RESUMO
The Drosophila light-activated Transient Receptor Potential (TRP) channel is the founding member of a large and diverse family of channel proteins. The Drosophila TRP (dTRP) channel, which generates the electrical response to light has been investigated in a great detail two decades before the first mammalian TRP channel was discovered. Thus, dTRP is unique among members of the TRP channel superfamily because its physiological role and the enzymatic cascade underlying its activation are established. In this article we outline the research leading to elucidation of dTRP as the light activated channel and focus on a major physiological property of the dTRP channel, which is indirect activation via a cascade of enzymatic reactions. These detailed pioneering studies, based on the genetic dissection approach, revealed that light activation of the Drosophila TRP channel is mediated by G-Protein-Coupled Receptor (GPCR)-dependent enzymatic cascade, in which phospholipase C ß (PLC) is a crucial component. This physiological mechanism of Drosophila TRP channel activation was later found in mammalian TRPC channels. However, the initial studies on the mammalian TRPV1 channel indicated that it is activated directly by capsaicin, low pH and hot temperature (>42 °C). This mechanism of activation was apparently at odds with the activation mechanism of the TRPC channels in general and the Drosophila light activated TRP/TRPL channels in particular, which are target of a GPCR-activated PLC cascade. Subsequent studies have indicated that under physiological conditions TRPV1 is also target of a GPCR-activated PLC cascade in the generation of inflammatory pain. The Drosophila light-activated TRP channel is still a useful experimental paradigm because its physiological function as the light-activated channel is known, powerful genetic techniques can be applied to its further analysis, and signaling molecules involved in the activation of these channels are available.
Assuntos
Proteínas de Drosophila , Canais de Potencial de Receptor Transitório , Animais , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismo , Proteínas de Drosophila/metabolismo , Fosfolipase C beta/metabolismo , Capsaicina/metabolismo , Drosophila/fisiologia , Mamíferos/metabolismoRESUMO
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.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Células Fotorreceptoras de Invertebrados/metabolismo , Terminações Pré-Sinápticas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Proteínas de Drosophila/genética , Teste de Complementação Genética , Testes Genéticos , Transdução de Sinal Luminoso/genética , Morfogênese/genética , Junção Neuromuscular/fisiologia , Células Fotorreceptoras de Invertebrados/patologia , Terminações Pré-Sinápticas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Retina/crescimento & desenvolvimento , Retina/patologia , Deleção de Sequência/genética , Transmissão Sináptica/genética , Vesículas Sinápticas/genética , Vesículas Sinápticas/patologiaRESUMO
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.
Assuntos
Proteínas de Drosophila/deficiência , Proteínas do Olho/metabolismo , Mutação/genética , Células Fotorreceptoras de Invertebrados/fisiologia , Proteínas de Ligação ao Retinol/metabolismo , Animais , Animais Geneticamente Modificados , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Eletrorretinografia , Proteínas do Olho/genética , Testes Genéticos , Proteínas de Ligação ao Retinol/deficiência , Transdução de Sinais/genéticaRESUMO
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.
Assuntos
Drosophila melanogaster/fisiologia , Eletrofisiologia/história , Modelos Animais , Mutação , Células Fotorreceptoras de Invertebrados/fisiologia , Visão Ocular/genética , Animais , Drosophila melanogaster/genética , Eletrorretinografia/história , História do Século XXRESUMO
A systematic forward genetic Drosophila screen for electroretinogram mutants lacking synaptic transients identified the fuseless (fusl) gene, which encodes a predicted eight-pass transmembrane protein in the presynaptic membrane. Null fusl mutants 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-type fusl transgene 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 Ca(2+) concentrations. These defects are causally linked with severe loss of the Cacophony voltage-gated Ca(2+) 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 Ca(2+) channel domains required for efficient coupling of the Ca(2+) influx and synaptic vesicle exocytosis during neurotransmission.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Drosophila/fisiologia , Exocitose/fisiologia , Terminações Pré-Sinápticas/metabolismo , Vesículas Sinápticas/fisiologia , Animais , Animais Geneticamente Modificados , Membrana Celular/fisiologia , Drosophila , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Eletrorretinografia/métodos , Embrião não Mamífero , Potenciais Evocados Visuais/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana/fisiologia , Análise em Microsséries , Mutação/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/fisiologia , Junção Neuromuscular/ultraestrutura , Técnicas de Patch-Clamp/métodos , Estimulação Luminosa/métodos , Terminações Pré-Sinápticas/ultraestrutura , Interferência de RNA/fisiologia , Transmissão Sináptica/fisiologia , Visão Ocular/genética , Vias Visuais/anatomia & histologia , Vias Visuais/metabolismoRESUMO
Drosophila visual signaling, a G-protein-coupled phospholipase Cbeta (PLCbeta)-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) (PLCbeta). 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.
Assuntos
Proteínas de Drosophila/fisiologia , Células Fotorreceptoras de Invertebrados/fisiologia , Proteína Fosfatase 2/fisiologia , Transdução de Sinais/fisiologia , Percepção Visual/fisiologia , Potenciais de Ação/fisiologia , Adaptação Ocular/fisiologia , Sequência de Aminoácidos , Animais , Catálise , Drosophila , Proteínas de Drosophila/metabolismo , Proteínas do Olho/metabolismo , Proteínas do Olho/fisiologia , Feminino , Masculino , Dados de Sequência Molecular , FosforilaçãoRESUMO
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.
Assuntos
Canais de Cálcio/metabolismo , Drosophila melanogaster/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Interferência de RNA , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Drosophila melanogaster/citologia , Eletrorretinografia , Humanos , Mutação , Células Fotorreceptoras de Invertebrados/citologia , Células Fotorreceptoras de Invertebrados/metabolismo , Canais de Cátion TRPC , TransgenesRESUMO
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.
