Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences and Lundbeck Foundation Center for Biomembranes in Nanomedicine, University of Copenhagen, 2200N Copenhagen, Denmark, and.
Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences and.
J Neurosci. 2014 Aug 6;34(32):10688-700. doi: 10.1523/JNEUROSCI.5132-13.2014.
Protein Interacting with C Kinase 1 (PICK1) is a Bin/Amphiphysin/Rvs (BAR) domain protein involved in AMPA receptor trafficking. Here, we identify a selective role for PICK1 in the biogenesis of large, dense core vesicles (LDCVs) in mouse chromaffin cells. PICK1 colocalized with syntaxin-6, a marker for immature granules. In chromaffin cells isolated from a PICK1 knockout (KO) mouse the amount of exocytosis was reduced, while release kinetics and Ca(2+) sensitivity were unaffected. Vesicle-fusion events had a reduced frequency and released lower amounts of transmitter per vesicle (i.e., reduced quantal size). This was paralleled by a reduction in the mean single-vesicle capacitance, estimated by averaging time-locked capacitance traces. EM confirmed that LDCVs were fewer and of markedly reduced size in the PICK1 KO, demonstrating that all phenotypes can be explained by reductions in vesicle number and size, whereas the fusion competence of generated vesicles was unaffected by the absence of PICK1. Viral rescue experiments demonstrated that long-term re-expression of PICK1 is necessary to restore normal vesicular content and secretion, while short-term overexpression is ineffective, consistent with an upstream role for PICK1. Disrupting lipid binding of the BAR domain (2K-E mutation) or of the PDZ domain (CC-GG mutation) was sufficient to reproduce the secretion phenotype of the null mutant. The same mutations are known to eliminate PICK1 function in receptor trafficking, indicating that the multiple functions of PICK1 involve a conserved mechanism. Summarized, our findings demonstrate that PICK1 functions in vesicle biogenesis and is necessary to maintain normal vesicle numbers and size.
蛋白相互作用激酶 1（PICK1）是一种参与 AMPA 受体运输的 Bin/Amphiphysin/Rvs（BAR）结构域蛋白。在这里，我们确定了 PICK1 在小鼠嗜铬细胞中大而致密核心囊泡（LDCV）生物发生中的选择性作用。PICK1 与 syntaxin-6 共定位，后者是不成熟颗粒的标志物。在 PICK1 敲除（KO）小鼠分离的嗜铬细胞中，胞吐作用的量减少，而释放动力学和 Ca2+敏感性不受影响。囊泡融合事件的频率降低，每个囊泡释放的递质量减少（即，量子大小降低）。这与通过平均时间锁定电容迹线估计的平均单个囊泡电容的减少相平行。EM 证实 PICK1 KO 中的 LDCV 数量减少且明显减小，表明所有表型都可以通过减少囊泡数量和大小来解释，而缺乏 PICK1 对生成囊泡的融合能力没有影响。病毒拯救实验表明，长期表达 PICK1 是恢复正常囊泡内容物和分泌所必需的，而短期过表达无效，这与 PICK1 的上游作用一致。BAR 结构域（2K-E 突变）或 PDZ 结构域（CC-GG 突变）的脂质结合的破坏足以再现缺失突变体的分泌表型。已知相同的突变消除了 PICK1 在受体运输中的功能，这表明 PICK1 的多种功能涉及保守的机制。综上所述，我们的研究结果表明，PICK1 参与囊泡生物发生，对于维持正常囊泡数量和大小是必需的。
