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The Drosophila Transient Receptor Potential (TRP) channel is the founding member of a large and diverse family of channel proteins. These channels are evolutionarily conserved from yeast to mammals and are found in many organisms and tissues. The TRP family is classified into seven subfamilies, while the most closely related to the Drosophila TRP are members of the TRPC (Canonical) subfamily. This review focuses on a comparison between properties of Drosophila TRP, discovered in the native photoreceptor cells, and that of mammalian TRPC channels. These properties include: (i) organization of TRP channels in multimolecular signaling complexes via PDZ-containing scaffold proteins, (ii) mutations causing constitutive activity of TRP channels and cell degeneration, (iii) regulation of TRP channels by phosphorylation, and (iv) hypoxia/anoxia-activation of TRP channels. Hence, we suggest that knowledge gained from studies of Drosophila may guide studies in mammals that attempt elucidating diverse types of diseases caused by TRPC channel malfunction.

Strains of Drosophila melanogaster with resistance to the insecticides spinosyn A, spinosad, and spinetoram were produced by chemical mutagenesis. These spinosyn-resistant strains were not cross-resistant to other insecticides. The two strains that were initially characterized were subsequently found to have mutations in the gene encoding the nicotinic acetylcholine receptor (nAChR) subunit Dalpha6. Subsequently, additional spinosyn-resistant alleles were generated by chemical mutagenesis and were also found to have mutations in the gene encoding Dalpha6, providing convincing evidence that Dalpha6 is a target site for the spinosyns in D. melanogaster. Although a spinosyn-sensitive receptor could not be generated in Xenopus laevis oocytes simply by expressing Dalpha6 alone, co-expression of Dalpha6 with an additional nAChR subunit, Dalpha5, and the chaperone protein ric-3 resulted in an acetylcholine- and spinosyn-sensitive receptor with the pharmacological properties anticipated for a native nAChR.

Ca2+ modulates the visual response in both vertebrates and invertebrates. In Drosophila photoreceptors, an increase of cytoplasmic Ca2+ mimics light adaptation. Little is known regarding the mechanism, however. We explored the role of the sole Drosophila Ca2+/calmodulin-dependent protein kinase II (CaMKII) to mediate light adaptation. CaMKII has been implicated in the phosphorylation of arrestin 2 (Arr2). However, the functional significance of Arr2 phosphorylation remains debatable. We identified retinal CaMKII by anti-CaMKII antibodies and by its Ca2+-dependent autophosphorylation. Moreover, we show that phosphorylation of CaMKII is greatly enhanced by okadaic acid, and indeed, purified PP2A catalyzes the dephosphorylation of CaMKII. Significantly, we demonstrate that anti-CaMKII antibodies co-immunoprecipitate, and CaMKII fusion proteins pull down the catalytic subunit of PP2A from fly extracts, indicating that PP2A interacts with CaMKII to form a protein complex. To investigate the function of CaMKII in photoreceptors, we show that suppression of CaMKII in transgenic flies affects light adaptation and increases prolonged depolarizing afterpotential amplitude, whereas a reduced PP2A activity brings about reduced prolonged depolarizing afterpotential amplitude. Taken together, we conclude that CaMKII is involved in the negative regulation of the visual response affecting light adaptation, possibly by catalyzing phosphorylation of Arr2. Moreover, the CaMKII activity appears tightly regulated by the co-localized PP2A.

A systematic forward geneticDrosophilascreen for electroretinogram mutants lacking synaptic transients identified thefuseless(fusl) gene, which encodes a predicted eight-pass transmembrane protein in the presynaptic membrane. Nullfuslmutants display >75% reduction in evoked synaptic transmission but, conversely, an approximately threefold increase in the frequency and amplitude of spontaneous synaptic vesicle fusion events. These neurotransmission defects are rescued by a wild-typefusltransgene targeted only to the presynaptic cell, demonstrating a strictly presynaptic requirement for Fusl function. Defects in FM dye turnover at the synapse show a severely impaired exo-endo synaptic vesicle cycling pool. Consistently, ultrastructural analyses reveal accumulated vesicles arrested in clustered and docked pools at presynaptic active zones. In the absence of Fusl, calcium-dependent neurotransmitter release is dramatically compromised and there is little enhancement of synaptic efficacy with elevated external Ca2+concentrations. These defects are causally linked with severe loss of the Cacophony voltage-gated Ca2+channels, which fail to localize normally at presynaptic active zone domains in the absence of Fusl. These data indicate that Fusl regulates assembly of the presynaptic active zone Ca2+channel domains required for efficient coupling of the Ca2+influx and synaptic vesicle exocytosis during neurotransmission.

RNA interference has been widely used to reduce the quantity of the proteins encoded by the targeted genes. A constitutively active, dominant allele of trp, TrpP365, causes massive degeneration of photoreceptors through a persistent and excessive Ca2+ influx. Here we show that a substantial reduction of the TRP channel protein by RNAi in TrpP365 heterozygotes completely rescues the neuronal degeneration and significantly improves the light-elicited responses of the eye. The reduction need not be complete, suggesting that rescue of degeneration may be possible with minimal side effects arising from overdepletion of the target protein.

