Using Fluorescent GAP Indicators to Monitor ER Ca.

The endoplasmic reticulum (ER) is the main reservoir of Ca of the cell. Accurate and quantitative measuring of Ca dynamics within the lumen of the ER has been challenging. In the last decade a few genetically encoded Ca indicators have been developed, including a family of fluorescent Ca indicators, dubbed GFP-Aequorin Proteins (GAPs). They are based on the fusion of two jellyfish proteins, the green fluorescent protein (GFP) and the Ca-binding protein aequorin. GAP Ca indicators exhibit a combination of several features: they are excitation ratiometric indicators, with reciprocal changes in the fluorescence excited at 405 and 470 nm, which is advantageous for imaging experiments; they exhibit a Hill coefficient of 1, which facilitates the calibration of the fluorescent signal into Ca concentrations; they are insensible to variations in the Mg concentrations or pH variations (in the 6.5-8.5 range); and, due to the lack of mammalian homologues, these proteins have a favorable expression in transgenic animals. A low Ca affinity version of GAP, GAP3 (K ≅ 489 µM), has been engineered to conform with the estimated [Ca] in the ER. GAP3 targeted to the lumen of the ER (erGAP3) can be utilized for imaging intraluminal Ca. The ratiometric measurements provide a quantitative method to assess accurate [Ca], both dynamically and at rest. In addition, erGAP3 can be combined with synthetic cytosolic Ca indicators to simultaneously monitor ER and cytosolic Ca. Here, we provide detailed methods to assess erGAP3 expression and to perform Ca imaging, either restricted to the ER lumen, or simultaneously in the ER and the cytosol. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Detection of erGAP3 in the ER by immunofluorescence Basic Protocol 2: Monitoring ER Ca Basic Protocol 3: Monitoring ER- and cytosolic-Ca Support Protocol: Generation of a stable cell line expressing erGAP3.