Measurement of secretory vesicle pH reveals intravesicular alkalinization by vesicular monoamine transporter type 2 resulting in inhibition of prohormone cleavage

1. The acidic interior of neuroendocrine secretory vesicles provides both a n energy gradient for amine-proton exchangers (VMATs) to concentrate small transmitter molecules, for example catecholamines, and an optimal pH for th e prohormone convertases which cleave hormone precursors. There is evidence that VMAT activity modulates prohormone cleavage, but in the absence of me asurements of pH in secretory vesicles in intact cells, it has not been pos sible to establish whether these effects are attributable to raised intrave sicular pH due to proton transport through VMATs. 2. Clones were generated of the hamster insulinoma cell line HIT-T15 expres sing a pH-sensitive form of green fluorescent protein (GFP-F64L/S65T) targe ted to secretory vesicles, with and with co-expression of VMAT2. In order t o study prohormone cleavage, further clones were generated that expressed p reprogastrin with and without co-expression of VMAT2. 3. Confocal microscopy of GFP fluorescence indicated that the pH in the sec retory vesicles was 5.6 in control cells, compared with 6.6 in cells expres sing VMAT2; the latter was reduced to 5.8 by the VMAT inhibitor reserpine. 4. Using a pulse-chase labelling protocol, cleavage of 34-residue gastrin ( G34) was found to be inhibited bg co-expression with VMAT2, and this was re versed by reserpine. Similar effects on vesicle pH and G34 cleavage were pr oduced by ammonium chloride. 5. We conclude that VMAT expression confers th e linked abilities to store biogenic amines and modulate secretory vesicle pH over a range influencing prohormone cleavage and therefore determining t he identity of regulatory peptide secretory products.