Oligomerization of KCC2 correlates with development of inhibitory neurotransmission

Abstract

The neuron-specific K+-Cl- cotransporter KCC2 extrudes Cl- and renders GABA and glycine action hyperpolarizing. Thus, it plays a pivotal role in neuronal inhibition. Development-dependent KCC2 activation is regulated at the transcriptional level and by unknown posttranslational mechanisms. Here, we analyzed KCC2 activation at the protein level in the developing rat lateral superior olive (LSO), a prominent auditory brainstem structure. Electrophysiology demonstrated ineffective KCC2-mediated Cl- extrusion in LSO neurons at postnatal day 3 (P3). Immunohistochemical analyses by confocal and electron microscopy revealed KCC2 signals at the plasma membrane in the somata and dendrites of both immature and mature neurons. Biochemical analysis demonstrated mature glycosylation pattern of KCC2 at both stages. Immunoblot analysis of the immature brainstem demonstrated mainly monomeric KCC2. In contrast, three KCC2 oligomers with molecular masses of approximately 270, approximately 400, and approximately 500 kDa were identified in the mature brainstem. These oligomers were sensitive to sulfhydryl-reducing agents and resistant to SDS, contrary to the situation seen in the related Na+-(K+)-Cl- cotransporter. In HEK-293 cells, coexpressed hemagglutinin-tagged KCC2 assembled with histidine-tagged KCC2, demonstrating formation of homomers. Based on these findings, we conclude that the oligomers represent KCC2 dimers, trimers, and tetramers. Finally, immunoblot analysis identified a development-dependent increase in the oligomer/monomer ratio from embryonic day 18 to P30 throughout the brain that correlates with KCC2 activation. Together, our data indicate that the developmental shift from depolarization to hyperpolarization can be determined by both increased gene expression and KCC2 oligomerization.

Figure 1.

Analysis of Cl⁻ extrusion capacity in the developing LSO. A, Current traces obtained from a P3 LSO neuron by focal GABA uncaging at the soma and at a proximal dendrite at different holding potentials. Horizontal bars indicate the time of the uncaging UV flash. Peak amplitudes of the GABA-evoked currents were used to generate the I–V relationship (right). E_GABA was −48 and −50 mV at the soma and at the dendrite, respectively. B, Current traces (left) and E_GABA determination (right) for a P12 LSO neuron. Horizontal bars indicate the time of the GABA uncaging UV flash. In contrast to the P3 neuron (A), E_{GABA,dendrite} (−65 mV) was clearly more negative than E_GABA,soma (−56 mV). C, At P3 (n = 5), E_GABA,soma (−54.6 ± 4.2 mV) was not different from E_{GABA,dendrite} (−56.2 ± 5.0 mV; p > 0.05). Under the same conditions, E_GABA,soma at P12 (−56.4 ± 2.3 mV) differed significantly from E_{GABA,dendrite} (−69.0 ± 6.2 mV; p < 0.01; n = 5). D, When the Cl⁻ load was increased by raising the Cl⁻ concentration in the patch pipette to 41 mm, this resulted in an E_GABA,soma of approximately −30 mV. Bumetanide was added to the bath (10 μm final concentration) to exclude an influence of NKCC1 on E_GABA. However, under these conditions, there was no difference between E_GABA,soma (−29.2 ± 2.4 mV) and −E_{GABA,dendrite} (−29.7 ± 2.9 mV; n = 10; p > 0.05) at P3. E, Schematic drawing of the experimental setup. Focal photolytic release of caged GABA was used to determine E_{GABA,dendrite} and E_GABA,soma under constant Cl⁻ load via the patch pipette. Flashes indicate sites of GABA uncaging. If E_{GABA,dendrite} < E_GABA,soma, an effective Cl⁻ extrusion mechanism is present.

Figure 2.

Analysis of anti-KCC2 antibody specificity. A–F, Anti-KCC2 antibodies directed against the N terminus (anti-nKCC2; A–D) or the C terminus (anti-cKCC2; E, F) were applied to coronal brainstem sections of P12 wild-type (A, C, E) or KCC2 knockdown (B, D, F) mice. A, For anti-nKCC2 in wild-type mice, a moderate IR was observed in sections containing the medial nucleus of the trapezoid body (MNTB) and the LSO, two of the main SOC nuclei. B, Only faint IR was present in KCC2 knockdown mice. C, E, High-magnification confocal images illustrated a similar labeling pattern for anti-nKCC2 and anti-cKCC2 in +/+ mice. D, F, Labeling of the presumed surface of LSO neuron somata (n) and transversely cut dendrites in the neuropil (np) is indicated by arrows and arrowheads, respectively. Both antibodies revealed only faint IR in the LSO of −/− mice. G, No immunoreactive signal was present in the LSO of wild-type mice incubated with secondary antibodies alone. Scale bar: A, B, 200 μm; C–G, 10 μm. H, Immunoblot analysis of anti-nKCC2. Membrane fractions of brains from wild-type (left) or KCC2 knockdown (right) mice were probed with anti-nKCC2. A strong signal in the range of the expected molecular weight was observed in +/+ mice but not in −/− mice. In addition, a weak signal was observed at lower molecular weight, independent of the genotype. 7n, Facial nerve; d, dorsal; l, lateral; M_r, relative molecular mass; +/+, wild-type mice; −/−, KCC2 knockdown mice.

