Fig. 5. Glutamate-evoked NKA activity is reduced in PRRT2-deficient neurons.

A Representative traces from WT (black; left) and PRRT2 KO (red; right) primary hippocampal neurons show the response to glutamate puffs performed under control conditions (ctrl) or under blockade of the NKA with 1 mM ouabain (oua). Black triangles show the time of the application of the glutamate puff (glu). Digital subtraction of the response obtained in the presence of ouabain from the control response yields the NKA activity (green traces). The integrated area under this ouabain-sensitive current (gray-shaded area) defines the NKA charge. B–D NKA charge (B), glutamate puff-induced current (C), and NKA charge normalized by the amplitude of the glutamate current (D) for WT and PRRT2 KO primary neurons (n = 29 and 28 neurons for WT and PRRT2 KO, respectively, from three independent neuronal preparations). E Primary hippocampal neurons were subjected to acute PRRT2 knockdown (KD) by RNA interference and to rescue of PRRT2 expression by transduction with a Sh-resistant PRRT2 construct. Representative traces recorded from neurons 7 days post-infection with Scramble/Control Cherry (Scr; black; left) or Sh4/Control Cherry (KD; red; middle) or Sh4/PRRT2-Cherry (KD + PRRT2; blue; right) upon glutamate puffs performed in the absence (ctrl) or presence of 1 mM ouabain (oua). Black triangles mark the time of glutamate application. Green traces indicate the NKA activity. F–H NKA charge (F), glutamate puff-induced current (G) and NKA charge normalized by the amplitude of the glutamate current (H) for Scr, PRRT2 KD, and KD + PRRT2 neurons (n = 52, 61, and 46 neurons for Scr, PRRT2 KD and KD + PRRT2, respectively, from six independent neuronal preparations). Bar plots show the means ± SEM with superimposed individual experimental points. *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA/Bonferroni’s tests.