Figure 5
From: A role for intracellular zinc in glioma alteration of neuronal chloride equilibrium
Intracellular Zn2+ modulates EGABA through Src and TrkB activation. (a) B ottom, EGABA is rapidly depolarized in cultured hippocampal neurons loaded with ~10 nM free Zn2+ through the patch pipette (dark red circles; t=0, −79.6±4.7 mV; t=5′, −58.8±4.8 mV; n= 17; *P<0.05; **P<0.01, paired t-test), but not with standard EGTA-containing intracellular solution (empty circles; free Zn2+=0.1 pM; P=0.54; n=8). Top, in cultures treated with the KCC2 blocker DIOA (20 μM, 1 h pre-application and during the experiment), EGABA was significantly depolarized at t=0 with both intracellular solutions (dark red squares, free Zn2 ~10 nM, −47.5±1.2 mV; n=5; empty squares, free+Zn2+=0.1 pM, −52.8±6.1 mV, n=6; ##P<0.01; respect to control, ANOVA ), and remained stable despite the intracellular Zn2+ loading. (b) TPEN application reverts Zn2+-induced EGABA shift. Top, sample current traces from 10 nM free Zn2+-loaded neuron, at start of recording (t=0), 5′ after and following TPEN application (20 μM; 15′). Bottom, Zn2+-induced EGABA depolarization at t= 0; 5′ and 20′ (n=7, **P<0.01, ANOVA); dashed column represents EGABA after 15′ TPEN application (following 5′ Zn2+ loading; n=8, ##P<0.01 versus 20′, ANOVA-Tukey test). (c) Impairment of Zn2+-induced EGABA depolarization (ΔEGABA at t=5′, 22.8±3.8 mV, n=9) in the presence of Src kinases inhibitor (PP2, 5 μM, n=9, P<0.01) or TrkB inhibitor (K252A, 200 nM; n=7, P<0.01) (versus control, ANOVA). EGABA and RMP were depolarized in control following, K252A or PP2 treatment as reported in Supplementary Table S4. (d) GCM increases Src phosphorylation. Top, typical immunoblot experiment; bottom, GCM treatment (15′) increases Src (n=10, P<0.01; ANOVA) phosphorylation (platelet-derived growth factor represent positive control). (e) TPEN application prevents GCM-induced Src phosphorylation; Top, typical immunoblot experiment; bottom, GCM treatment (15′) failed to increase Src (n=6, P=0.92; ANOVA) phosphorylation in cultures pre-treated with TPEN (20 μM, 15′; y-axis as in (d). (f) GCM increases TrkB phosphorylation. Top, typical immunoblot experiment; bottom, GCM treatment (15′) increases TrkB (n=3, P<0.05; ANOVA) phosphorylation (BDNF represents positive control)
