Rathje, Mette4; Fang, Huaqiang3; Bachman, Julia L3; Anggono, Victor3; Gether, Ulrik5; Huganir, Richard L3; Madsen, Kenneth L5
1 Neuropharm and Genetics Lab, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet2 Graduate School of Health and Medical Sciences, Faculty of Health and Medical Sciences, Københavns Universitet3 unknown4 Graduate School of Health and Medical Sciences, Faculty of Health and Medical Sciences, Københavns Universitet5 Neuropharm and Genetics Lab, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet
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.
Proceedings of the National Academy of Sciences of the United States of America, 2013, Vol 110, Issue 35, p. 14426-31