A large number of mutants in the norpA gene, which encodes the phospholipase C (PLC) involved in Drosophila phototransduction, is available for the investigation of the effects of specific amino acid substitutions in PLC on biochemical and electrophysiological properties of these mutants. Of the 47 norpA mutants screened for PLC protein content, all but one (H43) displayed drastically decreased amounts of the protein suggesting that almost any mutational alteration has a deleterious effect on the integrity of the protein. Three new amino acids were identified in the catalytic domains X and Y that are important for PLC catalytic activity and the generation of photoreceptor responses (ERG). One of them was found substituted in H43, which showed a low specific PLC activity, a pronounced decrease in ERG sensitivity, and a wild-type-like response termination time. The response termination times obtained from three mutants was found to be approximately inversely proportional to the amount of PLC. In addition, we show that (i) the specific PLC activity is a key factor determining the photoreceptor sensitivity; (ii) the catalytic activity and response termination are separable functions of PLC; and (iii) a mutation in the putative G alpha-interacting C2 domain causes a preferentially strong defect in latency.

Because almost everything we know about Drosophila phototransduction has come from studies based on genetic approaches, this review begins with a discussion of genetic approaches. We then present a brief overview of Drosophila phototransduction (section on Drosophila phototransduction: an overview) followed by a more detailed treatment of individual components of the transduction machinery (section on Components of the phototransduction machinery). Discussion of transduction mechanisms is presented under three headings: Mechanism(s) of channel excitation, Organization of the transduction proteins, and Regulatory mechanisms in phototransduction. Perhaps the most important unanswered question in this field is the mechanism(s) of activation and regulation of transduction channels. This question is explored in the section entitled Mechanism(s) of channel excitation. Identification of at least two of the proteins discussed was totally unexpected: the rhodopsin chaperone protein, ninaA, and the signal complex scaffold protein, INAD. They are discussed in the sections titled Requirement for a chaperone protein for Rh1 opsin, and: Formation of signaling complexes, respectively. One of the important developments in this field has been the discovery of mammalian homologs of many of the proteins identified in Drosophila. A brief discussion of the most extensively studied of these, the mammalian homologs of light-activated channel protein, trp, is presented in the section on Mammalian Homologs of trp. We conclude the review with Perspective, a brief look at the current status and the future outlook of the field.

The trp gene encodes subunits of a highly Ca(2+)-permeable class of light-activated channels of Drosophila photoreceptors. The recently characterized mutation in this gene, Trp(P365), is semidominant and causes massive degeneration of photoreceptors by making the TRP channel constitutively active. We show that a single amino acid change, Phe-550 to Ile, near the beginning of the fifth transmembrane domain of TRP channel subunits is necessary to induce, and sufficient to closely mimic, the original mutant phenotypes of Trp(P365). Hypotheses are presented as to why the amino acid residues at position 550 and its immediate vicinity might be important in influencing the regulation of the TRP channel and why the substitution of Phe for Ile at this position, in particular, could result in constitutive activity of the channel.

The trp and trpl genes are thought to encode two classes of light-activated ion channels in Drosophila . A previous report indicated that a null trpl mutant does not display any mutant phenotype. This lack of detectable mutant phenotypes made it difficult to suggest functions for the transient receptor potential-like (TRPL) channel in photoreceptor responses. Here, the properties of trpl photoreceptor responses were studied by using electroretinogram (ERG) and intracellular recording techniques in combination with light stimuli of relatively long durations. Distinct mutant phenotypes were detectable under these conditions. These consisted of a reduced sustained component, oscillations superimposed on the response, a poststimulus hyperpolarization, and altered adaptation properties to dim background light. Comparison of photoreceptor responses obtained from wild type, trp , and trpl showed that the responses obtained from the trp and trpl null mutants did not sum up to that of the wild-type response. To explain the nonlinear summation at the peak of the response, [Reuss et al. (1997)][1] proposed that Ca2+ions entering through the TRP channel modulate TRP and TRPL channel activities differentially. However, nonlinear summation was present not only at the peak but throughout the duration of response. Two lines of evidence are presented to suggest that, in addition to the interaction proposed by [Reuss et al. (1997)][1], there are other forms of interactions between TRP and TRPL channels, probably involving the channel proteins themselves. [1]: #ref-18

TheDrosophila trpgene encodes a light-activated Ca2+channel subunit, which is a prototypical member of a novel class of channel proteins. Previously identifiedtrpmutants are all recessive, loss-of-function mutants characterized by a transient receptor potential and the total or near-total loss of functional TRP protein. Although retinal degeneration does occur in these mutants, it is relatively mild and slow in onset. We report herein a new mutant,TrpP365, that does not display the transient receptor potential phenotype and is characterized by a substantial level of the TRP protein and rapid, semi-dominant degeneration of photoreceptors. We show that, in spite of its unusual phenotypes,TrpP365is atrpallele because aTrpP365transgene induces the mutant phenotype in a wild-type background, and a wild-typetrptransgene in aTrpP365background suppresses the mutant phenotype. Moreover, amino acid alterations that could cause theTrpP365phenotype are found in the transmembrane segment region of the mutant channel protein. Whole-cell recordings clarified the mechanism underlying the retinal degeneration by showing that the TRP channels ofTrpP365are constitutively active. Although several genes, when mutated, have been shown to cause retinal degeneration inDrosophila, the underlying mechanism has not been identified for any of them. The present studies provide evidence for a specific mechanism for massive degeneration of photoreceptors inDrosophila. Insofar as some human homologs of TRP are highly expressed in the brain, a similar mechanism could be a major contributor to degenerative disorders of the brain.

