Fig. 7

Intracellular alkalization rescues Ca²⁺ entry through two distinct mechanisms. a FM2-10 destaining confirms the defect in synaptic vesicle exocytosis. Hippocampal neurons were loaded with FM2-10 in bicarbonate-buffered media by stimulation at 10 Hz for 120 s, allowed to recover for 10 min, then stimulated again at 10 Hz for 150 s to unload the FM dye. b Neurons from the NHE9 KO loaded simultaneously with bafilomycin as well as FM2-10 do not differ from WT neurons in the rate of FM dye destaining in response to 10 Hz stimulation. c The initial rate of destaining shows that bafilomycin rescues the exocytosis defect of NHE9 KO neurons but does not affect the destaining of WT cells. **p < 0.01, ****p < 0.0001 by one-way ANOVA with Bonferroni comparison. WT, n = 19/15 coverslips/3 cultures; KO, n = 20/15 coverslips/3 cultures. d Cumulative frequency distribution of Ca²⁺ in boutons imaged using Fluo5F shows that bafilomycin loaded into the recycling synaptic vesicles substantially augments Ca²⁺ entry in both WT and KO neurons stimulated at 10 Hz. n = total number of boutons analyzed from 22 coverslips. WT, n = 22/21 coverslips/3 cultures; KO, n = 21/21 coverslips/3 cultures genotype/condition. e Cumulative frequency distribution (left) and mean peak (right) of the Ca²⁺ response to single action potentials before and after synaptic vesicle loading with bafilomycin. Bafilomycin does not change peak Ca²⁺ influx. **p < 0.01, ***p < 0.001 by one-way ANOVA with Bonferroni. n = 90–100 responses averaged from 30 boutons/coverslip across 14–15 coverslips and 3 cultures/genotype. f Cumulative frequency distribution (left) and mean peak (right) of the Ca²⁺ response to single action potentials in KO neurons transfected for 7 days with empty vector control (KO), NHE9 L236S mutant (+L236S) or wild-type NHE9 (+WT) plasmids. ***p < 0.001 by one-way ANOVA with Bonferroni. n = 96–108 responses averaged from 3 neurons/coverslip across 6 coverslips and 3 cultures/genotype. Data in c, e, and f indicate mean ± s.e.m. NS not significant