RESUMO
SNAREs (soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptors) are ubiquitous proteins that direct vesicular trafficking and exocytosis. In neurons, SNAREs act to mediate release of neurotransmitters, which is a carefully regulated process. Calcium influx has long been shown to be the key trigger of release. However, calcium alone cannot regulate the degree of vesicle content release. For example, only a limited number of docked vesicles releases neurotransmitters when calcium entry occurs; this suggests that exocytosis is regulated by other factors besides calcium influx. Regulation of the degree of release is best explained by looking at the many enzymatic proteins that interact with the SNARE complex. These proteins have been hypothesized to regulate the formation, stability, or disassembly of the SNARE complex and therefore may regulate neurotransmitter release. One group of enzymatic regulators is the protein kinases. These proteins phosphorylate sites on both SNARE proteins and proteins that interact with SNARE proteins. Recent research has identified some of the specific effects that phosphorylation (or dephosphorylation) at these sites can produce. Additionally, palmitoylation of SNAP-25, regulates the localization, and hence activity of this key SNARE protein. This review focuses on the location and effects of phosphorylation on SNARE regulation.
Assuntos
Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Proteínas SNARE/metabolismo , Sítios de Ligação , Exocitose/fisiologia , Proteínas Munc18/metabolismo , Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Fosforilação , Ligação Proteica , Proteínas Quinases/metabolismo , Proteínas SNARE/química , Proteína 25 Associada a Sinaptossoma/metabolismo , Sinaptotagminas/metabolismo , Proteínas de Transporte Vesicular/fisiologiaRESUMO
This review summarizes the types of evidence that can be invoked in order to demonstrate that a virally encoded protein possesses ion channel activity that is intrinsic to the life cycle of the virus. Ion channel activity has been proposed to be a key step in the life cycle of influenza virus, and the protein responsible for this activity has been proposed to be the M2 protein encoded by the virus. This review contrasts the evidence supporting the conclusion that the A/M2 protein of influenza A virus has intrinsic ion channel activity with the evidence that the 3AB protein encoded by the human rhinovirus possesses intrinsic ion channel activity.
Assuntos
Canais Iônicos/química , Orthomyxoviridae/fisiologia , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/fisiologia , Animais , Relação Dose-Resposta a Droga , Humanos , Íons , Modelos Biológicos , Rhinovirus/genética , Fatores de TempoRESUMO
BACKGROUND/AIM: Pancreatic acinar cells are involved in the secretion of digestive enzymes. Digestive enzymes in pancreatic acinar cells are stored in membrane-bound secretory vesicles called zymogen granules (ZGs). The swelling of ZGs is implicated in the regulation of the expulsion of intravesicular contents during secretion. The molecular mechanism of ZG swelling has been previously elucidated. It has been further demonstrated that the water channel aquaporin-1, the potassium channel IRK-8, and the chloride channel CLC-2, are present in the ZG membrane and involved in ZG swelling. However, a direct measurement of these ion channels at the ZG membrane in intact ZGs had not been performed. The aim of this study was to investigate the electrical activity of single ZGs and verify the types of channels found within their membrane. METHODS: ZGs from pancreatic acinar cells were isolated from the pancreas of Sprague-Dawley rats. Direct measurements of whole vesicle currents, in the presence and absence of ion channel blockers (quinine, glyburide and DIDS), were recorded following successful patching of single ZGs. CONCLUSION: In this study, we were able, for the first time, to patch single ZGs and study ion channels in their membrane. We were able to record currents across the ZG membrane and, utilizing ion channel blockers, confirm the presence of the chloride channels CLC-2 and the potassium channel IRK-8 (Kir6.1), and additionally demonstrate the presence of a second chloride channel CLC-3.
Assuntos
Precursores Enzimáticos/metabolismo , Canais Iônicos/metabolismo , Pâncreas/enzimologia , Técnicas de Patch-Clamp/métodos , Vesículas Secretórias/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Aquaporina 1 , Aquaporinas/metabolismo , Canais de Cloro CLC-2 , Canais de Cloreto/metabolismo , Canais Iônicos/antagonistas & inibidores , Canais KATP , Masculino , Microscopia de Força Atômica , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Ratos , Ratos Sprague-Dawley , Vesículas Secretórias/efeitos dos fármacosRESUMO
The involvement of secretory vesicle swelling has been proposed in secretion; however, little is known about its role. Using both the pancreatic acinar cell and neuronal model, we show secretory vesicle swelling in live cells. Our study reveals that vesicle swelling potentiates its fusion at the cell plasma membrane, and is required for expulsion of intravesicular contents. Since the extent of swelling is directly proportional to the amount of vesicular contents expelled, this provides cells with the ability to regulate release of secretory products. These direct observations of the requirement of secretory vesicle swelling in secretion, provides an understanding of the appearance of partially empty vesicles following the process.