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

Work addressing whether cystic fibrosistransmembrane conductance regulator (CFTR) plays a role in regulatingorganelle pH has remained inconclusive. We engineered a pH-sensitiveexcitation ratiometric green fluorescent protein (pHERP) and targetedit to the Golgi with sialyltransferase (ST). As determined byratiometric imaging of cells expressing ST-pHERP, Golgi pH(pH_G) of HeLa cells was 6.4, while pH_G ofmutant (F508) and wild-type CFTR-expressing (WT-CFTR) respiratoryepithelia were 6.7-7.0. Comparison of genetically matched F508and WT-CFTR cells showed that the absence of CFTR statisticallyincreased Golgi acidity by 0.2 pH units, though this small differencewas unlikely to be physiologically important. Golgi pH was maintainedby a H⁺ vacuolar (V)-ATPase countered by a H⁺leak, which was unaffected by CFTR. To estimate Golgi proton permeability (P_H⁺), we modeledtransient changes in pH_G induced by inhibiting the V-ATPaseand by acidifying the cytosol. This analysis required knowing Golgibuffer capacity, which was pH dependent. Our in vivo estimate is thatGolgi P_H⁺ = 7.5 × 10⁴ cm/s when pH_G = 6.5, andsurprisingly, P_H⁺ decreased aspH_G decreased.