Figure 3.

KCC2-IR in rat LSO neurons during maturation. A–D, Strong KCC2-IR was present in LSO neurons (n) at P0 (A), P4 (B), P12 (C), and P60 (D) using anti-nKCC2. Both the plasma membrane of somata (arrows) and the plasma membrane of transversely or longitudinally cut dendrites (arrowheads) were strongly KCC2 immunoreactive. The density of KCC2-IR in the neuropil (np) decreased with age. A weak labeling in the soma interior was obvious at all ages. Scale bar: (in A) AA–D, 10 μm.

Figure 4.

Ultrastructural localization of KCC2 in LSO neurons during maturation. Location of KCC2 molecules in the LSO of P4 (A, C, E, F) and P12 (B, D, G, H) rats, revealed with the preembedding immunogold technique. A, B, Semithin sections with labeled neurons in the LSO. Silver-intensified gold particles were found at the presumed surface of somatic and dendritic membranes (arrows). C, D, High-magnification electron micrographs illustrated labeled dendritic profiles with boutons (asterisks) making asymmetrical synapses (arrowheads) onto the dendrite. KCC2 was localized homogenously along the perisynaptic and extrasynaptic membranes at both ages. The postsynaptic thickening was almost devoid of KCC2 (arrowheads). E–H, High-magnification ultrastructural analysis showed even distribution of gold particles at perisynaptic sites (arrows) of excitatory (E, G) and inhibitory (F, H) synapses (asterisks). n, Neuronal soma; d, dendrite. Scale bars: (in A) A, B, 30 μm; (in C) C–H, 500 μm.

Figure 5.

A, B, Glycosylation pattern of KCC2 during maturation. Membrane fractions from P2 or P30 rat brainstems were left untreated or treated with PGNase F (A) or endo H (B) and then separated under reducing conditions by a linear 4–12% Bis–Tris NuPAGE system for 1.5 h. At both ages, KCC2 was PGNase sensitive and endo H insensitive.

Figure 6.

Effects of detergents on KCC2. Membranes from P30 rat brainstems were treated for 5 min at 40°C with no detergent (control), 4% Triton X-100, 0.5% SDS, 1% N-dodecyl-β-d-maltoside, or 0.5% N-laurylsarcosine. After adding sample buffer containing 0.5% LDS, samples were separated by a linear 4–12% Bis–Tris NuPAGE system. Immunoblot analysis revealed that, under all conditions, KCC2 was present mainly in molecular masses higher than that expected for the monomer. Asterisk denotes monomeric KCC2, and the bracket depicts high-molecular-mass KCC2-IR.

Figure 7.

Effect of sulfhydryl-reducing agents on KCC2 oligomers. A, Membranes from P30 rat brainstem were treated for 5 min at 40°C with increasing concentrations of β-mercaptoethanol, separated by a linear 3–8% Tris–acetate NuPAGE system for 3 h, and analyzed by immunoblot. In the absence of reducing agents, four distinct KCC2-immunoreactive bands were present, with molecular masses of ∼130–140, ∼270, ∼400, and ∼500 kDa. Asterisk denotes the monomeric KCC2, and arrows point to KCC2 oligomers. All KCC2 oligomers were β-mercaptoethanol sensitive yet resistant to 1% SDS. B, Analysis of oligomeric structures for the comigration with the Na⁺/K⁺-ATPase α subunit. According to A, P30 rat brainstem was analyzed for immunoreactivity against the Na⁺/K⁺-ATPase α subunit. Under all conditions, only monomeric Na⁺/K⁺-ATPase α subunits were observed, with a molecular mass of ∼100 kDa.

Figure 8.

