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.