Fig. 5

Loss of NHE9 impairs synaptic vesicle exocytosis. a Cultured hippocampal neurons from NHE9 KO mice show a smaller VGLUT1-pHluorin fluorescence response than WT cells to 10 Hz stimulation for 60 s (left panel). Middle panel indicates fluorescence at the end of stimulation. **p < 0.01 by unpaired Student’s t test. Right panel shows no change in the time constant of endocytosis (τ), p = 0.65. b Cumulative frequency distribution of the extent of exocytosis for all boutons shows a consistent shift to smaller values for the NHE9 KO relative to WT. p < 0.0001 by Kolmogorov–Smirnov. WT, n = 816 boutons; KO, n = 1060. c Hippocampal neurons expressing VGLUT1-pHluorin were stimulated at 10 Hz for 60 s, then stimulated again at 10 Hz for 150 s in the presence of bafilomycin. The fluorescence change in the presence of bafilomycin (left) was used to determine the initial rate of exocytosis (middle), which is reduced for NHE9 KO neurons relative to WT, p = 0.0047. The recycling pool size, determined by normalizing the peak fluorescence response in bafilomycin to the total fluorescence revealed in NH₄Cl (right), shows no change (p = 0.22). d The rate constant of endocytosis during stimulation was calculated by subtracting the fluorescence in the presence of bafilomycin from that in the absence. NHE9 KO neurons do not differ from WT in endocytosis during or after the stimulus. n = 17 (WT) or 21 (KO) coverslips/genotype for c and d. Bar graphs indicate mean ± s.e.m. e Response of VGLUT1-pHluorin to 10 Hz stimulation shows that 4 mM Ca²⁺ rescues the exocytosis defect in the NHE9 KO neurons observed at 2 mM Ca²⁺, **p < 0.01 by two-tailed unpaired t test. N = 20 WT, 18 KO coverslips