AMPA receptor pHluorin-GluA2 reports NMDA receptor-induced intracellular acidification in hippocampal neurons

Rathje, Mette, Fang, Huaqiang, Bachman, Julia L., Anggono, Victor, Gether, Ulrik, Huganir, Richard L. and Madsen, Kenneth L. (2013) AMPA receptor pHluorin-GluA2 reports NMDA receptor-induced intracellular acidification in hippocampal neurons. Proceedings of the National Academy of Sciences of the United States of America, 110 35: 14426-14431. doi:10.1073/pnas.1312982110


Author Rathje, Mette
Fang, Huaqiang
Bachman, Julia L.
Anggono, Victor
Gether, Ulrik
Huganir, Richard L.
Madsen, Kenneth L.
Title AMPA receptor pHluorin-GluA2 reports NMDA receptor-induced intracellular acidification in hippocampal neurons
Journal name Proceedings of the National Academy of Sciences of the United States of America   Check publisher's open access policy
ISSN 0027-8424
1091-6490
Publication date 2013-08
Year available 2013
Sub-type Article (original research)
DOI 10.1073/pnas.1312982110
Open Access Status DOI
Volume 110
Issue 35
Start page 14426
End page 14431
Total pages 6
Place of publication Washington, United States
Publisher NAtional Academy of Sciences
Collection year 2014
Language eng
Formatted abstract
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.
Keyword Internalization and recycling
Synaptic plasticity
Glutamate receptors
Brain ischemia
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Queensland Brain Institute Publications
Official 2014 Collection
 
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