RESUMO
Drosophila phototransduction occurs in light-sensitive microvilli arranged in a longitudinal structure of the photoreceptor, termed the rhabdomere. Rhodopsin (Rh), isomerized by light, couples to G-protein, which activates phospholipase C (PLC), which in turn cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG), inositol trisphosphate and H+. This pathway opens the light-dependent channels, transient receptor potential (TRP) and transient receptor potential like (TRPL). PLC and TRP are held together in a protein assembly by the scaffold protein INAD. We report that the channels can be photoactivated in on-cell rhabdomeric patches and in excised patches by DAG. In excised patches, addition of PLC-activator, m-3M3FBS, or G-protein-activator, GTP-γ-S, opened TRP. These reagents were ineffective in PLC-mutant norpA and in the presence of PLC inhibitor U17322. However, DAG activated TRP even when PLC was pharmacologically or mutationally suppressed. These observations indicate that PLC, G-protein, and TRP were retained functional in these patches. DAG also activated TRP in the protein kinase C (PKC) mutant, inaC, excluding the possibility that PKC could mediate DAG-dependent TRP activation. Labeling diacylglycerol kinase (DGK) by fusion of fluorescent mCherry (mCherry-DGK) indicates that DGK, which returns DAG to dark levels, is highly expressed in the microvilli. In excised patches, TRP channels could be light-activated in the presence of GTP, which is required for G-protein activation. The evidence indicates that the proteins necessary for phototransduction are retained functionally after excision and that DAG is necessary and sufficient for TRP opening. This work opens up unique possibilities for studying, in sub-microscopic native membrane patches, the ubiquitous phosphoinositide signaling pathway and its regulatory mechanisms in unprecedented detail.
Assuntos
Ativação do Canal Iônico/efeitos da radiação , Luz , Microvilosidades/metabolismo , Microvilosidades/efeitos da radiação , Células Fotorreceptoras de Invertebrados/citologia , Canais de Potencial de Receptor Transitório/metabolismo , Canais de Potencial de Receptor Transitório/efeitos da radiação , Animais , Diacilglicerol Quinase/biossíntese , Diglicerídeos/farmacologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/efeitos da radiação , Drosophila melanogaster , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Proteína Quinase C/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sulfonamidas/farmacologia , Canais de Potencial de Receptor Transitório/isolamento & purificação , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/genéticaRESUMO
The intercellular transport of lactate is crucial for the astrocyte-to-neuron lactate shuttle (ANLS), a model of brain energetics according to which neurons are fueled by astrocytic lactate. In this study we show that the Drosophila chaski gene encodes a monocarboxylate transporter protein (MCT/SLC16A) which functions as a lactate/pyruvate transporter, as demonstrated by heterologous expression in mammalian cell culture using a genetically encoded FRET nanosensor. chaski expression is prominent in the Drosophila central nervous system and it is particularly enriched in glia over neurons. chaski mutants exhibit defects in a high energy demanding process such as synaptic transmission, as well as in locomotion and survival under nutritional stress. Remarkably, locomotion and survival under nutritional stress defects are restored by chaski expression in glia cells. Our findings are consistent with a major role for intercellular lactate shuttling in the brain metabolism of Drosophila.
