Fusion-Activated Ca2+ Entry: An Active Zone'' of Elevated Ca2+ during the Postfusion Stage of Lamellar Body Exocytosis in Rat Type II Pneumocytes

Background: Ca2+ is essential for vesicle fusion with the plasma membrane in virtually all types of regulated exocytoses. However, in contrast to the well-known effects of a high cytoplasmic Ca2+ concentration ([Ca2+](c)) in the prefusion phase, the occurrence and significance of Ca2+ signals in the postfusion phase have not been described before. Methodology/Principal Findings: We studied isolated rat alveolar type II cells using previously developed imaging techniques. These cells release pulmonary surfactant, a complex of lipids and proteins, from secretory vesicles (lamellar bodies) in an exceptionally slow, Ca2+- and actin-dependent process. Measurements of fusion pore formation by darkfield scattered light intensity decrease or FM 1-43 fluorescence intensity increase were combined with analysis of [Ca2+](c) by ratiometric Fura-2 or Fluo-4 fluorescence measurements. We found that the majority of single lamellar body fusion events were followed by a transient (t(1/2) of decay - 3.2 s) rise of localized [Ca2+](c) originating at the site of lamellar body fusion. [Ca2+](c) increase followed with a delay of similar to 0.2-0.5 s (method-dependent) and in the majority of cases this signal propagated throughout the cell (at similar to 10 mm/s). Removal of Ca2+ from, or addition of Ni2+ to the extracellular solution, strongly inhibited these [Ca2+](c) transients, whereas Ca2+ store depletion with thapsigargin had no effect. Actin-GFP fluorescence around fused LBs increased several seconds after the rise of [Ca2+](c). Both effects were reduced by the non-specific Ca2+ channel blocker SKF96365. Conclusions/Significance: Fusion-activated Ca2+ entry (FACE) is a new mechanism that leads to [Ca2+](c) transients at the site of vesicle fusion. Substantial evidence from this and previous studies indicates that fusion-activated Ca2+ entry enhances localized surfactant release from type II cells, but it may also play a role for compensatory endocytosis and other cellular functions.