Drosophila visual signaling, a G-protein coupled PLCbeta4-mediated mechanism, is regulated by eye-PKC that promotes light adaptation and fast deactivation, most likely via phosphorylation of INAD and TRP. To reveal the critical phosphatases that dephosphorylate INAD, we utilized several biochemical analyses and identified PP2A as a candidate. Importantly, the catalytic subunit of PP2A, MTS, is co-purified with INAD, and an elevated phosphorylation of INAD by eye-PKC was observed in three mts heterozygotes. To explore whether PP2A (MTS) regulates dephosphorylation of INAD by counteracting eye-PKC (INAC) in vivo, we performed ERG recordings. We discover that inaCP209 is semi-dominant, because inaCP209 heterozygotes display abnormal light adaptation and slow deactivation. Interestingly, the deactivation defect of inaCP209 heterozygotes was rescued by the mtsXE2258 heterozygous background. In contrast, mtsXE2258 failed to modify the severe deactivation of norpAP16, indicating that MTS does not modulate NORPA (PLCbeta4). Together, our results strongly indicate that dephosphorylation of INAD is catalyzed by PP2A, and a reduction of PP2A can compensate for a partial loss of function in eye-PKC restoring the fast deactivation kinetics in vivo. We thus propose that the fast deactivation of the visual response is modulated in part by the phosphorylation of INAD.
A rapid electrical potential, which we have named the M-potential, can be obtained from the Drosophila eye using a high energy flash stimulus. The potential can be elicited from the normal fly, but it is especially prominent in the mutant norp AP12 (a phototransduction mutant), particularly if the eye color pigments are genetically removed from the eye. Several lines of evidence suggest that the M-potential arises from photoexcitation of long-lived metarhodopsin. Photoexcitation of rhodopsin does not produce a comparable potential. The spectral sensitivity of the M-potential peaks at about 575 nm. The M-potential pigment (metarhodopsin) can be shown to photoconvert back and forth with a "silent pigment(s)" absorbing maximally at about 485 nm. The silent pigment presumably is rhodopsin. These results support the recent spectrophotometric findings that dipteran metarhodopsin absorbs at much longer wavelengths than rhodopsin. The M-potential probably is related to the photoproduct component of the early receptor potential (ERP). Two major differences between the M-potential and the classical ERP are: (a) Drosophila rhodopsin does not produce a rapid photoresponse, and (b) an anesthetized or freshly sacrificed animal does not yield the M-potential. As in the case of the ERP, the M-potential appears to be a response associated with a particular state of the fly visual pigment. Therefore, it should be useful in in vivo investigations of the fly visual pigment, about which little is known.
Drosophila visual signaling, a G-protein-coupled phospholipase Cβ (PLCβ)-mediated mechanism, is regulated by eye-protein kinase C (PKC) that promotes light adaptation and fast deactivation, most likely via phosphorylation of inactivation no afterpotential D (INAD) and TRP (transient receptor potential). To reveal the critical phosphatases that dephosphorylate INAD, we used several biochemical analyses and identified protein phosphatase 2A (PP2A) as a candidate. Importantly, the catalytic subunit of PP2A, microtubule star (MTS), is copurified with INAD, and an elevated phosphorylation of INAD by eye-PKC was observed in three mts heterozygotes. To explore whether PP2A (MTS) regulates dephosphorylation of INAD by counteracting eye-PKC [INAC (inactivation no afterpotential C] in vivo , we performed ERG recordings. We discovered that inaC P209 was semidominant, because inaC P209 heterozygotes displayed abnormal light adaptation and slow deactivation. Interestingly, the deactivation defect of inaC P209 heterozygotes was rescued by the mts XE2258 heterozygous background. In contrast, mts XE2258 failed to modify the severe deactivation of norpA P16 , indicating that MTS does not modulate NORPA (no receptor potential A) (PLCβ). Together, our results strongly indicate that dephosphorylation of INAD is catalyzed by PP2A, and a reduction of PP2A can compensate for a partial loss of function in eye-PKC, restoring the fast deactivation kinetics in vivo . We thus propose that the fast deactivation of the visual response is modulated in part by the phosphorylation of INAD.
The recent identification and characterization of two genes, encoding histamine-gated chloride channel subunits from Drosophila melanogaster, has confirmed that histamine is a major neurotransmitter in the fruitfly. One of the cloned genes, hclA (synonyms: HisCl-alpha1; HisCl2), corresponds to ort (ora transientless), mutationsin which affect synaptic transmission in the Drosophila visual system. We identified a mutational change (a null mutation) in the genomic and RNA copies of hclA derived from mutants carrying the ort(1) allele. This correlates with new phenotypes observed in the mutant strain. We found hypersensitivity to the avermectin neurotoxins in both the ort(1) adult flies and third instar larvae compared to Oregon R wild-type animals. On the other hand, the mutation makes both male and female adult flies more resistant to treatment with diethyl ether, and the animals show substantially prolonged recovery from paralysis after diethylether anaesthesia, as well as from paralysis after mechanical shock, as revealed by the bang sensitivity test. Altogether, our data give direct evidence that in vivo a HCLA subunit-containing receptor has a distinct role in the neurotoxic action of the avermectins. They also provide new evidence for a function in the response to diethylether anaesthesia and, moreover, that HCLA function is not limited to the visual system.
