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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Fusion-activated Ca~(2+) entry via vesicular P2X_4 receptors promotes fusion pore opening and exocytotic content release in pneumocytes
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Fusion-activated Ca~(2+) entry via vesicular P2X_4 receptors promotes fusion pore opening and exocytotic content release in pneumocytes

机译：Fusion-activated Ca~(2+) entry via vesicular P2X_4 receptors promotes fusion pore opening and exocytotic content release in pneumocytes

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摘要

Ca~(2+) is considered a key element in multiple steps during regulated exocytosis. During the postfusion phase, an elevated cytoplasmic Ca~(2+) concentration (Ca~(2+))_c leads to fusion pore dilation. In neurons and neuroendocrine cells, this results from activation of voltage- gated Ca~(2+) channels in the plasma membrane. However, these channels are activated in the prefusion stage, and little is known about Ca~(2+) entry mechanisms during the postfusion stage. This may be particularly important for slow and nonexcitable secretory cells. We recently described a "fusion-activated" Ca~(2+) entry (FACE) mechanism in alveolar type II (ATII) epithelial cells. FACE follows initial fusion pore opening with a delay of 200-500 ms. The site, molecular mechanisms, and functions of this mechanism remain unknown, however. Here we show that vesicle-associated Ca~(2+) channels mediate FACE. Using RT-PCR, Western blot analysis, and immunofluorescence, we demonstrate that P2X_4 receptors are expressed on exocytotic vesicles known as lamellar bodies (LBs). Electrophysiological, pharmacological, and genetic data confirm that FACE is mediated via these vesicular P2X_4 receptors. Furthermore, analysis of fluorophore diffusion into and out of individual vesicles after exocytotic fusion provides evidence that FACE regulates postfusion events of LB exocytosis via P2X_4. Fusion pore dilation was clearly correlated with the amplitude of FACE, and content release from fused LBs was accelerated in fusions followed by FACE. Based on these findings, we propose a model for regulation of the exocytotic postfusion phase in nonexcitable cells in which Ca~(2+) influx via vesicular Ca~(2+) channels regulates fusion pore expansion and vesicle content release.

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