We have identified and analyzed a Drosophila melanogaster gene that encodes a chloride channel subunit (DrosGluCl-α) previously shown to function as a glutamategated chloride channel in an in vitro expression system. Sequence analysis of several cDNAs corresponding to the gene revealed sequence diversity in their open reading frames at seven specific sites. Site-specific A-to-G variations between cDNA and genomic sequences, consistent with RNA editing, were detected at five nucleotide positions. In addition, sequence variations among cDNA clones consistent with alternative splicing of mRNA were found at two different sites. In the 5′ region, two small adjacent exons, containing similar but distinct modular sequences, are alternatively incorporated into the mature mRNA. In the 3′ region, alternative splicing generates a variant encoding a protein with four additional amino acids just upstream of the fourth transmembrane domain. Combinations of RNA editing and alternative splicing can lead to extensive diversification of transcripts. These results give the first example of RNA editing in neurotransmitter-gated chloride channel genes or of alternative splicing in a glutamate-gated chloride channel gene of Drosophila.

Inositol phosphate signaling has been implicated in a wide variety of eukaryotic cellular processes. In Drosophila, the phototransduction cascade is mediated by a phosphoinositide-specific phospholipase C (PLC) encoded by the norpA gene. We have characterized eight norpA mutants by electroretinogram (ERG), Western, molecular, and in vitro PLC activity analyses. ERG responses of the mutants show allele-dependent reductions in amplitudes and retardation in kinetics. The mutants also exhibit allele-dependent reductions in in vitro PLC activity levels and greatly reduced or undetectable NorpA protein levels. Three carry a missense mutation and five carry a nonsense mutation within the norpA coding sequence. In missense mutants, the amino acid substitution occurs at residues highly conserved among PLCs. These substitutions reduce the levels of both the NorpA protein and the PLC activity, with the reduction in PLC activity being greater than can be accounted for simply by the reduction in protein. The effects of the mutations on the amount and activity of the protein are much greater than their effects on the ERG, suggesting an amplification of the transduction signal at the effector (NorpA) protein level. Transgenic flies were generated by germline transformation of a null norpA mutant using a P-element construct containing the wild-type norpA cDNA driven by the ninaE promoter. Transformed flies show rescue of the electrophysiological phenotype in R1-R6 photoreceptors, but not in R7 or R8. The degeneration phenotype of R1-R6 photoreceptors is also rescued.

The Drosophila ninaA gene encodes photoreceptor-specific cyclophilin thought to play a critical role in rhodopsin folding or transport during its synthesis or maturation in the most abundant subclass of photoreceptors. Cyclophilins comprise a highly conserved family of proteins which are the primary targets of the potent immunosuppressive drug, cyclosporin A (CsA), and which display peptidyl prolyl cis-trans-isomerase (PPIase) activity. In an attempt to identify mammalian cyclophilins with properties similar to the NinaA protein, a probe derived from the ninaA cDNA was used to screen bovine retinal cDNA libraries. The screen identified two major alternatively spliced forms of cDNA that would encode proteins containing a region of high homology to other cyclophilins and that are expressed specifically in the retina. These proteins represent a new class of cyclophilins with novel structural features and greatly reduced PPIase and CsA binding activities in comparison to other known cyclophilins. Tissue in situ hybridization and immunolocalization of the proteins showed that the RNA and protein products are expressed in photoreceptors as well as other retinal neurons. However, among photoreceptors, the proteins are found predominantly in cones. Thus, mammalian retinas do contain cyclophilins that are retina- specifically and photoreceptor class-preferentially expressed. The results suggest that, in cones, the main function of these proteins is, like the NinaA protein, to facilitate proper folding or intracellular transport of opsins.

We have isolated, by interspecies hybridization, two classes of Drosophila cDNA each encoding a different guanylate cyclase (GC). One of them encodes an alpha subunit homolog of soluble GC, designated DGC alpha 1, and the other encodes a receptor-type GC, designated DrGC. The dgc alpha 1 cDNA encodes a protein of 676 amino acids and maps to 99B. In situ hybridization to adult tissue sections showed that dgc alpha 1 mRNA is found mainly in the cell bodies of the optic lobe, central brain, and thoracic ganglia. The DGC alpha 1 protein was also localized primarily to the nervous system by immunocytochemical staining, consistent with results of in situ hybridization. However, no detectable expression of this protein was found in the retina. The other class of cDNA, drgc, maps to 76C and encodes a 1525-amino acid protein displaying structural features similar to other known receptor-type guanylate cyclases. However, it has a C-terminal 430 amino acid region that has no homology to any known proteins. drgc RNA is expressed at low levels throughout development and in adult heads and bodies. In situ hybridizations to adult tissue sections showed that drgc mRNA is expressed in a wide range of tissues, including the optic lobe, central brain, thoracic ganglia, digestive tract, and the oocyte.

