Distinct functions of kainate receptors in the brain are determined by the auxiliary subunit Neto1

Abstract

Ionotropic glutamate receptors principally mediate fast excitatory transmission in the brain. Among the three classes of ionotropic glutamate receptors, kainate receptors (KARs) have a unique brain distribution, which has been historically defined by (3)H-radiolabeled kainate binding. Compared with recombinant KARs expressed in heterologous cells, synaptic KARs exhibit characteristically slow rise-time and decay kinetics. However, the mechanisms responsible for these distinct KAR properties remain unclear. We found that both the high-affinity binding pattern in the mouse brain and the channel properties of native KARs are determined by the KAR auxiliary subunit Neto1. Through modulation of agonist binding affinity and off-kinetics of KARs, but not trafficking of KARs, Neto1 determined both the KAR high-affinity binding pattern and the distinctively slow kinetics of postsynaptic KARs. By regulating KAR excitatory postsynaptic current kinetics, Neto1 can control synaptic temporal summation, spike generation and fidelity.

Figure 1

Hippocampus-abundant Neto1 interacts with kainate receptors in vivo. (a) Distinct expression of Neto1 and Neto2 in the brain. Neto1 expressed strongly in hippocampus, whereas Neto2 expressed in all brain regions except hippocampus. (b) Neto1 and Neto2 co-immunoprecipitated with GluK2/3 and GluK5, but not with GluA1 or PSD95, from rat brain lysate. Asterisk indicates Ig heavy chain. (c) Total protein levels of Neto1 and GluK5 were reduced in hippocampus from GluK2 knockout (−/−) compared to heterozygous (+/−), demonstrating a genetic interaction between GluK2 and Neto1 together with GluK5. Signal intensities were measured and normalized by those from Neto1 heterozygous (n = 4). (d) To compare protein expression at the postsynaptic density (PSD), PSDs were fractionated from hippocampi of the indicated genotypes (n = 4 each). Data are given as mean ± s.e.m.; * P < 0.05, *** P< 0.005.

Figure 2

Neto 1 is highly expressed in the hippocampus CA3 pyramidal neurons and localizes at s. lucidum. (a) Endogenous Neto1 promoter activity was monitored by beta-galactosidase activity in the knockout (−/−), in which the Neto1 gene was replaced with beta-galactosidase. Strong beta-galactosidase activity was observed in the hippocampus CA3 pyramidal cells, whereas weak activity was observed in the hippocampus CA1 interneurons, cerebral cortex, and striatum. Boxes represent regions where the recordings were performed in Fig. 4a-c. (b) Localization of Neto1 protein shown by immunohistochemistry with an anti-Neto1 antibody. Neto1 localized strongly at the hippocampus s. lucidum, where mossy fiber and CA3 pyramidal cells form synapses, in the wild type (WT), but not in the Neto1-knockout (−/−). Scale bars: Left panel, 1mm; Right panel, 200 μm.

Figure 3

Distinct distribution of high-affinity kainate receptors is determined by Neto1 postsynaptically. (a) Kainate binding in coronal hippocampal sections were visualized using an autoradiographical technique with [³H]kainate (50 nM). A strong [³H]kainate signal was observed in the s. lucidum (St. L.) in the wild type, but not in the GluK2 knockout (−/−). On the other hand, the [³H]kainate signal was reduced in Neto1-knockout mice (−/−). Scale bar: 100 μm. (b) Binding of carious concentration of [³H]kainate to hippocampal membranes was measured. The binding curve was shifted to the right in the Neto1-knockout. (c,d) Comparison of channel properties in transfected heterologous cells using a piezo-driven fast-perfusion system in outside-out patches. GluK2 and GluK5 were co-expressed with empty plasmid (Mock) or Neto1 in tsA201 cells. Representative response to sustained 1mM kainate application (c) and a 1-ms pulse (d). (c) Co-expression of Neto1 slowed desensitization and increased the ratio of steady-state and peak currents of GluK2/5 heteromeric channels (n=7-12). (d) Neto1 slowed deactivation of GluK2/5 (n=7-9). Upper traces in c and d indicate the open-tip potential to confirm rate of solution exchange. Data are given as mean ± s.e.m.; * P < 0.05, **P < 0.01.

