Mechanism of activity-dependent downregulation of the neuron-specific K-Cl cotransporter KCC2

Abstract

GABA-mediated fast-hyperpolarizing inhibition depends on extrusion of chloride by the neuron-specific K-Cl cotransporter, KCC2. Here we show that sustained interictal-like activity in hippocampal slices downregulates KCC2 mRNA and protein expression in CA1 pyramidal neurons, which leads to a reduced capacity for neuronal Cl- extrusion. This effect is mediated by endogenous BDNF acting on tyrosine receptor kinase B (TrkB), with down-stream cascades involving both Shc/FRS-2 (src homology 2 domain containing transforming protein/FGF receptor substrate 2) and PLCgamma (phospholipase Cgamma)-cAMP response element-binding protein signaling. The plasmalemmal KCC2 has a very high rate of turnover, with a time frame that suggests a novel role for changes in KCC2 expression in diverse manifestations of neuronal plasticity. A downregulation of KCC2 may be a general early response involved in various kinds of neuronal trauma.

Figure 2.

Area-specific decrease of KCC2 immunofluorescent staining in slices exposed to the 0-Mg²⁺ solution. A, Slices superfused for 1 hr in control solution (Aa) and in the Mg²⁺-free solution (Ab). Note the pronounced reduction in staining intensity in CA1. so, Stratum oriens; sr, stratum radiatum; sl, stratum lucidum; slm, stratum lacunosum-moleculare; DG, dentate gyrus). B, Plot of optical intensities from the distinct areas marked in Aa and Ab after 1 and 3 hr of 0-Mg²⁺ superfusion. (t test; ^***p < 0.001; n = 10.) Scale bar, 400 μm.

Figure 4.

Decay of cell surface biotinylated KCC2. Aa, Western blot of streptavidin-precipitated B-KCC2 showing a very fast rate of fall of B-KCC2 protein under control conditions that was enhanced further by omission of extracellular Mg²⁺. The level of plasmalemmal B-TfR does not show any significant change within 90 min. Ab, The application of NBQX (10 μm) and dl-AP-5 (40 μm) blocked the enhancing effect of 0-Mg²⁺ on the rate of decline of B-KCC2. B, Plot of normalized B-KCC2 and B-TfR optical band intensities from experiments such as shown in A.

Figure 1.

Interictal-like activity leads to a downregulation of KCC2 protein and mRNA levels in acute hippocampal slices. A, Extracellular field potentials in CA1 stratum pyramidale at 0.5, 1, and 3 hr after withdrawal of Mg²⁺. B, RT-PCR shows that KCC2 mRNA is downregulated, whereas NF-LmRNA remains constant. C, Western blots show activity-dependent downregulation of the KCC2 protein (∼150 kDa band) after 1-3 hr in 0-Mg²⁺. KCC2 mRNA and protein are not downregulated in slices in which 0-Mg²⁺-induced activity was blocked with 10 μm NBQX and 40 μm dl-AP-5 (B, C), showing an activity-specific effect.α-Tubulin was used to monitor the amount of total protein. D, Statistical representation of optical densities from five experiments such as those in C, expressed as percentage of control (means ± SEM; t test; ^***p < 0.001; ^**p < 0.01).

Figure 3.

Exposure to the convulsant drug, 4-AP, leads to downregulation of KCC2. A, Nonradioactive free-floating in situ hybridization showing the distribution and expression intensity of KCC2 mRNA in acute hippocampal slices superfused in the absence (left) and presence (right) of 100 μm 4-AP for 1 hr. B, Western blot analysis from slices treated as in A show strong 4-AP-induced downregulation of KCC2 protein levels (two parallel experiments). α-Tubulin was used to monitor the amount of total protein. Scale bar, 900 μm.

Figure 5.