Our objective is to present a comprehensive view of the PDA (prolonged depolarizing afterpotential)-defective Drosophila mutants, nina's and ina's, from the discussion of the PDA and the PDA-based mutant screening strategy to summaries of the knowledge gained through the studies of mutants generated using the strategy. The PDA is a component of the light-evoked photoreceptor potential that is generated when a substantial fraction of rhodopsin is photoconverted to its active form, metarhodopsin. The PDA-based mutant screening strategy was adopted to enhance the efficiency and efficacy of ERG (electroretinogram)-based screening for identifying phototransduction-defective mutants. Using this strategy, two classes of PDA-defective mutants were identified and isolated, nina and ina, each comprising multiple complementation groups. The nina mutants are characterized by allele-dependent reduction in the major rhodopsin, Rh1, whereas the ina mutants display defects in some aspects of functions related to the transduction channel, TRP (transient receptor potential). The signaling proteins that have been identified and elucidated through the studies of nina mutants include the Drosophila opsin protein (NINAE), the chaperone protein for nascent opsin (NINAA), and the multifunctional protein, NINAC, required in multiple steps of the Drosophila phototransduction cascade. Also identified by the nina mutants are some of the key enzymes involved in the biogenesis of the rhodopsin chromophore. As for the ina mutants, they led to the discovery of the scaffold protein, INAD, responsible for the nucleation of the supramolecular signaling complex. Also identified by the ina mutants is one of the key members of the signaling complex, INAC (ePKC), and two other proteins that are likely to be important, though their roles in the signaling cascade have not yet been fully elucidated. In most of these cases, the protein identified is the first member of its class to be so recognized.

Activation of PI-PLC initiates two independent branches of protein phosphorylation cascades catalyzed by either PKC or Ca 2+ /calmodulin-dependent protein kinase (CaMK). We find that phosrestin I (PRI), a Drosophila homolog of vertebrate photoreceptor arrestin, undergoes light-induced phosphorylation on a subsecond time scale which is faster than that of any other protein in vivo. We determine that a CaMK activity is responsible for in vitro PRI phosphorylation at Ser 366 in the C-terminal tryptic segment, MetLysSer(P)IIeGIuGInHisArg, in which Ser(P) represents phosphoserine 366 . We also demonstrate that Ser366 is the phosphorylation site of PRI in vivo by identifying the molecular species resulting from in-gel tryptic digestion of purified phospho-PRI using HPLC-electrospray ionization tandem quadrupole mass spectroscopy. From these data, we conclude that the CaMK pathway, not the PKC pathway, is responsible for the earliest protein phosphorylation event following activation of PI-PLC in living Drosophila photoreceptors.

Histamine is known to be the neurotransmitter of insect photoreceptors. Histamine-like immunoreactivity is also found in a number of interneurons in the central nervous system of various insects. Here, we demonstrate by immunohistochemical techniques that, in Drosophila melanogaster (Acalypterae), most or all mechanosensory neurons of imaginal hair sensilla selectively bind antibodies directed against histamine. The histamine-like staining includes the cell bodies of these neurons as well as their axons, which form prominent fibre bundles in peripheral nerves, and their terminal projections in the central neuropil of head and thoracic ganglia. The specificity of the immunostaining is demonstrated by investigating a Drosophila mutant unable to synthesize histamine. Other mechanosensory organs, such as campaniform sensilla or scolopidial organs, do not stain. In the calypteran flies, Musca and Calliphora, we find no comparable immunoreactivity associated with either hair sensilla or the nerves entering the central nervous system, observations in agreement with earlier studies on Calliphora. Thus, histamine seems to be a major mechanosensory transmitter candidate of the adult nervous system of Drosophila, but apparently not of Musca or Calliphora.

A systematic Drosophila forward genetic screen for photoreceptor synaptic transmission mutants identified no-on-and-no-off transient C (nonC) based on loss of retinal synaptic responses to light stimulation. The cloned gene encodes phosphatidylinositol-3-kinase-like kinase (PIKK) Smg1, a regulatory kinase of the nonsense-mediated decay (NMD) pathway. The Smg proteins act in an mRNA quality control surveillance mechanism to selectively degrade transcripts containing premature stop codons, thereby preventing the translation of truncated proteins with dominant-negative or deleterious gain-of-function activities. At the neuromuscular junction (NMJ) synapse, an extended allelic series of Smg1 mutants show impaired structural architecture, with decreased terminal arbor size, branching and synaptic bouton number. Functionally, loss of Smg1 results in a ~50% reduction in basal neurotransmission strength, as well as progressive transmission fatigue and greatly impaired synaptic vesicle recycling during high-frequency stimulation. Mutation of other NMD pathways genes (Upf2 and Smg6) similarly impairs neurotransmission and synaptic vesicle cycling. These findings suggest that the NMD pathway acts to regulate proper mRNA translation to safeguard synapse morphology and maintain the efficacy of synaptic function.

This review recounts the early history of Drosophila phototransduction genetics, covering the period between approximately 1966 to 1979. Early in this period, the author felt that there was an urgent need for a new approach in phototransduction research. Through inputs from a number of colleagues, he was led to consider isolating Drosophila mutants that are defective in the electroretinogram. Thanks to the efforts of dedicated associates and technical staff, by the end of this period, he was able to accumulate a large number of such mutants. Particularly important in this effort was the use of the mutant assay protocol based on the "prolonged depolarizing afterpotential." This collection of mutants formed the basis of the subsequent intensive investigations of the Drosophila phototransduction cascade by many investigators.