Figure 4

Neto1 modulates kainate receptor function in the hippocampus. (a-c) Representative examples of inward currents elicited by agonist (3 μM kainate) from Neto1-knockout and wild-type littermate mice recorded in the whole-cell voltage clamp configuration in CA3 pyramidal cells (a), CA1 s. radiatum interneurons (b), and cerebellar Purkinje cells (c). Kainate-evoked inward currents were recorded in the presence of (in μM) 30 GYKI 53655, 50 d-APV, 100 picrotoxin, and 0.5 TTX. Each bar shows the mean peak current amplitude in each neuron from the numbers of cells;animals indicated. (d) To compare cell surface expression of KARs, acute hippocampal slices were prepared and biotinylated with cell-impermeable Sulfo-NHS-SS-biotin. After solubilization, biotinylated proteins were precipitated with Neutravidin-beads to isolate proteins at the cell surface. Most GluK2/3 was detected in the “Surface” fraction, whereas a cytosolic protein, tubulin, was detected in the “Internal” fraction. (e) No obvious change in surface expression of GluK2/3 or GluK5 was observed in acute hippocampal slices from wild-type and Neto1-knockout mice (n = 6). Scale bars (a-c): 50 μm. Data are given as mean ± s.e.m.; * P < 0.05, * P < 0.01.

Figure 5

The slow decay of kainate receptor–mediated synaptic transmission is determined by Neto1. (a,b) Representative normalized kainate receptor (KAR)-mediated EPSCs showing faster EPSC decay time constant (tau) (a) and rise time (b) in Neto1 knockout mice compare to wild littermates. (c,d) Representative traces of AMPA receptor (AMPAR)-mediated EPSCs (elicited by stimulation of associational/commissural fibers), and NMDA receptor (NMDAR)-mediated EPSCs (elicited by mossy fiber stimulation) showing no difference between WT and Neto1 knockout littermates. (e) Representative example of a KAR/NMDAR experiment. Mossy fiber-evoked mixed KAR and NMDAR responses were recorded at +30 mV in the presence of 30 μM GYKI 53655, 100 μM picrotoxin, and 3 μM CGP 55845. After a baseline was acquired, 50 μM MK-801 was washed in to isolate pure KAR-EPSCs, which were subsequently abolished in the presence of 10 μM NBQX. Inset traces depict the residual KAR-EPSC following MK-801 wash-in for Neto1 knockout and WT littermates. (f) Representative normalized AMPAR/NMDAR-EPSCs from wild-type and Neto1 knockout mice.

Figure 6

Localization of kainate receptors in the brain is independent of Neto1 and its PDZ binding domain. (a) GluK2/3 protein was observed by immunohistochemistry in s. lucidum, and no obvious difference was detected between the wild type (WT) and Neto1-knockout(−/−). Scale bars: 200 μm. High-magnification confocal microscopy in s. lucidum showed that GluK2/3 immunoreactivity (green) was unchanged in the Neto1-knockout, but was not detected in the GluK2 knockout (−/−). A presynaptic marker protein, synaptophysin (Sph; red), showed no difference among the three genotypes. Scale bars: 20 μm. (b) Relative fluorescence intensity (GluK2/Sph) from randomly selected CA3 areas was measured (n=6). (c). Biochemical fractionation of hippocampi showed enrichment of Neto1, Neto2, GluK2/3, and GluK5 in the PSD fraction together with PSD-95 and Shank1 as markers for the PSD and s. lucidum glomerulus-type synapse, respectively. (d) Protein expression in the PSD fraction. No change in protein expression of ionotropic glutamate receptors and Neto2 was observed in the Neto1-knockout. For analysis of GluN2A and GluN2B, comparison between wild type (+/+) and knockout littermates is shown (n=4). Data are given as mean ± s.e.m.; * P < 0.05, *** P < 0.005.

Figure 7

Neto1 modulates KAR-driven temporal summation and spike fidelity in CA3 pyramidal neurons. (a) Neto1 significantly contributes to the charge transfer of KAR-EPSCs elicited by brief bursts (5 pulses) of mossy fiber stimulation at 3, 10, and 30 Hz. Representative averaged traces are normalized to the peak of the first response. (b) Summary data indicate a significant attenuation of the KAR-EPCS charge transfer between wild-type and Neto1-knockout littermates. Normalized charge transfer was calculated by integrating the area under the curve for traces that were normalized to the peak amplitude of the first response. (c) Presynaptic facilitation is unchanged between wild-type and Neto1-knockout littermates. Pulse 5/Pulse 1 current amplitudes were calculated across the frequency range tested, and no significant difference was observed. (d) Neto1 can significantly impact spiking output of CA3 pyramidal cells driven by synaptic KARs. KAR-EPSPs were elicited by short bursts of MF stimulation (5 pulses) while recording from CA3 pyramidal cells in whole-cell current clamp mode. Stimulation intensity was adjusted such that a spike was elicited following the fourth stimulation 50% of the time. (e) Summary data of all experiments as in (d) (7 cells, 3 animals for WT and KO). (f) Even in the absence of glutamate receptor antagonists, spike probability during the fifth pulse was also significantly lower in Neto1 knockout animals than in their WT littermates (6 cells, 3 animals for both genotypes). Data are given as mean ± s.e.m.; * P < 0.05.