Assuntos
Proteínas de Membrana Transportadoras/genética , Transportadores de Ácidos Monocarboxílicos/genética , Neuroglia/metabolismo , Estresse Fisiológico , Sequência de Aminoácidos , Animais , Linhagem Celular , Sobrevivência Celular , Pareamento Cromossômico , Drosophila , Humanos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Transportadores de Ácidos Monocarboxílicos/química , Transportadores de Ácidos Monocarboxílicos/metabolismo , Neurônios/metabolismoRESUMO
BACKGROUND: CLCA is a family of metalloproteases that regulate Ca2+-activated Cl- fluxes in epithelial tissues. In HEK293 cells, CLCA1 promotes membrane expression of an endogenous Anoctamin 1 (ANO1, also termed TMEM16A)-dependent Ca2+-activated Cl- current. Motif architecture similarity with CLCA2, 3 and 4 suggested that they have similar functions. We previously detected the isoform CLCA4L in rat olfactory sensory neurons, where Anoctamin 2 is the principal chemotransduction Ca2+-activated Cl- channel. We explored the possibility that this protein plays a role in odor transduction. RESULTS: We cloned and expressed CLCA4L from rat olfactory epithelium in HEK293 cells. In the transfected HEK293 cells we measured a Cl--selective Ca2+-activated current, blocked by niflumic acid, not present in the non-transfected cells. Thus, CLCA4L mimics the CLCA1 current on its ability to induce the ANO1-dependent Ca2+-activated Cl- current endogenous to these cells. By immunocytochemistry, a CLCA protein, presumably CLCA4L, was detected in the cilia of olfactory sensory neurons co-expressing with ANO2. CONCLUSION: These findings suggests that a CLCA isoform, namely CLCA4L, expressed in OSN cilia, might have a regulatory function over the ANO2-dependent Ca2+-activated Cl- channel involved in odor transduction.
Assuntos
Cálcio/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Sequência de Aminoácidos , Animais , Anoctaminas/metabolismo , Canais de Cloreto/genética , Cílios/metabolismo , Clonagem Molecular , Células HEK293 , Humanos , Íons/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Alinhamento de Sequência , TransfecçãoRESUMO
Giardia intestinalis is a common gastrointestinal protozoan worldwide, but its effects on childhood growth in developing countries are not clearly understood. The authors aimed to describe its effects on child growth. They followed 220 Peruvian children daily for diarrhea, weekly for stool samples, and monthly for anthropometry. The authors modeled the effect of nutritional status on the risk of Giardia infection and the risk of diarrhea attributable to Giardia using negative binomial regression. They modeled the effects of Giardia infection on growth using linear regression, with 85% of children becoming infected with Giardia and 87% of these becoming reinfected. In multivariable analysis, the risk of Giardia infection did not vary with weight for age (relative risk = 1.00, 95% confidence interval: 0.89, 1.12) or height for age (relative risk = 0.92, 95% confidence interval: 0.82, 1.04). Giardiasis did not affect growth at 1 or 2 months following the first infection at any age interval. The longitudinal prevalence of Giardia between 6 and 24 months of age was not associated with height gain in that interval (p = 0.981). Giardia was not associated with an increased risk of diarrhea at any age interval. Study results question the importance of Giardia as a childhood pathogen in developing countries where giardiasis is hyperendemic.
Assuntos
Diarreia Infantil/parasitologia , Giardia lamblia/isolamento & purificação , Giardíase/complicações , Transtornos do Crescimento/etiologia , Vigilância da População/métodos , Animais , Pré-Escolar , Diarreia Infantil/complicações , Diarreia Infantil/epidemiologia , Feminino , Giardíase/epidemiologia , Transtornos do Crescimento/classificação , Transtornos do Crescimento/epidemiologia , Humanos , Lactente , Recém-Nascido , Modelos Lineares , Estudos Longitudinais , Masculino , Peru/epidemiologia , PrevalênciaRESUMO
Odour-mediated signal transduction is a complex process that occurs in the cilia of olfactory sensory neurons. To gain insight in to the molecular organization of the odour transduction machinery, we developed a procedure to purify olfactory cilia membranes by differential centrifugation of rat olfactory epithelium extracts. We tested whether known scaffolding proteins that might participate in the assembly of the complex chemotransduction apparatus are present in the purified membrane fraction. Utilizing immunoblotting and immunohistochemistry, we show that the multidomain scaffolding proteins ProSAP/Shanks and calcium/calmodulin-dependent serine protein kinase CASK are present in the olfactory cilia. Ion channels involved in chemotransduction could be reconstituted into planar lipid bilayers for electrophysiological recordings. Our procedure should allow the identification of further chemotransduction-related proteins.