Five different, well-characterized mutants of the R1-6 rhodopsin gene (ninaE), which corresponds to the rod opsin gene of vertebrates, have been examined morphologically as a function of age (up to 9 weeks) to determine whether or not the photoreceptors degenerate and to assess the pattern of degeneration. Structural deterioration of R1-6 photoreceptors with age has been found in all five mutants. The structural pattern of degeneration is similar in the five mutants, but the time course of degeneration is allele dependent and varies greatly among the five, with the strongest alleles causing the fastest degeneration. The degeneration appears to be independent of either the illumination cycle to which the animals are exposed or the presence of screening pigments in the eye. Although the degeneration first appears in R1-6 photoreceptors, eventually R7/8 photoreceptors, which correspond to cones of vertebrates, are also affected. In many of these mutants, striking proliferations of membrane processes have been observed in the subrhabdomeric region of R1-6 photoreceptors. It is hypothesized that (1) this accumulation of membranes may be caused by the failure of newly synthesized membranes that are inserted into the base of microvilli to be assembled into R1-6 rhabdomeres and (2) this failure may be caused by the extremely low concentration of normal R1-6 rhodopsin in the ninaE mutants.

Optic Atrophy 1 (OPA1) is a ubiquitously expressed dynamin-like GTPase in the inner mitochondrial membrane. It plays important roles in mitochondrial fusion, apoptosis, reactive oxygen species (ROS) and ATP production. Mutations of OPA1 result in autosomal dominant optic atrophy (DOA). The molecular mechanisms by which link OPA1 mutations and DOA are not fully understood. Recently, we created a Drosophila model to study the pathogenesis of optic atrophy. Heterozygous mutation of Drosophila OPA1 (dOpa1) by P-element insertion results in no obvious morphological abnormalities, whereas homozygous mutation is embryonic lethal. In eye-specific somatic clones, homozygous mutation of dOpa1 causes rough (mispatterning) and glossy (decreased lens deposition) eye phenotypes in adult Drosophila. In humans, heterozygous mutations in OPA1 have been associated with mitochondrial dysfunction, which is predicted to affect multiple organs. In this study, we demonstrated that heterozygous dOpa1 mutation perturbs the visual function and an ERG profile of the Drosophila compound eye. We independently showed that antioxidants delayed the onset of mutant phenotypes in ERG and improved larval vision function in phototaxis assay. Furthermore, heterozygous dOpa1 mutation also caused decreased heart rate, increased heart arrhythmia, and poor tolerance to stress induced by electrical pacing. However, antioxidants had no effects on the dysfunctional heart of heterozygous dOpa1 mutants. Under stress, heterozygous dOpa1 mutations caused reduced escape response, suggesting abnormal function of the skeletal muscles. Our results suggest that heterozygous mutation of dOpa1 shows organ-specific pathogenesis and is associated with multiple organ abnormalities in an age-dependent and organ-specific manner.

Mutations in the norpA gene drastically affect the phototransduction process in Drosophila. To study the biochemical characteristics of the norpA protein and its cellular and subcellular distributions, we have generated antisera against the major gene product of norpA. The antisera recognize an eye-specific protein of 130-kDa relative molecular mass that is present in wild-type head extracts but not in those of strong norpA mutants. The protein is associated with membranes and can be extracted with high salt. Immunohistochemical analysis at the light and electron microscopic levels indicates that the protein is expressed in all adult photoreceptor cells and specifically localized within the rhabdomeres, preferentially adjacent to, but not within, the rhabdomeric membranes. The results of the present study strongly support the previous suggestion that the norpA gene encodes the major phosphoinositol-specific phospholipase C in the photoreceptors. Moreover, insofar as the rhabdomeres are specialized structures for photoreception and phototransduction, specific localization of the norpA protein within these structures, in close association with the membranes, is consistent with the proposal that it has an important role in phototransduction.

Drosophilavisual 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 threemtsheterozygotes. 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 thatinaCP209was semidominant, becauseinaCP209heterozygotes displayed abnormal light adaptation and slow deactivation. Interestingly, the deactivation defect ofinaCP209heterozygotes was rescued by themtsXE2258heterozygous background. In contrast,mtsXE2258failed to modify the severe deactivation ofnorpAP16, 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 kineticsin vivo. We thus propose that the fast deactivation of the visual response is modulated in part by the phosphorylation of INAD.

SummaryIn Drosophila, a phospholipase C-mediated signaling cascade links photoexcitation of rhodopsin to the opening of the TRP/TRPL channels. A lipid product of the cascade, diacylglycerol (DAG) and its metabolite(s), polyunsaturated fatty acids (PUFAs), have both been proposed as potential excitatory messengers. A crucial enzyme in the understanding of this process is likely to be DAG lipase (DAGL). However, DAGLs that might fulfill this role have not been previously identified in any organism. In this work, the Drosophila DAGL gene, inaE, has been identified from mutants that are defective in photoreceptor responses to light. The inaE-encoded protein isoforms show high sequence similarity to known mammalian DAG lipases, exhibit DAG lipase activity in vitro, and are highly expressed in photoreceptors. Analyses of norpA inaE double mutants and severe inaE mutants show that normal DAGL activity is required for the generation of physiologically meaningful photoreceptor responses.

