Reply to Wilkinson et al.: Concerning the use of pHluorin-tagged GluA2 as a reporter for NMDA-induced AMPA receptor recycling

Abstract

As pointed out in the letter by Wilkinson et al. (1), we question in our recent paper by Rathje et al. (2) the use of pHluorin-GluA2 (pH-GluA2) as a reporter of NMDA- and AMPA-induced endocytosis because application of the agonists NMDA and AMPA results in intracellular acidification of hippocampal neurons. This conclusion rests on two important key observations: (i) the pHluorin response to NMDA treatment was almost intact after removal of surface-exposed pHluorin by thrombin cleavage (figure 1 of Rathje et al.) (2) and (ii) NMDA stimulation reduces fluorescence of both ER-retained pH-GluA2 and cytosolic pHluorin, in agreement with intracellular acidification, which was further supported by measurements using the pH-sensitive dye SNARF-1 (figures 2 and 3 of Rathje et al.) (2).