Sensores de pH genéticamente modificados dirigidos a microdominios celulares

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

Jornada; Jornadas de Investigación Facultad de Cs. Médicas UNLP; 2022

Introduction: Intra- and extracellular pH regulation is a key functionof all cells and tissues and a prerequisite for normal physiologicalfunction. Spatial nonuniformity of intracellular pH is generated due todifferential subcellular distribution of Na+/H+ exchanger (NHE1) andthe Na+/HCO3- cotransporter (NBC).Aim: To study proton microdomains we generated a proton sensorfluorescent protein (pHluorin2) fused with canonical targeting signalsto the endoplasmic reticulum, mitochondrial matrix and junctionalcleft of cardiac myocytes (cleft-targeted pH sensor were generatedby fusing pHluorin2 to FKBP12.6).Methods and Results: We initially corroborated the localization of thesensor in HEK cells with confocal microscopy. We used the ammonia-prepulse technique to create an intracellular acid load. pH recoverieswere obtained from cytosol and endoplasmic reticulum lumen.NHE inhibition by amiloride drastically reduced the pH recovery suggestingthe 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