Intracellular zinc inhibits KCC2 transporter activity

Abstract

We report that KCC2 activity, monitored with wide-field fluorescence, is inhibited by intracellular Zn 2+ , a key component of neuronal injury. Zn 2+ -mediated KCC2 inhibition produced a depolarizing shift of GABA A reversal potentials in rat neurons. Moreover, oxygen-glucose deprivation attenuated KCC2 activity in a Zn 2+ -dependent manner. The link between Zn 2+ and KCC2 activity provides a novel target for neuroprotection and may be important in activity-dependent regulation of inhibitory synaptic transmission. KeywordsZinc; potassium-chloride co-transporter; ischemiaThe 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 excitatory 1,2,3 , a process that has been tightly linked to neurodegeneration 4 . Another important contributor to neuronal injury and death is an increase of cytosolic Zn 2+ concentrations ([Zn 2+ ] i ) 5 . Although both rise in [Zn 2+ ] i and decrease in KCC2 have been associated with neuronal injury, whether Zn 2+ itself can influence KCC2 activity is unknown.To test whether [Zn 2+ ] i modulates KCC2 activity we first monitored KCC2-mediated ion transport in HEK 293T cells expressing the co-transporter 6 . NH 4 + 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 NH 4 + -induced acidification rate than cells transfected with empty vector (Fig. 1a,b). Increasing [Zn 2+ ] i by a 2 min application of Zn 2+ with the Zn 2+ ionophore pyrithione (ZnPyr) was immediately followed by a decrease of KCC2-mediated acidification rate, with an IC 50 of ~50 µM Zn 2+ (Fig. 1a-c (Fig. 1b). Moreover, TPEN alone increased KCC2 activity (Fig. 1a,b (Fig. 1d). Increasing [Zn 2+ ] i with ZnPyr blocked the Cl − influx (Fig 1d,e), while TPEN enhanced it (Fig. 1d,e), similar to the results observed with NH 4 + -induced acidification.We then tested whether KCC2 inhibition by Zn 2+ 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, NH 4 + 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, NH 4 + -induced acidification was pronounced. Importantly, in mature neurons ZnPyr also effectively attenuated KCC2 activity (Fig. 1f).The Zn 2+ -dependent changes in KCC2 activity should affect the Cl − gradient and thereby the reversal potential of GABA A receptor-mediated currents (E GABA ). We measured E GABA using the gramicidin perforated patch technique, which leaves intracellular Cl − undisturbed. A 2 min ZnPyr treatment produced a gradual positive shift in E GABA as, presumably, the...

Redox Regulation of Intracellular Zinc: Molecular Signaling in the Life and Death of Neurons

“…Diverse Zn 2þ -dependent cell death signaling events have been described, including regulation of Zn 2þ -dependent transcription factors, induction of p75 neurotrophin receptor-mediated pathways, and activation of kinases and poly-ADP-ribose polymerase (PARP-1) (110). Recent work has also illuminated a previously unknown link between Zn 2þ and the K þ =Cl À cotransporter 2 (KCC2) after oxygen-glucose deprivation (40). Here, we highlight some recent examples of neuronal cell death pathways that critically involve an accumulation of free Zn 2þ .…”

Downregulation of Potassium Chloride Cotransporter KCC2 After Transient Focal Cerebral Ischemia

“…1,2 Various mechanisms by which KCC2 activity is regulated are a main focus of current research. 3,4 KCC2 is responsible for an inwardly directed electrochemical gradient for chloride that leads to hyperpolarizing GABA A receptormediated responses in the adult brain. 2,5 Soon after birth, KCC2 expression, especially in the hippocampus and the cortex, is low but developmentally upregulated to adult levels during the first 2 postnatal weeks.…”

Differences in the Large Extracellular Loop between the K+-Cl− Cotransporters KCC2 and KCC4

“…Accordingly, null mice die perinatally (2), and loss of the splice variant KCC2b leads to generalized seizures (6). KCC2 is further involved in neuroprotection (7), stress response (8), and neuropathic pain (9). In contrast, truncation of KCC3 causes a severe peripheral neuropathy, often accompanied by agenesis of the corpus callosum (10,11).…”