Upregulation of KCC2 activity by zinc-mediated neurotransmission via the mZnR/GPR39 receptor

Abstract

Vesicular Zn(2+) regulates postsynaptic neuronal excitability upon its corelease with glutamate. We previously demonstrated that synaptic Zn(2+) acts via a distinct metabotropic zinc-sensing receptor (mZnR) in neurons to trigger Ca(2+) responses in the hippocampus. Here, we show that physiological activation of mZnR signaling induces enhanced K(+)/Cl(-) cotransporter 2 (KCC2) activity and surface expression. As KCC2 is the major Cl(-) outward transporter in neurons, Zn(2+) also triggers a pronounced hyperpolarizing shift in the GABA(A) reversal potential. Mossy fiber stimulation-dependent upregulation of KCC2 activity is eliminated in slices from Zn(2+) transporter 3-deficient animals, which lack synaptic Zn(2+). Importantly, activity-dependent ZnR signaling and subsequent enhancement of KCC2 activity are also absent in slices from mice lacking the G-protein-coupled receptor GPR39, identifying this protein as the functional neuronal mZnR. Our work elucidates a fundamentally important role for synaptically released Zn(2+) acting as a neurotransmitter signal via activation of a mZnR to increase Cl(-) transport, thereby enhancing inhibitory tone in postsynaptic cells.

Figure 1.

Extracellular Zn²⁺ upregulates NH₄⁺-dependent influx mediated by KCC2 activity. A, To assess KCC2 transport activity, we monitored BCECF fluorescence in acute mouse hippocampal slices using the NH₄⁺ transport paradigm. Application of NH₄Cl (5 mm) induces initial alkalinization (due to passive diffusion of NH₃ through the cell membrane). Subsequent NH₄⁺ influx via KCC2, acting in reverse mode, induces acidification of the cells, and the initial rate of acidification represents KCC2 activity (see Materials and Methods). This paradigm was applied to slices obtained from 6- or 14-d-old mice (n = 4 slices). Inset shows the signal in the region marked by the box, where the linear regression curve was fitted to represent KCC2 activity. Note that for clarity of the inset only every second measurement is shown. Averaged rates of NH₄⁺ influx were 0.0006 ± 0.0004 (R/R₀/s) in P6 slices and −0.0004 ± 0.0003 (R/R₀/s) in P14 slices (*p < 0.05). B, Intracellular Zn²⁺ concentration in slices loaded with FluoZin-3, following (at indicated times) electrical stimulation (Besser et al., 2009), addition of Zn²⁺ (200 μm) and depolarization using ACSF containing 50 mm KCl (replacing NaCl) in the presence of Zn²⁺ (200 μm). Only following depolarization, in the presence of Zn²⁺, a rise in fluorescence was observed (n = 3 slices). C, Averaged traces from BCECF-loaded slices (n = 8 slices), imaged with or without application of NH₄Cl (5 mm) in the presence or absence of DIOA (100 μm). Slices were pretreated with extracellular Zn²⁺ (200 μm, 2 min) or maintained in ACSF (vehicle) and imaged within 2 min. In the box, the signal from the region marked in the graph is depicted, and the linear regression curve that was fitted to represent KCC2 activity is shown. Note that for clarity only every second measurement is shown. Inset shows bright-field (left) and BCECF fluorescence (right) images of the CA3 region that was monitored; SO stratum oriens, SL stratum lucidum. D, Averaged rate of acidification due to steady-state NH₄⁺ influx ±SEM (n = 8 slices, *p < 0.05 compared with vehicle control). The rate of NH₄⁺ influx is enhanced following pretreatment with extracellular Zn²⁺, and is blocked by DIOA (100 μm) or furosemide (100 μm) but not by the Na⁺/K⁺/Cl⁻ cotransporter inhibitor bumetanide (1 μm). E, Shown are representative traces from slices exposed to NH₄Cl that were calibrated to pH_i, using the calibration curve (see Materials and Methods). Each trace is an average of 7–12 ROIs within the somatic CA3 region from a single slice. Slices were treated with Zn²⁺ (200 μm, 2 min) or maintained in ACSF (vehicle) as marked.

Figure 2.

