Figure 3

Biphasic changes in pHluorin fluorescence induced by application of acidic solution (pH _o = 5.4). A, trace showing averaged time course of the pHluorin-GluR-A fluorescence quenching (normalized to the fluorescent intensity at the pre-application level). The initial, fast phase of fluorescence quenching is marked by green circles, the subsequent slow phase by red circles. Green and red lines represent best fitting curves for fast phase (exponential decay) and slow phase (linear regression), respectively. B, averaged time course of the cytosolic pHluorin (pHluorin-CS) quenching induced by application of acidic solution. The values are normalized to the fluorescence intensity at the pre-application level. Red line represents best linear fit for the plot. C, averaged time course of quenching of the pHluorin trapped in endoplasmic reticulum (pHluorin-ER) induced by application of acidic solution. Values are normalized to the fluorescence intensity at the pre-application level. Red line represents best linear fit for the plot. Note that the kinetics are similar both to these of pHluorin-CS (B) and to the slow phase of pHluorin-GluR-A fluorescence quenching (red circles in A). Schematic representation of the cellular localization of pHluorin-tagged molecules for each of the construct is shown at the bottom of each panel.