RT info:eu-repo/semantics/article
T1 Using fluorescent GAP indicators to monitor ER Ca2+
A1 Rojo Ruiz, Jonatan
A1 Sánchez Rabadán, Cinthia
A1 Calvo Rodríguez, Belén
A1 García-Sancho Martín, Francisco Javier
A1 Alonso Alonso, María Teresa
K1 aequorin
K1 Ca2+ calibration
K1 fluorescence
K1 GFP
K1 imaging
K1 2302 Bioquímica
K1 2415 Biología Molecular
AB 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 (KD ≅ 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.
PB Wiley
SN 2691-1299
YR 2024
FD 2024
LK https://uvadoc.uva.es/handle/10324/75165
UL https://uvadoc.uva.es/handle/10324/75165
LA eng
NO Current Protocols, 2024, vol. 4, n. 6, e1060
NO Producción Científica
DS UVaDOC
RD 03-mar-2025