SUMMARY Division of labor in honey bee colonies is influenced by the foraging gene (Amfor), which encodes a cGMP-dependent protein kinase (PKG). Amfor upregulation in the bee brain is associated with the age-related transition from working in the hive to foraging for food outside, and cGMP treatment (which increases PKG activity)causes precocious foraging. We present two lines of evidence in support of the hypothesis that Amfor affects division of labor by modulating phototaxis. We first show that a subset of worker bees involved in the removal of corpses from the hive had forager-like brain levels of Amfor brain expression despite being middle aged; age-matched food-handlers, who do not leave the hive to perform their job, had low levels of Amforexpression. This finding suggests that occupations that involve working outside the hive are associated with high levels of Amfor in brain. Secondly, foragers were much more positively phototactic than hive bees in a laboratory assay, and cGMP treatment caused a precocious onset of positive phototaxis. The cGMP effect was not due to a general increase in behavioral activity; cGMP treatment had no effect on locomotor activity under either constant darkness or a light:dark regime. The cGMP effect also was not due to changes in circadian rhythmicity; cGMP treatment had no effect on age at onset of locomotor circadian rhythmicity or the period of rhythmicity. The effects of Amfor on phototaxis are not related to peripheral processing;electroretinogram analysis revealed no effect of cGMP treatment on photoreceptor activity and no differences between untreated hive bees and foragers. The cAMP/PKA pathway does not appear to be playing a similar role to cGMP/PKG in the honey bee; cAMP treatment did not affect phototaxis and gene expression analysis revealed task-related differences only for the gene encoding the regulatory subunit, but not the catalytic subunit, of PKA. Our findings implicate one neural process associated with honey bee division of labor that can be affected by naturally occurring changes in the expression of Amfor.

Because almost everything we know about Drosophila phototransduction has come from studies based on genetic approaches, this review begins with a discussion of genetic approaches. We then present a brief overview of Drosophila phototransduction (section on Drosophila phototransduction: an overview) followed by a more detailed treatment of individual components of the transduction machinery (section on Components of the phototransduction machinery). Discussion of transduction mechanisms is presented under three headings: Mechanism(s) of channel excitation, Organization of the transduction proteins, and Regulatory mechanisms in phototransduction. Perhaps the most important unanswered question in this field is the mechanism(s) of activation and regulation of transduction channels. This question is explored in the section entitled Mechanism(s) of channel excitation. Identification of at least two of the proteins discussed was totally unexpected: the rhodopsin chaperone protein, ninaA, and the signal complex scaffold protein, INAD. They are discussed in the sections titled Requirement for a chaperone protein for Rh1 opsin, and: Formation of signaling complexes, respectively. One of the important developments in this field has been the discovery of mammalian homologs of many of the proteins identified in Drosophila. A brief discussion of the most extensively studied of these, the mammalian homologs of light-activated channel protein, trp, is presented in the section on Mammalian Homologs of trp. We conclude the review with Perspective, a brief look at the current status and the future outlook of the field.

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.

We discuss in this chapter the role of Ca2+ homeostasis in maintaining the structural integrity of photoreceptor cells in Drosophila. Both insufficient and excessive amounts of Ca2+ in photoreceptor cells appear to lead to cell degeneration. Because one of the two classes of light-sensitive channels in Drosophila photoreceptors is highly Ca2+-permeable, how well this class of channels functions can profoundly affect Ca2+ homeostasis. We will begin by reviewing Drosophila phototransduction, emphasizing what is known about the mechanism of activation of light-sensitive channels. We will then describe Ca2+ entry through light-sensitive channels and the presumed mechanisms by which too little and too much Ca2+ entry can both cause photoreceptor degeneration. We will conclude the chapter with discussions of two examples of mutations known to cause unregulated Ca2+ entry through light-sensitive channels, leading to massive photoreceptor degeneration.

The Drosophila norpA gene encodes a phosphatidylinositol-specific phospholipase C (PI-PLC) expressed predominantly in photoreceptors and involved in phototransduction. However, no direct role for a phospholipase C in vertebrate phototransduction has been identified to date. Recently, we reported the isolation and characterization of bovine cDNAs encoding PI-PLC isoforms expressed predominantly in the retina and with higher homology to the NorpA protein than to any other known PI-PLC. Here, we present evidence that the norpA-homologous bovine retinal PI-PLCs, although found in other retinal neurons as well, are found in cones but not in rods. The results suggest that the phototransduction cascade in cones may utilize phospholipase C in addition to phosphodiesterase.

We discuss in this chapter the role of Ca2+homeostasis in maintaining the structural integrity of photoreceptor cells inDrosophila.Both insufficient and excessive amounts of Ca2+in photoreceptor cells appear to lead to cell degeneration. Because one of the two classes of light-sensitive channels inDrosophilaphotoreceptors is highly Ca2+-permeable, how well this class of channels functions can profoundly affect Ca2+homeostasis. We will begin by reviewingDrosophilaphototransduction, emphasizing what is known about the mechanism of activation of light-sensitive channels. We will then describe Ca2+entry through light-sensitive channels and the presumed mechanisms by which too Iittle and too much Ca2+entry can both cause photoreceptor degeneration. We will conclude the chapter with discussions of two examples of mutations known to cause unregulated Ca2+entry through light-sensitive channels, leading to massive photoreceptor degeneration.

The trp gene of Drosophila encodes a subunit of a class of Ca 2+ -selective light-activated channels that carry the bulk of the phototransduction current. Transient receptor potential (TRP) homologs have been identified throughout animal phylogeny. In vertebrates, TRP-related channels have been suggested to mediate “store-operated Ca 2+ entry,” which is important in Ca 2+ homeostasis in a wide variety of cell types. However, the mechanisms of activation and regulation of the TRP channel are not known. Here, we report on the Drosophila inaF gene, which encodes a highly eye-enriched protein, INAF, that appears to be required for TRP channel function. A null mutation in this gene significantly reduces the amount of the TRP protein and, in addition, specifically affects the TRP channel function so as to nearly shut down its activity. The inaF mutation also dramatically suppresses the severe degeneration caused by a constitutively active mutation in the trp gene. Although the reduction in the amount of the TRP protein may contribute to these phenotypes, several lines of evidence support the view that inaF mutations also more directly affect the TRP channel function, suggesting that the INAF protein may have a regulatory role in the channel function.

