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.

Figure 1

A Construction design for the expression of SEP-BTX in P. pastoris. The DNA coding sequence for the SEP-BTX fusion protein was inserted into the EcoRI site of the expression vector pPIC9. It was cloned downstream and in frame with the alpha factor signal sequence (ss) of S. cerevisiae and upstream of the coding sequences for the recognition peptide for tobacco etch virus protease (TEV) and a 6 × His tag (6His). 5′AOX1, TT and 3′AOX1: promoter region, transcription terminator and 3′ region of P. pastoris alcohol Oxidase 1 gene, respectively. HIS4: Histidinol dehydrogenase gene for selection of transformants in minimal medium. B Western blot of recombinant SEP-BTX purification fractions revealed with anti-His tag (left) or anti-GFP (right) antibodies. Culture supernatant (C) was concentrated by ultrafiltration with a 30 kDa Amicon Ultra (A30) and SEP-BTX was purified and eluted (E) by gravity flow Ni–NTA affinity chromatography. Marker: Colorplus Prestained Protein Marker (New England Biolabs). The expected molecular weight of the fusion protein is around 37 kDa. A smaller fragment was also detected by the anti-GFP antibody (*). The presence of this fragment did not prevent proper binding of SEP-BTX to the acetylcholine receptor. C Confocal image of a levator auris NMJ junction incubated with the SEP-BTX fusion protein and captured with Nomarsky optics (488 nM wavelength light excitation, 510 nM emission). Fluorescence and transmission images were merged.

Figure 2

A The fluorescence image shows the NMJ incubated with SEP-BTX fusion protein bathed with 10 mM HEPES saline buffered at pH 6, 7, and 8. B Calibration curve (average of n = 3) obtained by measuring the fluorescence intensity of the NMJ incubated at different pHs. The value at pH 6 and 9 were considered the minimal and maximal fluorescence outputs, respectively (for calibration curve, see main text).

Figure 3

A Left: examples of NMJ fluorescence intensity changes before, during and after nerve stimulation at 100, 300 Hz and 300 Hz plus 100 µM DAP. The red line indicates the interval during which the nerve was stimulated. Right: the average changes in NMJ fluorescence intensity were calculated for each type of nerve stimulation. Letters indicate significant differences (Student’s t-test): a. p < 0.01 compared to stimulation at 100 Hz. b. p < 0.01 compared to stimulation at 300 Hz. B Left: comparison of NMJ fluorescence intensity changes during nerve stimulation at 50 Hz at the indicated HEPES buffer concentrations in the presence of 100 µM DAP. Right: the average changes in NMJ fluorescence intensity were calculated for each type of nerve stimulation. * p < 0.01 compared to stimulation in 20 mM HEPES.