Photolysis of caged Ca2+ facilitates and inactivates but does not directly excite light-sensitive channels in Drosophila photoreceptors

Abstract

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.