Photolysis of caged Ca2+ facilitates and inactivates but does not directly excite light-sensitive channels in Drosophila photoreceptors.
Substantial evidence implicates the phosphoinositide cascade in invertebrate phototransduction, but the final pathway of excitation remains obscure. In order to test the hypothesis that Ca2+ is the excitatory messenger rapid concentration jumps of cytosolic Ca2+ were achieved in dissociated Drosophila photoreceptors by flash photolysis of the caged Ca2+ compounds DM-nitrophen and nitr-5. Both compounds were introduced via patch pipettes used to record whole-cell currents. Calibrations using INDO-1 and Mag-INDO-1 indicated that photolysis of DM-nitrophen (5 mM loaded with 4 mM Ca2+), raised Ca, to ca. 20-50 microM, and nitr-5 (same loading) to ca. 1-2 microM. In mutants lacking light responses (ora, lacking rhodopsin; norpA, lacking phospholipase C; trp, which is inactivated by conditioning lights), the only current evoked by photolysis of DM-nitrophen was a small inward current with no detectable latency. This current did not reverse at +80 mV and was blocked by substitution of external Na+ for Li+, suggesting it represents activation of an electrogenic Na+/Ca2+ exchanger. A similar current was also the only current elicited by caged Ca2+ during the 5 msec latent period in wild type (WT) photoreceptors. To investigate possible modulatory effects of caged Ca2+ on the light-activated conductance, cells were first stimulated with a saturating light stimulus, itself incapable of releasing significant Ca2+, and then the photolytic flash was discharged during the response. During the rising phase of the response, photolysis of DM-nitrophen (but not nitr-5) induced a pronounced facilitation in WT photoreceptors. When photolysed during the plateau phase both DM-nitrophen and nitr-5 induced a rapid inactivation of the light-induced current. By contrast, in trp photoreceptors, which lack one class of Ca2+ permeable light-sensitive channel, photolysis of DM-nitrophen induced a significant facilitation during the falling phase of the response, but during the rising phase photolysis significantly depressed the overall response. In conclusion, caged Ca2+ failed to activate any channels in Drosophila photoreceptors but profoundly affected the light-dependent channels once they have been activated.
Hardie
R C
RC
Cambridge University, Department of Zoology, United Kingdom.
D000085:Acetates / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004330:Drosophila / Q000235:genetics; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D005029:Ethylenediamines / Q000378:metabolism*; D007473:Ion Channels / Q000378:metabolism* / Q000528:radiation effects; D008027:Light; D009154:Mutation; D010782:Photolysis; D017956:Photoreceptor Cells, Invertebrate / Q000378:metabolism*; D011930:Reaction Time
D000085:Acetates; D002614:Chelating Agents; D005029:Ethylenediamines; D007473:Ion Channels; C052455:nitr 5; C057014:DM-nitrophen; D004533:Egtazic Acid; D002118:Calcium