In insects, histamine is found both in the peripheral nervous system (PNS) and in the CNS and is known to function as a fast neurotransmitter in photoreceptors that have been shown to express selectively thehdcgene. This gene codes for histidine decarboxylase (HDC), the enzyme for histamine synthesis. Fast neurotransmission requires the efficient removal of the transmitter from the synaptic cleft. Here we identify inDrosophilaphoto- and mechanoreceptors a histamine uptake mechanism that can restore the function of these receptors in mutants unable to synthesize histamine. When apparent null mutants for thehdcgene imbibe aqueous histamine solution or are genetically “rescued” by a transgene ubiquitously expressing histidine decarboxylase under heat-shock control, sufficient amounts of histamine selectively accumulate in photo- and mechanoreceptors to generate near-normal electrical responses in second-order visual interneurons and qualitatively to restore wild-type visual and mechanosensory behavior. This strongly supports the proposal that histamine functions as a fast neurotransmitter also in a certain class of mechanoreceptors. A set of CNS-intrinsic neurons that in the wild type contain high concentrations of histamine apparently lacks this uptake mechanism. We therefore speculate that histamine of intrinsic neurons may function as a neuromodulator rather than as a fast transmitter.

The cellular mechanism of photoreceptor cell death in inherited retinal degeneration is not yet understood. Mutations in photoreceptor-specifically expressed genes, for example the rhodopsin gene, have been identified as primary genetic defects. Using transgenic Drosophila as a model system, we investigated whether mutations in distinct amino acids which are conserved within the cytoplasmic domains throughout the rhodopsin family, namely Leu81, Asn86 or Glu271, may cause inherited retinal degeneration. Substitutions at these sites are shown to interfere with two rhodopsin functions: (i) Rhodopsin biosynthesis is partially blocked, leading to a lowered amount of functional rhodopsin in photoreceptor cells, (ii) Photoreceptor cells expressing mutant rhodopsins undergo age-dependent degeneration in a recessive manner. Degeneration starts with a deterioration of the membranes constituting the photoreceptive cell compartment. In later states of degeneration, photoreceptor cell bodies and the extracellular interphotoreceptor space are filled with remnants of the photoreceptive membrane. Retinal degeneration is interpreted to result from alterations in rhodopsin’s cytoplasmic surface which destabilize protein-protein interactions required in maintaining the architecture of the photoreceptive membrane compartment.

The role of histamine as a fast neurotransmitter of imaginal insect photoreceptors is firmly established. In adult Drosophila, histamine is also found in mechanosensory receptors of cuticular hair sensilla and in a small number of nonreceptor neurons in head and body ganglia. Here we investigate the function of histamine by immunohistochemical and behavioral analysis of mutants deficient in the hdc gene that codes for histidine decarboxylase. The allele hdcJK910 appears to be a null mutation, as histamine immunoreactivity is almost entirely eliminated. Homozygous flies are blind in various behavioral paradigms. Mutant larvae, on the other hand, show normal photokinetic responses. Thus, adult Drosophila photoreceptors most likely utilize only a single substance, histamine, as a neurotransmitter, whereas larval photoreceptors apparently employ a different transmitter. With the alleles hdcP211, hdcP217, and hdcP218, variable amounts of histamine are found in photoreceptors and mechanoreceptors, but no histamine could be detected in any of the nonreceptor neurons. These mutants show various degrees of visual and mechanosensory impairment, as determined by quantitative behavioral assays. We conclude that histamine is required for normal function of cuticular hair sensilla and for efficient grooming of the body surface. Thus, in Drosophila, histamine represents a major functional neurotransmitter for mechanosensory receptors.

The Drosophila receptor oscillation A (rosA) mutations, which cause electroretinogram (ERG) defects, including oscillations, were localized to the 24F4-25A2 region of chromosome 2L. Genomic fragments from this region, isolated from bacteriophage P1 clones, included those that detect transcriptional defects in rosA mutants in RNA blot experiments. One of these genomic fragments was used to screen a head cDNA library. The largest cDNA clone (3.6 kb) isolated was shown to rescue a rosA mutant in P element-germline transformation experiments. The ROSA protein deduced from the open reading frame in the 3.6 kb rosA cDNA is 943 amino acids long and is 36-41% identical to members of the superfamily of Na+/Cl(-)-dependent neurotransmitter transporters, with no indication of higher sequence identity to any one subgroup within the superfamily. RNA blot experiments revealed multiple transcripts in various developmental stages, the most abundant one being a 3.7 kb transcript, particularly in the adult head. Tissue in situ experiments identified the rosA transcript to be localized to many tissues, with higher levels of hybridization in the nervous system and digestive tract. The results demonstrate that the rosA gene encodes a novel Na+/Cl(-)-dependent transporter important for normal response properties of the photoreceptor.

