RESUMO
The absence of the chloride channel CLC-3 in Clcn3(-/-) mice results in hippocampal degeneration with a distinct temporal-spatial sequence that resembles neuronal loss in temporal lobe epilepsy. We examined how the loss of CLC-3 might affect GABAergic synaptic transmission in the hippocampus. An electrophysiological study of synaptic function in hippocampal slices taken from Clcn3(-/-) mice before the onset of neurodegeneration revealed a substantial decrease in the amplitude and frequency of miniature inhibitory postsynaptic currents compared with those in wild-type slices. We found that CLC-3 colocalized with the vesicular GABA transporter VGAT in the CA1 region of the hippocampus. Acidification of inhibitory synaptic vesicles induced by Cl(-) showed a marked dependence on CLC-3 expression. The decrease in inhibitory transmission in Clcn3(-/-) mice suggests that the neurotransmitter loading of synaptic vesicles was reduced, which we attribute to defective vesicular acidification. Our observations extend the role of Cl(-) in inhibitory transmission from that of a postsynaptic permeant species to a presynaptic regulatory element.
Assuntos
Canais de Cloreto/fisiologia , Hipocampo/metabolismo , Inibição Neural/fisiologia , Terminações Pré-Sinápticas/metabolismo , Transmissão Sináptica/genética , Ácido gama-Aminobutírico/fisiologia , Animais , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/ultraestrutura , Canais de Cloreto/deficiência , Canais de Cloreto/genética , Hipocampo/ultraestrutura , Concentração de Íons de Hidrogênio , Potenciais Pós-Sinápticos Inibidores/genética , Camundongos , Camundongos Knockout , Inibição Neural/genética , Técnicas de Cultura de Órgãos , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Wistar , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/fisiologiaRESUMO
Alveolar macrophages (AMs) play a major role in host defense against microbial infections in the lung. To perform this function, these cells must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Recently, we demonstrated that murine alveolar macrophages employ the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel as a determinant in lysosomal acidification (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Lysosomes and phagosomes in murine cftr(-/-) AMs failed to acidify, and the cells were deficient in bacterial killing compared with wild type controls. Cystic fibrosis is caused by mutations in CFTR and is characterized by chronic lung infections. The information about relationships between the CFTR genotype and the disease phenotype is scarce both on the organismal and cellular level. The most common disease-causing mutation, DeltaF508, is found in 70% of patients with cystic fibrosis. The mutant protein fails to fold properly and is targeted for proteosomal degradation. G551D, the second most common mutation, causes loss of function of the protein at the plasma membrane. In this study, we have investigated the impact of CFTR DeltaF508 and G551D on a set of core intracellular functions, including organellar acidification, granule secretion, and microbicidal activity in the AM. Utilizing primary AMs from wild type, cftr(-/-), as well as mutant mice, we show a tight correlation between CFTR genotype and levels of lysosomal acidification, bacterial killing, and agonist-induced secretory responses, all of which would be expected to contribute to a significant impact on microbial clearance in the lung.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/mortalidade , Lisossomos/metabolismo , Macrófagos Alveolares/metabolismo , Fagossomos/metabolismo , Animais , Linhagem Celular , Fibrose Cística/genética , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Lisossomos/genética , Lisossomos/patologia , Macrófagos Alveolares/patologia , Camundongos , Camundongos Endogâmicos CFTR , Camundongos Knockout , Mutação , Fagossomos/genética , Fagossomos/patologiaRESUMO
Insulin secretion from pancreatic beta cells is dependent on maturation and acidification of the secretory granule, processes necessary for prohormone convertase cleavage of proinsulin. Previous studies in isolated beta cells revealed that acidification may be dependent on the granule membrane chloride channel ClC-3, in a step permissive for a regulated secretory response. In this study, immuno-EM of beta cells revealed colocalization of ClC-3 and insulin on secretory granules. Clcn3(-/-) mice as well as isolated islets demonstrate impaired insulin secretion; Clcn3(-/-) beta cells are defective in regulated insulin exocytosis and granular acidification. Increased amounts of proinsulin were found in the majority of secretory granules in the Clcn3(-/-) mice, while in Clcn3(+/+) cells, proinsulin was confined to the immature secretory granules. These results demonstrate that in pancreatic beta cells, chloride channels, specifically ClC-3, are localized on insulin granules and play a role in insulin processing as well as insulin secretion through regulation of granular acidification.