Extracellular Zn²⁺ upregulates Cl⁻-dependent influx mediated by KCC2 activity. A, Representative traces from slices loaded with the Cl⁻-sensitive dye MQAE (Hershfinkel et al., 2009), each trace is an average of 7–12 ROIs within the somatic CA3 region of one slice. Slices were treated with Zn²⁺ (200 μm, 2 min) or maintained in ACSF (vehicle). Within 2 min of the Zn²⁺ treatment slices were imaged and 10 mm KCl was added as indicated. B, Averaged traces from slices treated with or without 10 mm KCl. Shown are traces from control slices (vehicle, n = 7 slices), slices pretreated with extracellular Zn²⁺ (200 μm, 2 min, n = 7 slices), slices treated with Zn²⁺ in the presence of the KCC2 inhibitor DIOA (100 μm, n = 10 slices) or control slices, without KCl (n = 9 slices). Inset shows the CA3 region that was imaged (left, bright-field; right, fluorescent images); SO stratum oriens, SL stratum lucidum. In the box, the signal from the region marked in the graph is depicted, and the linear regression curve that was fitted to represent KCC2 activity is shown. Note that for clarity every second measurement is shown. C, Averaged rates ± SEM (n = 7 slices for vehicle or Zn²⁺-treated, and 10 slices for Zn²⁺ + DIOA) of Cl⁻ influx as monitored in B (*p < 0.05, **p < 0.01 compared with vehicle control). D, The effect of intracellular Zn²⁺ rise on KCC2 activity was determined in MQAE-loaded slices, as in A. Shown are averaged traces from control slices (vehicle) or slices pretreated with Zn²⁺ (100 μm) in the presence of its ionophore pyrithione (5 μm), marked as ZnPyr (n = 5 slices). Averaged rates of Cl⁻ influx were 0.033 ± 0.004 (−%F/F₀/s) in vehicle control slices and 0.017 ± 0.002 (−%F/F₀/s) in the ZnPyR-treated slices (*p < 0.05).

Figure 3.

Extracellular Zn²⁺ induces a hyperpolarizing shift in GABA_A reversal potential. A, In cell-attached recordings with 5 μm GABA in a patch pipette, GABA_A channel reversal potential was measured by delivering slow depolarizing voltage ramps from 10 to 73 mV relative to the membrane resting potential (V_r +10 to V_r +73 mV) every 15 s (n = 4 cells). Shown are two consecutive ramp current sweeps from a patch which contained GABA_A channels, under control conditions (top) and the same patch 5 min after addition of Zn²⁺ (200 μm, 2 min) to the bath (bottom), with the respective extrapolated E_GABA values for each ramp to the left. Sweeps are leak-subtracted and digitized at 20 kHz through a low-pass filter of 2 kHz (−3 dB). Dashed lines (red) are drawn through the closed and single-channel open state. B, Effect of Zn²⁺ application on I–V relationships of the current through a single GABA_A channel-containing patch. Amplitudes of channel opening plotted against voltage in control (vehicle treated, black) and following Zn²⁺ application (red), from a representative cell. The straight lines are linear fits of the data. Notice that the channel slope conductance remains unchanged (19 pS), the extrapolated E_GABA, however, shifted from the membrane resting potential from −2 to −16 mV following Zn²⁺ application. C, Mean unitary current amplitudes binned with a step of 2 mV and averaged over all patches. Note that the x-axis intercepts of the two fitted lines (indicated by arrows) give the reversal potential values of V_r −2.3 ± 0.5 mV in control and V_r −17 ± 2 mV following Zn²⁺ application.

Figure 4.

KCC2 activity is upregulated by synaptic Zn²⁺ released from mossy fibers. A, KCC2 activity measured in BCECF-loaded slices, as in Figure 1. Slices from ZnT3 KO mice lacking synaptic Zn²⁺ (open symbols) or WT mice (filled symbols) were monitored under control nonstimulated conditions or 2 min following electrical stimulation of the mossy fibers (a train of 10 pulses at 66 Hz) that induces release of synaptic Zn²⁺ (n = 6), or following application of Zn²⁺ (200 μm, 2 min, n = 6 slices). B, Averaged rates of NH₄⁺ influx ± SEM as monitored in A (n = 6 slices, **p < 0.01 compared with WT control). C, KCC2-dependent Cl⁻ transport monitored in MQAE-loaded slices, as in Figure 2, using 10 mm KCl to reverse KCC2 activity. Slices from ZnT3 KO mice (open symbols) or WT mice (filled symbols) were monitored following stimulation of the mossy fibers (a train of 10 pulses at 66 Hz) or following application of Zn²⁺ (200 μm, 2 min) (n = 5 slices). D, Averaged rates ± SEM of Cl⁻ influx as monitored in C (n = 5 slices, **p < 0.01 compared with the stimulated ZnT3 KO). The rate of ion influx, using the NH₄⁺ or Cl⁻ transport, is enhanced following mossy fiber stimulation in slices from WT mice compared with ZnT3 KO mice, indicating that KCC2 activity is upregulated by the release of synaptic Zn²⁺.

Figure 5.

