Fusion-activated Ca ²⁺ entry via vesicular P2X ₄ receptors promotes fusion pore opening and exocytotic content release in pneumocytes

Abstract

Ca2+ 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.egulated secretion is a fundamental cellular process in many different types of eukaryotic cells, with Ca 2+ -triggered exocytosis being the key element (1-4). Multiple Ca 2+ -dependent steps have been elucidated that ultimately lead to fusion of exocytic vesicles with the plasma membrane, resulting in formation of an aqueous channel, the fusion pore, through which vesicle contents are released (5-8). Although the molecular composition of the fusion pore remains elusive, there is a general acceptance that fusion pores are not merely passive structures, but that their opening and closure are highly regulated and control, or even fine-tune, vesicle content secretion (9-14). Voltage-gated Ca 2+ channels are not present (25). After LB fusion with the plasma membrane, surfactant, a water-insoluble bulky complex, largely remains entrapped within the fused vesicles (26) in which the fusion pores behave as regulated valves or mechanical barriers for release (16,27). As a result, in vitro full content release can be delayed for minutes up to hours (28).We recently reported a "fusion-activated" Ca 2+ entry (FACE) mechanism as a phenomenon in the postfusion phase of surfactant secretion (29). Given that this Ca 2+ signal occasionally spreads throughout the cell, we speculated that it might be important for triggering...

Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels and cAMP-Dependent Modulation of Exocytosis in Cultured Rat Lactotrophs

“…1d), is in agreement with previous studies on other secretory cells, where very low amounts of the transcripts were detected (El-Kholy et al, 2007). Similarly, the subcellular localization of HCN channels in lactotrophs is consistent with previous findings (Noam et al, 2010;Fig. 1).…”

“…It has recently been shown that vesicular calcium channels regulate exocytosis by facilitating calcium entry through the fusion pore. This generates microdomains of increased [Ca 2ϩ ] i around the fused vesicles, which leads to the expansion of the fusion pore (Miklavc et al, 2011). Previous studies on lactotrophs showed a strong correlation between an increase in [Ca 2ϩ ] i and an increase exocytic activity (Vardjan et al, 2007;.…”

“…1cii). In neurons, HCN channels are also located in the cytoplasmic vesicular structures (Noam et al, 2010). To check whether HCN2-containing channels are colocalized with PRLcontaining vesicles, we immunolabeled lactotrophs with antibodies against HCN2 and PRL (Fig.…”

Actin coating and compression of fused secretory vesicles are essential for surfactant secretion: a role for Rho, formins and myosin II

“…This can involve regulating the size of the initial fusion pore or fusion pore dilation (Albillos et al, 1997;Babich et al, 2008;Obermüller et al, 2005;Perrais et al, 2004;Tsuboi et al, 2004) or active extrusion of vesicle cargos. In ATII cells, fusion pores do expand in a Ca 2+ -dependent manner (Haller et al, 2001a;Miklavc et al, 2011), with most LBs exhibiting relatively large fusion pores that are stable for long periods (Haller et al, 2001a). This is in line with recent findings that fusion pores are the subjects of stabilization and that stable fusion pore diameters depend on vesicle sizes (Jorgačevski et al, 2010).…”

“…At the beginning of the experiment, cells were stimulated with 100 mM ATP, a potent agonist for LB fusion and surfactant secretion . LB fusions with the PM were indicated by rapid decay of Lysotracker Red (LTR) fluorescence in fused vesicles (Haller et al, 2001a;Miklavc et al, 2011) (see also Fig. 1).…”