Assuntos
Cálcio/metabolismo , Proteínas de Drosophila , Luz , Células Fotorreceptoras de Invertebrados/fisiologia , Animais , Canais de Cálcio/genética , Canais de Cálcio/fisiologia , Drosophila , Proteínas de Insetos/genética , Proteínas de Insetos/fisiologia , Microscopia Eletrônica , Mutação , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/ultraestrutura , Fatores de Tempo , Canais de Potencial de Receptor TransitórioRESUMO
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.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Resistência a Medicamentos/genética , Inseticidas/farmacologia , Macrolídeos/farmacologia , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Animais , Chaperoninas/genética , Chaperoninas/metabolismo , Drosophila melanogaster , Combinação de Medicamentos , Resistência a Medicamentos/efeitos dos fármacos , Expressão Gênica , Mutação , Oócitos/citologia , Oócitos/metabolismo , Xenopus laevisRESUMO
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.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Heterozigoto , Proteínas de Membrana/genética , Mutação , Animais , Antioxidantes/farmacologia , Eletrorretinografia , Modelos Animais , Visão Ocular/efeitos dos fármacosRESUMO
Ca(2+) modulates the visual response in both vertebrates and invertebrates. In Drosophila photoreceptors, an increase of cytoplasmic Ca(2+) mimics light adaptation. Little is known regarding the mechanism, however. We explored the role of the sole Drosophila Ca(2+)/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 Ca(2+)-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.
Assuntos
Arrestinas/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/química , Catálise , Domínio Catalítico , Citosol/metabolismo , Drosophila , Eletrorretinografia/métodos , Heterozigoto , Fenótipo , Fosforilação , Estrutura Terciária de ProteínaRESUMO
In 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.
Assuntos
Proteínas de Drosophila/metabolismo , Lipase Lipoproteica/metabolismo , Células Fotorreceptoras de Invertebrados/fisiologia , Canais de Cátion TRPC/fisiologia , Animais , Drosophila , Proteínas de Drosophila/genética , Ativação Enzimática/fisiologia , Lipase Lipoproteica/genética , Estimulação Luminosa/métodosRESUMO
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, porin(G2294), 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 <10% of the normal level of wild-type (WT) porin transcripts and 16.5% of WT level of the PORIN protein. The P-element insertion affects only the expression of Class I transcript but not Class II transcript of the porin gene. Unlike in WT, the mutant displays an ERG (electroretinogram) that is not maintained during a prolonged light stimulus. The revertant obtained from remobilization of the P-element in the mutant produces the WT level of porin transcripts and PORIN protein, and shows a normal ERG response. Our data suggest that the PORIN protein is important in maintaining a photoreceptor response during prolonged stimulation.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Mitocôndrias/genética , Mutação/genética , Células Fotorreceptoras de Invertebrados/fisiologia , Visão Ocular/genética , Canais de Ânion Dependentes de Voltagem/genética , Potenciais de Ação/genética , Animais , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Eletrorretinografia , Metabolismo Energético/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Estimulação Luminosa , Isoformas de Proteínas/genética , Processamento de Proteína Pós-Traducional/genética , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
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.
Assuntos
Drosophila melanogaster/genética , Transdução de Sinais , Visão Ocular/fisiologia , Animais , Drosophila melanogaster/fisiologia , Potenciais Evocados VisuaisRESUMO
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.
Assuntos
Anestésicos Inalatórios/farmacologia , Canais de Cloreto/fisiologia , Drosophila melanogaster/efeitos dos fármacos , Histamina/fisiologia , Ivermectina/análogos & derivados , Animais , Canais de Cloreto/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Resistência a Medicamentos , Éter/farmacologia , Feminino , Ativação do Canal Iônico/fisiologia , Ivermectina/farmacologia , Masculino , Mutação , FenótipoRESUMO
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.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Mutação/genética , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Proteínas de Drosophila/deficiência , Drosophila melanogaster/enzimologia , Eletrofisiologia , Eletrorretinografia , Ativação Enzimática/genética , Dados de Sequência Molecular , Fosfolipase C beta , Células Fotorreceptoras de Invertebrados/fisiologia , Tempo de Reação/genética , Homologia de Sequência de Aminoácidos , Fosfolipases Tipo C/deficiênciaRESUMO
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.
Assuntos
Canais de Cálcio/química , Células Fotorreceptoras/patologia , Degeneração Retiniana/etiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Canais de Cálcio/fisiologia , Drosophila , Microscopia Confocal , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Relação Estrutura-Atividade , Canais de Cátion TRPCRESUMO
By screening Drosophila mutants that are potentially defective in synaptic transmission between photoreceptors and their target laminar neurons, L1/L2, (lack of electroretinogram on/off transients), we identified ort as a candidate gene encoding a histamine receptor subunit on L1/L2. We provide evidence that the ort gene corresponds to CG7411 (referred to as hclA), identified in the Drosophila genome data base, by P-element-mediated germ line rescue of the ort phenotype using cloned hclA cDNA and by showing that several ort mutants exhibit alterations in hclA regulatory or coding sequences and/or allele-dependent reductions in hclA transcript levels. Other workers have shown that hclA, when expressed in Xenopus oocytes, forms histamine-sensitive chloride channels. However, the connection between these chloride channels and photoreceptor synaptic transmission was not established. We show unequivocally that hclA-encoded channels are the channels required in photoreceptor synaptic transmission by 1) establishing the identity between hclA and ort and 2) showing that ort mutants are defective in photoreceptor synaptic transmission. Moreover, the present work shows that this function of the HCLA (ORT) protein is its native function in vivo.