Proton leak and CFTR in regulation of Golgi pH in respiratory epithelial cells

Work addressing whether cystic fibrosis tranmembrane conductance regulator (CFTR) plays. a role in regulating organelle pH has remained inconclusive. We engineered a pH-sensitive excitation ratiometric green fluorescent prote in (pHERP) and targeted it to the Golgi with sialyltransferase (ST). As det ermined by ratiometric imaging of cells expressing ST-pHERP, Golgi pH (pH(G )) Of HeLa cells was 6.4, while pH(G), of mutant (Delta F508) and wildtype CFTR-expressing (WT-CFTR) respiratory epithelia were 6.7-7.0. Comparison of genetically matched Delta F508 and WT-CFTR cells showed that the absence o f CFTR statistically increased Golgi acidity by 0.2 pH units, though this s mall difference was unlikely to be physiologically important. Golgi pH was maintained by a H+ vacuolar (V)-ATPase countered by a H+ leak, which was un affected by CFTR. To estimate Golgi proton permeability (PH+), we modeled t ransient changes in pHG induced by inhibiting the V-ATPase and by acidifyin g the cytosol. This analysis required knowing Golgi buffer capacity, which was pH dependent. Our in vivo estimate is that Golgi PH+ = 7.5 x 10(-4) cm/ s when pH(G) = 6.5, and surprisingly, PH+ decreased as pH(G) decreased.