Fig. 4

Aβ potentiates astrocytic PMCA via cAMP signaling. a Elevation of astrocytic cAMP level increased PMCA-mediated Ca²⁺ extrusion. cAMP was elevated by forskolin (100 µM) and sub-cellular Ca²⁺ level imaged with Lck-GCaMP3 and TIRFM. Ca²⁺ diminution was blocked by the PMCA blocker La³⁺ (50 µM; n = 9–13 cells per condition). b Intracellular cAMP level was monitored with the FRET sensor GFP^nd-EPAC(dDEP)-mCherry and dual-color TIRFM. Left, representative dual-color recording of fluorescence change for EGFP and mCherry of the FRET sensor. Right, averaged astrocytic cAMP rise induced by Aβ25–35 (6 µM; n = 6). c PMCA-mediated astrocytic Ca²⁺ diminution was coupled with H⁺ influx. Dual-color TIRFM recorded concomitant cytosolic Ca²⁺ and pH diminution, by expressing the red genetically encoded Ca²⁺ sensor GECO-R and the pH-sensitive GFP protein in same astrocytes. Both effects were abolished by chelating astrocyte cytoplasmic Ca²⁺ with BAPTA AM (100 µM; n = 7–9 cells per condition). Scale bar, 5 µm