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 protein (pHERP) and targeted it to the Golgi with sialyltransferase (ST). As determined 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 of CFTR statistically increased Golgi acidity by 0.2 pH units, though this small difference was unlikely to be physiologically important. Golgi pH was maintained by a H+ vacuolar (V)-ATPase countered by a H+ leak, which was unaffected by CFTR. To estimate Golgi proton permeability (PH+), we modeled transient changes in pHG induced by inhibiting the V-ATPase and by acidifying 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.