GENETICALLY ENCODED pH SENSORS TARGETING CELLULAR MICRODOMAINS

SOFÍA CIARROCCHI; DELFINA GALLO; ROMINA DI MATTÍA; ERNESTO ALEJANDRO AIELLO; ALEJANDRO ORLOWSKI

Mar del Plata, Buenos Aires

Congreso; Reunion Anual (2022) de la Sociedad Argentina de Fisiología (SAFIS); 2022

Sociedad Argentina de Fisiología (SAFIS)

Introduction: Intra- and extracellular pH regulation is a key function of all cells and tissues and a prerequisite for normal physiological function. Spatial nonuniformity of intracellular pH is generated due to differential subcellular distribution of Na+/H+ exchanger (NHE1) and the Na+/HCO3- cotransporter (NBC).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).Methods and Results: 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 and endoplasmic reticulum lumen. NHE inhibition by amiloride drastically reduced the pH recovery suggesting the presence of NHE in reticulum (A.U. cito: 0.144±0.004, n=24; cito+amiloride: 0.066±0.005*, n=23; ER:0.098±0.005 n=24; ER+amiloride: 0.011±0.005* n=24; *p