A new tool to sense pH changes at the neuromuscular junction synaptic cleft

MATÍAS BLAUSTEIN, SONIA WIRTH, GUSTAVO SALDAÑA, ANA PAULA PIANTANIDA MARÍA EUGENIA BOGETTI, MARÍA EUGENIA MARTIN, ALEJANDRO COLMAN‑LERNER OSVALDO D. UCHITEL

NATURE

NATURE PUBLISHING GROUP

Lugar: Londres; Año: 2020

0028-0836

Synaptic transmission triggers transient acidification of the synaptic cleft. Recently, it has been shownthat pH affects the opening of postsynaptic channels and therefore the production of tools that allowto study these behaviors should result of paramount value. We fused α-bungarotoxin, a neurotoxinderived from the snake Bungarus multicinctus that binds irreversibly to the acetylcholine receptorextracellular domain, to the pH sensitive GFP Super Ecliptic pHluorin, and efficiently expressed itin Pichia pastoris. This sensor allows synaptic changes in pH to be measured without the need ofincorporating transgenes into animal cells. Here, we show that incubation of the mouse levatorauris muscle with a solution containing this recombinant protein is enough to fluorescently labelthe endplate post synaptic membrane. Furthermore, we could physiologically alter and measurepost synaptic pH by evaluating changes in the fluorescent signal of pHluorin molecules bound toacetylcholine receptors. In fact, using this tool we were able to detect a drop in 0.01 to 0.05 pH unitsin the vicinity of the acetylcholine receptors following vesicle exocytosis triggered by nerve electricalstimulation. Further experiments will allow to learn the precise changes in pH during and aftersynaptic activation