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Priming of insulin secretory granules for release requires intragranular acidification and depends on vesicular Cl(-)-fluxes, but the identity of the chloride transporter/ion channel involved is unknown. We tested the hypothesis that the chloride transport protein ClC-3 fulfills these actions in pancreatic beta cells. In ClC-3(-/-) mice, insulin secretion evoked by membrane depolarization (high extracellular K(+), sulfonylureas), or glucose was >60% reduced compared to WT animals. This effect was mirrored by a approximately 80% reduction in depolarization-evoked beta cell exocytosis (monitored as increases in cell capacitance) in single ClC-3(-/-) beta cells, as well as a 44% reduction in proton transport across the granule membrane. ClC-3 expression in the insulin granule was demonstrated by immunoblotting, immunostaining, and negative immuno-EM in a high-purification fraction of large dense-core vesicles (LDCVs) obtained by phogrin-EGFP labeling. The data establish the importance of granular Cl(-) fluxes in granule priming and provide direct evidence for the involvement of ClC-3 in the process.

Original publication

DOI

10.1016/j.cmet.2009.08.011

Type

Journal article

Journal

Cell Metab

Publication Date

10/2009

Volume

10

Pages

309 - 315

Keywords

Animals, Calcium, Chloride Channels, Chlorides, Cytoplasmic Granules, Glucagon-Like Peptide 1, Glucose, Insulin, Insulin-Secreting Cells, Mice, Mice, Knockout, RNA Interference, Sulfonylurea Compounds