Molecular Neuropharmacology Laboratory and Lundbeck Foundation Center for Biomembranes in Nanomedicine, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, The Panum Institute 18.6, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Proc Natl Acad Sci U S A. 2013 Aug 27;110(35):14426-31. doi: 10.1073/pnas.1312982110. Epub 2013 Aug 12.
NMDA receptor activation promotes endocytosis of AMPA receptors, which is an important mechanism underlying long-term synaptic depression. The pH-sensitive GFP variant pHluorin fused to the N terminus of GluA2 (pH-GluA2) has been used to assay NMDA-mediated AMPA receptor endocytosis and recycling. Here, we demonstrate that in somatic and dendritic regions of hippocampal neurons a large fraction of the fluorescent signal originates from intracellular pH-GluA2, and that the decline in fluorescence in response to NMDA and AMPA primarily describes an intracellular acidification, which quenches the pHluorin signal from intracellular receptor pools. Neurons expressing an endoplasmic reticulum-retained mutant of GluA2 (pH-GluA2 ΔC49) displayed a larger response to NMDA than neurons expressing wild-type pH-GluA2. A similar NMDA-elicited decline in pHluorin signal was observed by expressing cytosolic pHluorin alone without fusion to GluA2 (cyto-pHluorin). Intracellular acidification in response to NMDA was further confirmed by using the ratiometric pH indicator carboxy-SNARF-1. The NMDA-induced decline was followed by rapid recovery of the fluorescent signal from both cyto-pHluorin and pH-GluA2. The recovery was sodium-dependent and sensitive to Na(+)/H(+)-exchanger (NHE) inhibitors. Moreover, recovery was more rapid after shRNA-mediated knockdown of the GluA2 binding PDZ domain-containing protein interacting with C kinase 1 (PICK1). Interestingly, the accelerating effect of PICK1 knockdown on the fluorescence recovery was eliminated in the presence of the NHE1 inhibitor zoniporide. Our results indicate that the pH-GluA2 recycling assay is an unreliable assay for studying AMPA receptor trafficking and also suggest a role for PICK1 in regulating intracellular pH via modulation of NHE activity.
N-甲基-D-天冬氨酸（NMDA）受体的激活促进了 AMPA 受体的内吞作用，这是长时程突触抑制的重要机制。将 pH 敏感型 GFP 变体 pHluorin 融合到 GluA2 的 N 端（pH-GluA2）已被用于检测 NMDA 介导的 AMPA 受体内吞作用和循环。在这里，我们证明在海马神经元的体部和树突区，荧光信号的很大一部分来自细胞内 pH-GluA2，并且对 NMDA 和 AMPA 的荧光下降主要描述了细胞内酸化，这猝灭了细胞内受体池的 pHluorin 信号。与表达野生型 pH-GluA2 的神经元相比，表达内质网保留的 GluA2 突变体（pH-GluA2 ΔC49）的神经元对 NMDA 的反应更大。单独表达细胞质 pHluorin（无 GluA2 融合）而不表达内源性 GluA2 时，也观察到类似的 NMDA 诱导的 pHluorin 信号下降。通过使用比率型 pH 指示剂羧基-SNARF-1 进一步证实了 NMDA 诱导的细胞内酸化。NMDA 诱导的荧光下降随后伴随着来自 cyto-pHluorin 和 pH-GluA2 的荧光信号的快速恢复。恢复依赖于钠离子并对钠氢交换体（NHE）抑制剂敏感。此外，在 shRNA 介导的 GluA2 结合 PDZ 结构域蛋白相互作用蛋白激酶 1（PICK1）敲低后，恢复更快。有趣的是，在存在 NHE1 抑制剂 zoniporide 的情况下，PICK1 敲低对荧光恢复的加速作用被消除。我们的结果表明，pH-GluA2 循环测定不是研究 AMPA 受体运输的可靠测定方法，并且还表明 PICK1 通过调节 NHE 活性在调节细胞内 pH 中起作用。
