Genetically encoded pH sensors targeting cellular microdomains

CIARROCCHI S; GALLO D; DI MATTÍA RA; AIELLO EA; ORLOWSKI A

Congreso; Reunión Anual de Sociedades de Biociencias 2022; 2022

Genetically encoded pH sensors targeting cellular microdomainsCiarrocchi S, Gallo D, Di Mattia RA, Aiello EA, Orlowski A Centro de Investigaciones Cardiovasculares “Dr. Horacio Cingolani”, Facultad de Ciencias Médicas, Universidad Nacional de La Plata-CONICET. La Plata, Argentina.Intra- and extracellular pH regulation is a key function of all cells and tissues and a prerequisite for normal physiological function. Cells constantly produce H+ due to its metabolism and energy supply. In particular, cardiac myocytes rely on two mechanisms to regulate intracellular pH, the Na+/H+ exchanger (NHE1) and the Na+/HCO3- cotransporter (NBC). Spatial nonuniformity of intracellular pH is generated due to differential subcellular distribution of these transporters. With the aim to study proton microdomains we generated a proton sensor fluorescent protein (pHluorin2) fused with canonical targeting signals to the endoplasmic reticulum, mitochondrial matrix and junctional cleft of cardiac myocytes (cleft-targeted pH sensor were generated by fusing pHluorin2 to FKBP12.6). We initially corroborated the localization of the sensor in HEK cells with confocal microscopy. We used the ammonia-prepulse technique to create an intracellular acid load. pH recoveries were obtained from cytosol, mitochondrial matrix and endoplasmic reticulum lumen. NHE inhibition by amiloride drastically reduced the pH recovery suggesting the presence of NHE in reticulum and mitochondria. Cytosolic, mitochondrial, and endoplasmic pH decreased during cytosolic Ca2+ elevation trigger by histamine. Next, we generated a cardiotropic adeno-associated virus (AAV9) to express the pH sensors into cardiac ventricular myocytes. AAV9-pHluorin2 and AAV9-pHluorin2-FKBP were injected in 3 months rats, after 28 days cardiac myocytes were isolated and pH sensors expression were corroborated by confocal microscopy. pHluorin2-FKBP12.6 express in a striated pattern with intensity maxima spaced ~2 μm apart, this strongly supports the conclusion that the FKBP12.6-tagged sensors are targeted to cardiac ryanodine receptor (RyR) at the z-line. Proton dynamics in cardiac cells are poorly understood. Thus, it is important to highlight that herein we generated for the first time a fluorescent pH sensor targeting different cellular microdomains as a tool for proton microdomains studies.