Upregulation of KCC2 activity is mediated by mZnR signaling pathway and enhanced KCC2 surface expression. A, KCC2 activity was monitored in slices from WT mice loaded with BCECF, following stimulation of the mossy fibers in control slices (vehicle) or in slices pretreated with a Gαq inhibitor (YM-254890, 1 μm), a PLC inhibitor (U73122, 1 μm) or an ERK1/2 inhibitor (U0126, 1 μm); n = 7 slices. B, Averaged rate of NH₄⁺ influx as monitored in A. Dashed line indicates the rate of transport in nonstimulated control slices taken from Figure 4A (n = 7 slices, **p < 0.01 compared with vehicle control without stimulation). C, Top, Slices were biotinylated, control slices were maintained in ACSF, and loaded with the intracellular Ca²⁺-sensitive dye, Fura-2. Then Zn²⁺ (200 μm) was applied and the Ca²⁺ response is shown in control and biotinylated slices. Bottom, Surface expression level of KCC2, monitored using surface biotinylation followed by immunoblotting of KCC2 or transferrin receptor (TrfR), which is a nonrelated membrane transporter used as control. Surface expression in vehicle-control slices or in slices treated with Zn²⁺ in the presence or absence of YM-254890 (1 μm). Densitometry analysis of KCC2 surface expression is shown to the right, normalized to TrfR expression (n = 4 slices, *p < 0.05 compared with vehicle control).

Figure 6.

GPR39 mediates mZnR-dependent Ca²⁺ response in CA3 hippocampal neurons. A, Analysis of GPR39 transcripts was determined using PCR. The WT allele gives a 311 bp and the targeted GPR39 KO allele gives a 262 bp PCR product. This identifies GPR39^−/− (KO) or GPR39^+/+ (WT) mice. B, Synaptic Zn²⁺ release was determined using the nonpermeant form of Newport Green (Frederickson et al., 2006). Extracellular Zn²⁺-dependent changes in Newport Green (2 μm) fluorescence were monitored in the CA3 region following stimulation of the mossy fibers in slices from WT mice, GPR39 KO mice or ZnT3 KO mice, lacking synaptic Zn²⁺. Inset is a schematic model of the experimental setup. C, Averaged stimulation-dependent rise of Newport Green fluorescence, normalized to the initial fluorescence, monitored in slices from WT, GPR39 KO mice (n = 6 slices) or ZnT3 KO mice (n = 4 slices, **p < 0.01 compared with the GPR39 WT). D, Slices from WT or GPR39 KO mice were loaded with Fura-2, an intracellular Ca²⁺-sensitive dye. Intracellular Ca²⁺ rise was monitored following stimulation of the mossy fibers (a train of 10 pulses at 66 Hz, at the indicated time) in the presence of ACSF solution (vehicle) or ACSF containing the extracellular Zn²⁺ chelator CaEDTA (100 μm) (n = 7 slices). E, Averaged change of Ca²⁺ rises as monitored in D (n = 7 slices, **p < 0.01 compared with stimulated WT in ACSF). Note that the residual Ca²⁺ response in the slices from GPR39 KO mice is not attenuated further by CaEDTA and is similar to the response observed in the presence of CaEDTA in slices from WT mice.

Figure 7.

GPR39 mediates Zn²⁺-dependent upregulation of KCC2 activity. A, KCC2 activity was studied using the BCECF paradigm. NH₄⁺ influx rate was monitored in slices from WT (filled symbols) or GPR39 KO (open symbols) mice loaded with BCECF. BCECF fluorescence changes were monitored in slices following mossy fiber stimulation (a train of 10 pulses at 66 Hz) or in control nonstimulated slices (n = 6 slices). B, Slices from WT (filled symbols) or GPR39 KO (open symbols) mice (as in A) were pretreated with extracellular Zn²⁺ (200 μm, 2 min) or controls (n = 6 slices) and NH₄⁺ influx rate was monitored. C, Averaged rates of NH₄⁺ influx as monitored in A-B (n = 6 slices, *p < 0.05 compared with WT control). In slices from GPR39 KO mice, application of exogenous Zn²⁺ or synaptic Zn²⁺ release by mossy fiber stimulation did not affect KCC2 activity, which was similar to the level of the transporter activity in control slices obtained from WT mice. D, SHSY-5Y cells were transfected with siRNA constructs for silencing GPR39 (Besser et al., 2009) or a nonrelated G-protein-coupled receptor (siT1R3, control) and subjected to the MQAE paradigm to monitor KCC2 activity. Cells were treated with or without Zn²⁺ (200 μm, 2 min) and KCC2 activity was monitored in the presence or absence of DIOA (n = 5, *p < 0.05 compared with vehicle siT1R3, control). Zn²⁺-dependent Ca²⁺ rise was monitored using Fura-2 (see inset) and was attenuated in the siGPR39 transfected cells.