- The K+/Cl− co-transporter-2 (KCC2) is the major Cl− outward transporter in neurons, creating a Cl− equilibrium potential negative to the resting membrane voltage and rendering GABA inhibitory. However, following acute and chronic neuronal injury, KCC2 activity is decreased and GABA becomes excitatory1,2,3, a process that has been tightly linked to neurodegeneration4. Another important contributor to neuronal injury and death is an increase of cytosolic Zn2+ concentrations ([Zn2+]i)5. Although both rise in [Zn2+]i and decrease in KCC2 have been associated with neuronal injury, whether Zn2+ itself can influence KCC2 activity is unknown. To test whether [Zn2+]i modulates KCC2 activity we first monitored KCC2-mediated ion transport in HEK 293T cells expressing the co-transporter6. NH4+ was used as a surrogate ion for K+ and changes in intracellular pH were monitored using the fluorescent dye BCECF (see Supplemental methods online). KCC2-expressing cells showed a 3–fold faster NH4+-induced acidification rate than cells transfected with empty vector (Fig. 1a,b). Increasing [Zn2+]i by a 2 min application of Zn2+ with the Zn2+ ionophore pyrithione (ZnPyr) was immediately followed by a decrease of KCC2–mediated acidification rate, with an IC50 of ~50 µM Zn2+ (Fig. 1a–c). Application of Zn2+ without pyrithione did not change KCC2 activity (not shown), indicating that KCC2 is inhibited by intracellular Zn2+. Chelating [Zn2+]i with N,N,N’,N’–tetrakis–(2–pyridylmethyl)– ethylenediamine (TPEN) reversed the effects of ZnPyr (Fig. 1b). Moreover, TPEN alone increased KCC2 activity (Fig. 1a,b), indicating that endogenous levels of [Zn2+]i tonically inhibit KCC2. Figure 1 Increase in [Zn2+]i inhibits KCC2 activity Next, we determined changes in intracellular chloride [Cl−]i directly with the Cl− sensitive dye N-(ethoxycarbonylmethyl)6–methoxyquinolinium bromide (MQAE; see Supplemental Methods online). Application of 5 mM KCl reverses KCC2 transport leading to accumulation of intracellular Cl− (Fig. 1d). Increasing [Zn2+]i with ZnPyr blocked the Cl− influx (Fig 1d,e), while TPEN enhanced it (Fig. 1d,e), similar to the results observed with NH4+-induced acidification. We then tested whether KCC2 inhibition by Zn2+ is also observed in cortical neurons, which endogenously express the co-transporter. In BCECF-loaded immature neurons (7 days in vitro, DIV), expressing low levels of KCC2, NH4+ had little effect on intracellular acidification, similar to empty vector-transfected HEK 293T cells (Fig. 1f). In contrast, in mature neurons (>25 DIV), which express high levels of KCC2, NH4+–induced acidification was pronounced. Importantly, in mature neurons ZnPyr also effectively attenuated KCC2 activity (Fig. 1f). The Zn2+-dependent changes in KCC2 activity should affect the Cl− gradient and thereby the reversal potential of GABAA receptor–mediated currents (EGABA). We measured EGABA using the gramicidin perforated patch technique, which leaves intracellular Cl− undisturbed. A 2 min ZnPyr treatment produced a gradual positive shift in EGABA as, presumably, the intracellular Cl− concentration decreased with KCC2 inhibition (Fig. 2a). EGABA stabilized within 6–8 minutes after the ZnPyr application to a level ~20 mV positive to control (Fig. 2b,c,d). ZnPyr did not affect EGABA under whole-cell recording conditions with equal extracellular and intracellular chloride (Fig. 2d). The Zn2+-induced shift in EGABA reversed within 30 minutes after removing ZnPyr (Fig. 2d), indicating that neurons can tightly regulate [Zn2+]i under non-injurious conditions. Figure 2 Effects of [Zn2+]i on EGABA, and KCC2 inhibition by OGD in neurons Cerebral ischemia induces acute Zn2+ dysregulation in neurons7. Therefore, we investigated if KCC2 is inhibited by the sustained increases in neuronal [Zn2+]i following oxygen-glucose deprivation in mature neurons (OGD; Fig. 2e). Application of an extracellular Zn2+ chelator did not attenuate the [Zn2+]i rise following OGD (Fig. 2e), strongly suggesting that it is liberated from intracellular binding sites8. Importantly, in ischemic neurons NH4+-induced acidification rates were markedly decreased (Fig. 2f). Moreover, neurons exposed to TPEN during OGD had normal rates of acidification (Fig. 2f), indicating that the rise in [Zn2+]i following ischemic insult led to the inhibition of KCC2. Our results reveal a novel link between [Zn2+]i and KCC2 activity. KCC2 inhibition by Zn2+ was rapid, suggesting that Zn2+ may directly interact with intracellular cysteine or histidine residues of KCC2. Alternatively, Zn2+ could contribute to the known regulation of KCC2 activity by phosphorylation9 leading to changes in its expression9,10, or by oligomerization11. Zn2+, however, did not induce changes in KCC2 transcription, surface expression, or oligomeric organization (see Supplemental Figs. 1 and 2 online), some of which have been observed in other neuronal injury models2,3,4,9,10. Changes in KCC2 activity without changes in expression have also been reported in a deafness model12. The downregulation of KCC2 activity following OGD-induced Zn2+ rise may account for acute seizure activity following ischemia13. A major factor in neuronal injury following OGD is intracellular release of Zn2+ and it was recently shown that it is also critical for spreading depression triggered by OGD14. Interestingly, a decrease in GABA inhibition has been proposed to be a mechanism contributing to neuronal death following cerebral ischemia4. Our results indicate that Zn2+-dependent inhibition of KCC2 may be an important contributor to neuronal injury. We also suggest that influx of synaptically-released Zn2+ or its intracellular release, particularly during periods of intense synaptic activity15, may dynamically regulate inhibition by modulating KCC2 activity.