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

Abstract

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

Postsynaptic Calcium Extrusion at the Mouse Neuromuscular Junction Alkalinizes the Synaptic Cleft

“…2 A , D , E ). As Blaustein et al (2020) saw acidification at the synapse, we took a closer look at the 250 ms stimulation condition. Increasing our temporal resolution, we imaged every 20 ms (50 Hz) to match the previous report.…”

“…Only one stimulation condition (i.e., 20 s trains, 5 s trains, or 250 ms trains) was introduced in a preparation. An additional assay was also conducted to partially replicate the work in Blaustein et al (2020) under physiological conditions. In short, preparations were incubated with the nAChR inhibitor tubocurarine and stimulated for 250 ms at 50 Hz.…”

“…The BHC and µCono in the static bath were recycled to maintain inhibition at 2.7 u m BHC or 16.7 n m uCono, respectively. While maintaining the perfusion and temperature of the bath, the LAL was stimulated at 50 Hz for various durations and imaged over the course of 600 s. Tubocurarine was purchased from MilliporeSigma (catalog #T2379; Blaustein et al, 2020 ; Zhang et al, 2010 ), acetazolamide was purchased from MilliporeSigma (catalog #A6011; Bertone et al, 2017 ), and caffeine was purchased from MilliporeSigma (catalog #C0750; Neyroud et al, 2019 ). Caloxin 1b1 was purchased from AnaSpec (catalog #AS-64236; Pande et al, 2006 , 2011 ).…”

Basal lamina: A novel pH regulator at the neuromuscular junction

“…Interestingly, other effects were seen that suggest the existence of more regulatory elements that were not considered originally. For example, there were only marginal presynaptic pH changes when muscle depolarization was blocked, 5,7 illustrating a disagreement between extracellular pH differences predicted by intracellular pH transients in the motor neuron terminal and what was observed on the endplate membrane of the muscle. 6 Further, the contributions of the muscle measured at the cleft, although reciprocal of what was found in motor neuron terminals, were 2-6 times larger than what would be predicted to be in the cleft based on presynaptic data.…”

“… 6 However, the cleft only shows slight acidification under strenuous stimulation when presynaptic activity was isolated, and measurements were made from the postsynaptic muscle membrane at the endplate. 7 While some differences between vertebrate and invertebrate NMJs could be expected due to evolution and different transmitter types (acetylcholine versus glutamate, respectively), the large discrepancy between the intracellular and cleft pH of the mammalian NMJ was surprising.…”