Using Fluorescent GAP Indicators to Monitor ER Ca²

The endoplasmic reticulum (ER) is the main reservoir of Ca²⁺ of the cell. Accurate and quantitative measuring of Ca²⁺ dynamics within the lumen of the ER has been challenging. In the last decade a few genetically encoded Ca²⁺ indicators have been developed, including a family of fluorescent Ca²⁺ indicators, dubbed GFP-Aequorin Proteins (GAPs). They are based on the fusion of two jellyfish proteins, the green fluorescent protein (GFP) and the Ca²⁺-binding protein aequorin. GAP Ca²⁺ 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 Ca²⁺ concentrations; they are insensible to variations in the Mg²⁺ 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 Ca²⁺ affinity version of GAP, GAP3 (K_D ≅ 489 µM), has been engineered to conform with the estimated [Ca²⁺] in the ER. GAP3 targeted to the lumen of the ER (erGAP3) can be utilized for imaging intraluminal Ca²⁺. The ratiometric measurements provide a quantitative method to assess accurate [Ca²⁺]_ER, both dynamically and at rest. In addition, erGAP3 can be combined with synthetic cytosolic Ca²⁺ indicators to simultaneously monitor ER and cytosolic Ca²⁺. Here, we provide detailed methods to assess erGAP3 expression and to perform Ca²⁺ 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 Ca²⁺ Basic Protocol 3: Monitoring ER- and cytosolic-Ca²⁺ Support Protocol: Generation of a stable cell line expressing erGAP3.