Cyclophilins are ubiquitous and abundant proteins that exhibit peptidyl prolyl cis-trans isomerization (PPlase) activity in vitro. Their functions in vivo, however, are not well understood. Two new retinal cyclophilin isoforms, types I and II, are highly expressed in cone photoreceptors of the vertebrate retina. Type-II cyclophilin is identical to RanBP2, a large protein that binds the GTPase Ran. Here we report that two contiguous domains in RanBP2, Ran-binding domain 4 (RBD4) and cyclophilin, act in concert as a chaperone for the opsin molecule of the red/green-sensitive visual pigment of a dichromatic vertebrate. In Drosophila, the cyclophilin NinaA is expressed in all photoreceptors and is required for the expression of only a subset of opsins. The molecular basis of these photoreceptor class-specific effects and the functions of NinaA and other cyclophilins in vivo remain unclear. Unlike NinaA, which forms a stable complex with opsin from retinular cells R1-6, we find that the cyclophilin domain of RanBP2 does not bind opsin directly; rather, it augments and stabilizes the interaction between red/green (R/G) opsin and the RBD4 domain. This involves a cyclophilin-mediated modification of R/G opsin, possibly involving proline isomerization. The RBD4-cyclophilin supradomain of RanBP2, therefore, is a form of vertebrate chaperone of defined substrate specificity, which may be involved in the processing and/or transport of long-wavelength opsin in cone photoreceptor cells.

In invertebrates, a large body of evidence about the molecular mechanisms governing the visual cascade has been derived from the molecular genetic dissection of the components of the Drosophila photoreceptor machinery (1 – 5). As the best genetically characterized metazoan system, Drosophila provides an advantage in some respects over the mammalian system in that the genes can be both molecularly and genetically dissected facilitating the association of a particular gene product with its in vivo cellular function. Several of the Drosophila mutants identified with changes in the light evoked responses also lead to photoreceptor degeneration. These are of particular interest, as retinal degeneration is a major cause of inherited blindness in humans. In addition, electrophysiological studies in Limulus photoreceptors cells have also provided us with an important insight into the physiology of the invertebrate visual cascade (6).

The Drosophila norpA gene encodes a phospholipase C involved in phototransduction. However, phospholipase C apparently is not directly involved in phototransduction in vertebrate photoreceptors, although light-activated phospholipase C activity has been reported in vertebrate rod outer segments. Conserved regions of norpA cDNA were used to isolate bovine cDNAs that would encode four alternative forms of phospholipase C of the beta class that are highly homologous to the norpA protein and expressed preferentially in the retina. Two of the variants are highly unusual in that they lack much of the N-terminal region present in all other known phospholipases C. The sequence conservation between these proteins and the norpA protein is higher than that between any other known phospholipases C. GTPase sequence motifs found in proteins of the GTPase superfamily are found conserved in all four variants of the bovine retinal protein as well as the norpA protein but not in other phospholipases C. Results suggest that these proteins together with the norpA protein constitute a distinctive subfamily of phospholipases C that are closely related in structure, function, and tissue distribution. Mutations in the norpA gene, in addition to blocking phototransduction, cause light-dependent degeneration of photoreceptors. In view of the strong similarity in structure and tissue distribution, a defect in these proteins may have similar consequences in the mammalian retina.

Drosophila mutants of a single complementation group with defective on-/off-transients of the electroretinogram (ERG) were found to be deficient in synthesis of the photoreceptor transmitter, histamine, in a gene-dosage dependent manner, suggesting that the gene identified by the mutants (hdc) might be the structural gene for Drosophila histidine decarboxylase (HDC). A rat HDC cDNA was used to isolate a Drosophila homolog which shows approximately 60% sequence identity with mammalian HDCs over a region of 476 amino acids. In RNA blots, the Drosophila homolog detects four transcripts that are expressed primarily in the eye and are severely reduced in hdc mutants. The cloned Drosophila cDNA hybridizes to the 46F region of the chromosome, to which hdc mutations have been mapped, and rescues the hdc mutant phenotype in transgenic flies generated by P element-mediated germline transformation. The results thus show that the Drosophila homolog corresponds to the histidine decarboxylase gene, identified by the hdc mutants, and that mutations in the gene disrupt photoreceptor synaptic transmission.

Mitochondrial porins, also know as VDACs (voltage-dependent anion channels), play an important role in regulating energy metabolism, apoptosis, and the transport of metabolites across the mitochondrial outer membrane. So far three distinct isoforms of VDAC (VDAC1-3) have been reported in vertebrates, but their functions remain unknown. The annotation database of the Drosophila melanogaster genome sequence has identified four genes (porin, CG17137, CG17139, and CG17140) encoding different isoforms of VDACs. We identified post-translational modifications of PORIN that are specific to D. melanogaster eyes. We also identified the P-element insertion in the porin gene, porinG2294, that is homozygous viable whereas all the porin mutants previously reported are homozygous lethal at the pupal stage. The mutant does not show any defects in fly morphology, survival, and photoreceptor structure. The mutant, however, produces

Using a calcium indicator dye (arsenazo III), we detected a reversible rise in [Ca2−]i to about 2 mM in Limulus ventral photoreceptors when the cells were poisoned with the metabolic inhibitor DNP. Our results provide direct evidence for the hypothesis linking the action of metabolic inhibitors to an increase in [Ca2−]i in invertebrate photoreceptors.

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.

An intense blue light stimulus induces a prolonged depolarizing after-potential (PDA) in the peripheral retinula cells, but not in the central retinula cells, of theDrosophila ommatidia, providing the fly has been dark or red adapted and the screening pigments have been genetically removed from the compound eye. Thus, the PDA saturates only the peripheral retinula cells, allowing one to isolate and study extracellularly the summed receptor potentials (SR) of the central retinula cells (Fig. 1). The following lines of evidence support these ideas