0-Mg²⁺-induced activity leads to a decrease in the efficacy of Cl^- extrusion in CA1 pyramidal neurons. The IPSP_A reversal potential (E_IPSP-A) was measured in the presence of NBQX plus AP-5 using the 0.5 m Cl^--containing microelectrodes from control slices (A) and after a 1-3 hr period of 0-Mg²⁺-induced activity (B). Pharmacologically isolated IPSP_As were evoked by electrical stimulation (asterisk) at various levels of membrane potential (V_m; superimposed top traces) set by constant current pulses (bottom traces). The amplitudes of the IPSP_As measured within two time windows (the time of stimulus taken as 0) from the recordings in A and B have been plotted against membrane potential in C for detection of E_IPSP-A. The insets in C show individual IPSP_As evoked from the resting membrane potential (r.p.) level, and the arrowheads indicate the two successive time windows used for E_IPSP-A analysis (only the beginning of stimulus artifact is shown in the insets to indicate the timing of stimulation). D shows a summary of the results under steady-state conditions in the presence of the electrode-induced Cl^- load. Data are means ± SEM of driving forces of IPSPs (E_IPSP-A - V_m) obtained from control cells (n = 8), from cells exposed to 0-Mg²⁺ in the presence of NBQX plus AP-5 (to block interictal-like activity; n = 3), and from cells (n = 11) of slices that had undergone a 1-3 hr period of 0-Mg²⁺-induced activity before the intracellular recordings.

Figure 6.

Endogenous BDNF mediates the activity-dependent downregulation of KCC2 in hippocampal slices. Aa, RT-PCR analysis showing block of 0-Mg²⁺-induced KCC2 downregulation by the tyrosine kinase inhibitor K252a. NSE was used to confirm equal loading of total RNA. Ab, K252a was also able to block downregulation of KCC2 protein levels as evidenced by Western blot analysis. α-Tubulin was used to monitor an equal amount of total protein per lane. B, Scavenging endogenous BDNF with TrkB-Fc also inhibited the 0-Mg²⁺-induced downregulation of KCC2 mRNA (Ba) and protein (Bb) levels.

Figure 7.

TrkB is a major mediator of 0-Mg²⁺-induced CREB phosphorylation in hippocampal slices. A, Immunofluorescent staining showing 0-Mg²⁺-induced increase in CREB phosphorylation. After 1 hr in 0-Mg²⁺ solution, slices showed robust increase in anti-pCREB nuclear immunoreactivity in the CA1 region and dentate gyrus as compared with parallel control slices. After 3 hr in 0-Mg²⁺ solution, the level of activated CREB increased further and reached the CA3 region. Scale bar, 900 μm. B, Comparison of KCC2 and pCREB expression by Western blot analysis of 0-Mg²⁺-superfused (3 hr) hippocampal slices in the presence or absence of TrkB-Fc showing that scavenging of endogenous BDNF blocks CREB phosphorylation.α-Tubulin was used to monitor the amount of total protein.

Figure 8.

Both PLCγ and Shc docking sites of TrkB are required for BDNF-TrkB-mediated KCC2 downregulation, whereas the Shc site acting in isolation mediates an upregulation of KCC2. A, Schematic representation of the mutant TrkB receptors with a point mutation at either the tyrosine 515 or Shc site in the juxtamembrane region (Minichiello et al., 1998) or at the tyrosine 816 or PLCγ site in the C-terminal region. The trkB^W/W is a control-strategy receptor for the trkB^PLC/PLC mutant receptor (Minichiello et al., 2002). In the trkB^PLC/PLC and trkB^SHC/SHC mutant receptors, tyrosine (Y816) and tyrosine (Y515) were replaced by phenylalanine (F816) and phenylalanine (F515), respectively. B, Western blot analysis of BDNF-exposed (3-4 hr) acute slices from the point-mutant mice. In the control trkB^W/W mice, BDNF induced downregulation of KCC2 as expected. On the contrary, KCC2 was not downregulated in the trkB^SHC/SHC mutants, indicating that the Shc/FRS-2-coupled pathway is important for KCC2 downregulation. Strikingly, KCC2 was upregulated by BDNF in the trkB^PLC/PLC mutant mice. C, Western blot analysis of slices from the knock-in mice exposed for 3 hr to the 0-Mg²⁺ solution shows effects similar to those mediated by BDNF: the 0-Mg²⁺-induced KCC2 downregulation was blocked in the trkB^SHC/SHC mutant slices, and a clear upregulation was seen in trkB^PLC/PLC. α-Tubulin was used to monitor the amount of total protein. D, Plot of band intensities from experiments in B and C (means ± SEM; t test; ^***p < 0.001; ^**p < 0.01). E, Scheme interpreting the data obtained with the trkB mutant mice. Activation of both Shc and PLCγ cascades is required for trkB-mediated downregulation of KCC2, whereas activation of the Shc pathway in the absence of PLCγ activation leads to an upregulation of KCC2.