Physical association between differentially tagged KCC2 proteins. A, Wild-type KCC2 was transfected in HEK-293 cells. After expression, the membrane fraction was isolated and separated by a 3–8% Tris–acetate NuPAGE system for 3 h and analyzed by immunoblot. Four distinct KCC2-immunoreactive bands were present, with molecular masses of ∼130–140, ∼270, ∼400, and ∼500 kDa, similar to the KCC2 oligomers in the P30 brainstem. No KCC2-IR was observed in untransfected HEK-293. B1, Protein lysates from HEK-293 cells expressing either HA–KCC2 and His–KCC2 (lanes 1, 2) or HA–KCC2 with untagged KCC2 (lanes 3, 4) were subjected to purification by Ni²⁺ beads and resolved by 8% SDS-PAGE. Proteins were detected by immunoblotting with anti-HA or anti-His₆ antibody. HA–KCC2 can be isolated by Ni²⁺ beads only in the presence of coexpressed His–KCC2. Asterisk denotes the monomeric KCC2, and arrowheads indicates high-molecular-mass KCC2 immunoreactivity. Control staining with His tag demonstrates presence of His–KCC2 only in the Ni²⁺ eluate of HA–KCC2/His–KCC2 double-transfected cells. B2, Schematic drawing of the experimental design. If HA–KCC2 form dimers with His–KCC2, both will be purified by Ni²⁺ beads. C, Immunoblot of HEK-293 cells transfected with either His–KCC2 or HA–KCC2. Anti-KCC2 detects both fusion proteins, and anti-HA detects only HA–KCC2 and not His–KCC2. This demonstrates that the antibody is not cross-reacting.

Figure 9.

KCC2 oligomerization during brainstem development. A, Membranes isolated from P2 or P30 rat brainstem were prepared in the presence (+) or absence (−) of 8% iodoacetamide. After separation by a linear 3–8% Tris–acetate NuPAGE system for 3 h, immunoblot analysis was performed. In the presence of iodoacetamide, a prominent monomeric structure and low-abundant oligomeric structures were detected at P2. At P30, oligomeric structures were dominant. In the absence of iodoacetamide, oligomeric KCC2 structures became more prominent at P2 but not at P30. The mass shift induced by iodoacetamide is attributable to its binding to KCC2 thiol groups. Images show representative examples of four independent experiments. B, Relative abundance of the monomeric and the oligomeric structures compared with the whole amount of KCC2 at P1/P2 and in rats aged between P30 and P60. The optical density of the four KCC2-immunoreactive bands was quantified from densitometric scans of eight lanes from three independent preparations for rats aged P1/P2 and six lanes from two independent preparations for rats aged between P25 and P60. Both the decrease in the monomer fraction (87 ± 4 to 32 ± 1%) and the increase in the oligomer fraction (13 ± 4 to 68 ± 1%) were highly significant (p < 0.0001). C, The monomer/oligomer ratio decreased from 7.10 ± 2.60 at P1/P2 to 0.5 ± 0.03 in animals aged between P25 and P60.

Figure 10.

KCC2-IR throughout the developing brain. A, Montage forming a sagittal section of a P4 rat brain. Moderate to strong KCC2-IR was observed in various brain regions, including the olfactory bulb (OB), the dorsorostral border of the caudate–putamen (CPu), the thalamus (Th), the hypothalamus (Hy), the superior colliculus (SC), the inferior colliculus (IC), the pons (Po), and the medulla oblongata (MO). A dorsocaudal region of the cortex (Cx) showed also KCC2-IR. In the cerebellum (Cb), KCC2-IR is only weak in the rostral region but moderate in the ventrocaudal region. B, At P12, KCC2-IR was moderate throughout the brain, including the hippocampus (Hp) and the cortex, which showed only faint IR at P4. Signal intensity in the cerebellum was stronger than at P4, but a rostrocaudal gradient was still obvious. Scale bar: A, B, 1 mm. 7, Facial nucleus; MNTB, medial nucleus of the trapezoid body; PN, pontine nuclei.

Figure 11.

KCC2 oligomerization during development of the nervous system. Indicated brain areas were dissected at E18, P1, or P30, and the membrane fraction was prepared. After separation by a linear 3–8% Tris–acetate NuPAGE for 3 h, immunoblot analysis was performed. KCC2 is detected as early as E18 in all brain areas. During development, the monomer/oligomer ratio decreases in all areas analyzed. Asterisk denotes the monomeric KCC2, and the bracket indicates high-molecular-mass KCC2 immunoreactivity.

Figure 12.

Model of KCC2 activation in the LSO. In immature LSO neurons, monomeric KCC2 is present in the plasma membrane. Because the monomeric KCC2 is transport inactive, LSO neurons exhibit a relative high [Cl⁻]_i. Development-dependent oligomerization activates KCC2, thereby lowering [Cl⁻]_i. This change in [Cl⁻]_i results in a developmental shift from depolarization to hyperpolarization (D/H-shift). Hatched ovals represent glycine receptors (GlyR), and filled ovals represent individual KCC2 polypeptides.