Instituto Teófilo Hernando, IIS del Hospital Universitario de Princesa, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain.
Am J Physiol Cell Physiol. 2011 Jul;301(1):C86-98. doi: 10.1152/ajpcell.00440.2010. Epub 2011 Mar 30.
Calcium (Ca(2+))-dependent endocytosis has been linked to preferential Ca(2+) entry through the L-type (α(1D), Ca(V)1.3) of voltage-dependent Ca(2+) channels (VDCCs). Considering that the Ca(2+)-dependent exocytotic release of neurotransmitters is mostly triggered by Ca(2+) entry through N-(α(1B), Ca(V)2.2) or PQ-VDCCs (α(1A), Ca(V)2.1) and that exocytosis and endocytosis are coupled, the supposition that the different channel subtypes are specialized to control different cell functions is attractive. Here we have explored this hypothesis in primary cultures of bovine adrenal chromaffin cells where PQ channels account for 50% of Ca(2+) current (I(Ca)), 30% for N channels, and 20% for L channels. We used patch-clamp and fluorescence techniques to measure the exo-endocytotic responses triggered by long depolarizing stimuli, in 1, 2, or 10 mM concentrations of extracellular Ca(2+) (Ca(2+)). Exo-endocytotic responses were little affected by ω-conotoxin GVIA (N channel blocker), whereas ω-agatoxin IVA (PQ channel blocker) caused 80% blockade of exocytosis as well as endocytosis. In contrast, nifedipine (L channel blocker) only caused 20% inhibition of exocytosis but as much as 90% inhibition of endocytosis. Conversely, FPL67146 (an activator of L VDCCs) notably augmented endocytosis. Photoreleased caged Ca(2+) caused substantially smaller endocytotic responses compared with those produced by K(+) depolarization. Using fluorescence antibodies, no colocalization between L, N, or PQ channels with clathrin was found; a 20-30% colocalization was found between dynamin and all three channel antibodies. This is incompatible with the view that L channels are coupled to the endocytotic machine. Data rather support a mechanism implying the different inactivation rates of L (slow-inactivating) and N/PQ channels (fast-inactivating). Thus a slow but more sustained Ca(2+) entry through L channels could be a requirement to trigger endocytosis efficiently, at least in bovine chromaffin cells.
钙（Ca（2+））依赖性内吞作用与通过电压依赖性钙（Ca（2+））通道（VDCC）的 L 型（α（1D），Ca（V）1.3）优先进入 Ca（2+）有关。考虑到神经递质的 Ca（2+）依赖性胞吐释放主要是由通过 N-（α（1B），Ca（V）2.2）或 PQ-VDCC（α（1A），Ca（V）2.1）的 Ca（2+）进入触发的，并且胞吐作用和内吞作用是偶联的，因此假设不同的通道亚型专门用于控制不同的细胞功能是有吸引力的。在这里，我们在牛肾上腺嗜铬细胞瘤的原代培养物中探索了这一假设，其中 PQ 通道占 Ca（2+）电流（I（Ca））的 50％，N 通道占 30％，L 通道占 20％。我们使用膜片钳和荧光技术测量了在 1、2 或 10 mM 浓度的细胞外 Ca（2+）（[Ca（2+）]（e））下长去极化刺激触发的内吞作用。内吞作用对外源性囊泡素 GVIA（N 通道阻滞剂）的影响很小，而 ω-阿加托毒素 IVA（PQ 通道阻滞剂）导致胞吐作用和内吞作用的 80％阻断。相比之下，硝苯地平（L 通道阻滞剂）仅引起胞吐作用的 20％抑制，但内吞作用的抑制高达 90％。相反，FPL67146（L VDCC 的激活剂）显著增强了内吞作用。光释放的笼状 Ca（2+）引起的内吞作用响应明显小于 K（+）去极化产生的内吞作用响应。使用荧光抗体，未发现 L、N 或 PQ 通道与网格蛋白之间的共定位；发现动力蛋白与所有三种通道抗体之间存在 20-30％的共定位。这与 L 通道与内吞机器偶联的观点不一致。数据更支持一种机制，该机制暗示 L（缓慢失活）和 N / PQ 通道（快速失活）的不同失活速率。因此，通过 L 通道的缓慢但更持续的 Ca（2+）进入可能是有效触发内吞作用的要求，至少在牛肾上腺嗜铬细胞瘤中是这样。
