AIM It is widely accepted that the exocytosis of synaptic and secretory vesicles is triggered by Ca2+ entry through voltage-dependent Ca2+ channels. However, there is evidence of an alternative mode of exocytosis induced by membrane depolarization but lacking Ca2+ current and intracellular Ca2+ increase. In this work we investigated if such a mechanism contributes to secretory vesicle exocytosis in mouse chromaffin cells. METHODS Exocytosis was evaluated by patch-clamp membrane capacitance measurements, carbon fibre amperometry and TIRF. Cytosolic Ca2+ was estimated using epifluorescence microscopy and fluo-8 (salt form). RESULTS Cells stimulated by brief depolatizations in absence of extracellular Ca+2 show moderate but consistent exocytosis, even in presence of high cytosolic BAPTA concentration and pharmacological inhibition of Ca+2 release from intracellular stores. This exocytosis is tightly dependent on membrane potential, is inhibited by neurotoxin Bont-B (cleaves the v-SNARE synaptobrevin), is very fast (saturates with time constant <10 ms), it is followed by a fast endocytosis sensitive to the application of an anti-dynamin monoclonal antibody, and recovers after depletion in <5 s. Finally, this exocytosis was inhibited by: (i) ω-agatoxin IVA (blocks P/Q-type Ca2+ channel gating), (ii) in cells from knock-out P/Q-type Ca2+ channel mice, and (iii) transfection of free synprint peptide (interferes in P/Q channel-exocytic proteins association). CONCLUSION We demonstrated that Ca2+ -independent and voltage-dependent exocytosis is present in chromaffin cells. This process is tightly coupled to membrane depolarization, and is able to support secretion during action potentials at low basal rates. P/Q-type Ca2+ channels can operate as voltage sensors of this process.

中文翻译：

---

小鼠嗜铬细胞中Ca2 +依赖性和电压依赖性胞吐作用。

目的众所周知，突触小泡和分泌小泡的胞吐作用是由Ca2 +通过依赖电压的Ca2 +通道进入而触发的。但是，有证据表明，通过膜去极化诱导胞吐的另一种模式，但缺乏Ca2 +电流和细胞内Ca2 +增加。在这项工作中，我们调查了这种机制是否有助于小鼠嗜铬细胞中的分泌性小泡胞吐作用。方法通过膜片钳膜电容测量，碳纤维安培法和TIRF评估胞吐作用。使用落射荧光显微镜和fluo-8（盐形式）评估胞浆中的Ca2 +。结果在缺乏细胞外Ca + 2的情况下，通过短暂的去极化作用刺激的细胞显示出中等但一致的胞吐作用，即使在细胞质BAPTA浓度高和细胞内Ca + 2释放的药理抑制作用下也是如此。这种胞吐作用紧密依赖于膜电位，被神经毒素Bont-B抑制（裂解v-SNARE突触素），非常快（以时间常数<10 ms饱和），随后发生快速内吞作用，对内毒素的应用敏感一种抗动力素单克隆抗体，耗时<5 s即可恢复。最后，这种胞吐作用受到以下抑制：（i）ω-抗毒素IVA（阻断P / Q型Ca2 +通道门控），（ii）敲除P / Q型Ca2 +通道小鼠的细胞和（iii）转染突触肽的合成（干扰P / Q通道-胞外蛋白的缔合）。结论我们证明了在嗜铬细胞中存在不依赖Ca 2+和依赖电压的胞吐作用。该过程与膜去极化紧密耦合，并且能够以低基础速率在动作电位期间支持分泌。P / Q型Ca2 +通道可以用作此过程的电压传感器。