varying concentrations of Ca('++), Na('+), or K('+) while the LCRP was recorded intracellularly from a single photoreceptor. For the PDA the same experiments were performed but extracellular recordings were used. Raising the extracellular sodium concentration or lowering the extracellular calcium concentration increased the apparent sodium current in the wild type LCRP. A PDA inducing stimulus delivered to an isolated retina in the presence of high extracellular sodium or low extracellular calcium elicited an afterpotential in the mutant retinas resembling the afterpotential in wild type retinas in normal saline. However, the afterpotential was not reversed by applying metarhodopsin photoconverting stimuli or by returning the retina to the control saline. Control experiments were performed to show that this irreversible afterpotential was not due to osmotic damage by chloride ion fluxes and not due to the ionic treatment per se on the photoreceptor membrane. Ionic substitution experiments substituting Li('+) or Cs('+) for Na('+) in the perfusate showed that ina phenotype changed to a phenotype resembling wild type but that the phenotype of nina mutant photoreceptors did not change. Therefore it is possible that the ina mutation has affected the ionic mechanism for PDA generation. The nina mutation, on the other hand, has probably affected some process(es) preceding the ionic mechanism. If nina has affected the ionic mechanism, it does so in a different way from the ina mutation.

Degree

Ph.D.

Subject Area

Biophysics

COinS