Robarts Research Institute, University of Western Ontario, London, Ontario, Canada.
Neuroscience. 2010 May 19;167(3):765-73. doi: 10.1016/j.neuroscience.2010.02.026. Epub 2010 Feb 16.
Cholinergic neurons rely on the sodium-dependent choline transporter CHT to provide choline for synthesis of acetylcholine. CHT cycles between cell surface and subcellular organelles, but little is known about regulation of this trafficking. We hypothesized that activation of protein kinase C with phorbol ester modulates choline uptake by altering the rate of CHT internalization from or delivery to the plasma membrane. Using SH-SY5Y cells that stably express rat CHT, we found that exposure of cells to phorbol ester for 2 or 5 min significantly increased choline uptake, whereas longer treatment had no effect. Kinetic analysis revealed that 5 min phorbol ester treatment significantly enhanced V(max) of choline uptake, but had no effect on K(m) for solute binding. Cell-surface biotinylation assays showed that plasma membrane levels of CHT protein were enhanced following 5 min phorbol ester treatment; this was blocked by protein kinase C inhibitor bisindolylmaleimide-I. Moreover, CHT internalization was decreased and delivery of CHT to plasma membrane was increased by phorbol ester. Our results suggest that treatment of neural cells with the protein kinase C activator phorbol ester rapidly and transiently increases cell surface CHT levels and this corresponds with enhanced choline uptake activity which may play an important role in replenishing acetylcholine stores following its release by depolarization.
胆碱能神经元依赖于钠依赖性胆碱转运体 CHT 来提供胆碱用于合成乙酰胆碱。CHT 在细胞表面和亚细胞细胞器之间循环，但对这种运输的调节知之甚少。我们假设通过改变 CHT 从细胞膜内化或递送到细胞膜的速率来激活蛋白激酶 C 用佛波酯调节胆碱摄取。使用稳定表达大鼠 CHT 的 SH-SY5Y 细胞，我们发现细胞暴露于佛波酯 2 或 5 分钟可显著增加胆碱摄取，而较长时间的处理则没有效果。动力学分析表明，5 分钟佛波酯处理显著增强了胆碱摄取的 V(max)，但对溶质结合的 K(m)没有影响。细胞表面生物素化测定表明，5 分钟佛波酯处理后 CHT 蛋白的质膜水平增强；这被蛋白激酶 C 抑制剂双吲哚马来酰亚胺-I 阻断。此外，佛波酯可减少 CHT 的内化并增加 CHT 向质膜的转运。我们的结果表明，用蛋白激酶 C 激活剂佛波酯处理神经细胞可快速和短暂地增加细胞表面 CHT 水平，这与增强的胆碱摄取活性相对应，这可能在去极化释放乙酰胆碱后补充乙酰胆碱储存中发挥重要作用。
