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, 12 (6), 725-7

Intracellular Zinc Inhibits KCC2 Transporter Activity


Intracellular Zinc Inhibits KCC2 Transporter Activity

Michal Hershfinkel et al. Nat Neurosci.


We found that K(+)/Cl(-) co-transporter 2 (KCC2) activity, monitored with wide-field fluorescence, was inhibited by intracellular Zn(2+), a major component of neuronal injury. Zn(2+)-mediated KCC2 inhibition produced a depolarizing shift of GABA(A) reversal potentials in rat cortical neurons. Moreover, oxygen-glucose deprivation attenuated KCC2 activity in a Zn(2+)-dependent manner. The link between Zn(2+) and KCC2 activity provides a previously unknown target for neuroprotection and may be important in activity-dependent regulation of inhibitory synaptic transmission.


Figure 1
Figure 1. Increase in [Zn2+]i inhibits KCC2 activity
(a) KCC2 activity was monitored with the pH-sensitive dye BCECF in HEK 293T cells tranfected with a KCC2 or empty vector. Cells were incubated for 2 min with vehicle, Zn2+ (100 µM) with pyrithione (5 µM) (ZnPyr), or TPEN (10 µM) and the rate of intracellular pH change following application of NH4Cl (10 mM) was monitored (see Supplemental Methods online). (b) NH4+-mediated acidification rates (mean ± SEM) in control and KCC2-expressing HEK 293T cells treated with vehicle (n=17), ZnPyr (n=14), TPEN (n=14), or ZnPyr followed by TPEN (n=10); *p<0.05, **p<0.005 ANOVA/Tukey, compared to KCC2-expressing NH4Cl only group. (c) Concentration-inhibition curve of KCC2 activity by Zn2+ (n=11 for each concentration, mean ± SEM). (d) KCC2 activity monitored with the Cl sensitive dye MQAE. KCl (10 mM) was applied to KCC2-expressing HEK 293T cells and the rate of Cl dependent quenching of the signal was determined. (e) Summary of Cl transport rates; ZnPyr, or TPEN, compared to KCl alone (n=6 for each treatment; mean ± SEM); **p<0.005 ANOVA/Tukey. (f) Endogenous KCC2 activity monitored with BCECF in immature and mature cortical neurons.
Figure 2
Figure 2. Effects of [Zn2+]i on EGABA, and KCC2 inhibition by OGD in neurons
(a) Representative gramicidin-perforated whole-cell currents (Vhold −50 mV) in response to 10 µM GABA before and after a 2 min treatment with ZnPyr (100 µM/5 µM). Scale bars: 300 pA, 1 s. (b) Gramicidin-perforated whole-cell currents before and after inhibition of KCC2 by ZnPyr. Scale bars: 300 pA, 1 s. (c) Current-voltage relationships of peak currents shown in (b). ZnPyr shifted EGABA from −54 to −33 mV. (d) EGABA measured with gramicidin-patch in control neurons (n=6), at <1, and 6–10 min after ZnPyr (n=10), and after 30 min rinse (n=3). EGABA measured in whole-cell (equal extracellular and intracellular chloride conditions) under control conditions (n=10), and after ZnPyr (n=5; mean ± SEM ***p<0.001; ANOVA/Tukey) (e) OGD-induced increase in neuronal [Zn2+]i inhibits KCC2. Neurons were exposed to OGD for 90 min and loaded with the Zn2+-sensitive dye FluoZin3. A significant Zn2+ rise, sensitive to TPEN (20 µM), was monitored at 1 hour following OGD. This increase in cytoplasmic Zn2+, was insensitive to the extracellular Zn2+ chelator tricine (1 mM), suggesting the source of the metal was intracellular (n=7–9; mean ± SEM ; *p<0.05; ANOVA/Bonferroni). (f) One hour following OGD treatment, neuronal KCC2 activity was monitored with NH4+/BCECF. TPEN treatment during OGD abolished KCC2 inhibition (n=5–14; mean ± SEM ; **p<0.005; ANOVA/Tukey).

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