Neto1 is an auxiliary subunit of native synaptic kainate receptors

Abstract

Ionotropic glutamate receptors of AMPA, NMDA, and kainate receptor (KAR) subtypes mediate fast excitatory synaptic transmission in the vertebrate CNS. Auxiliary proteins have been identified for AMPA and NMDA receptor complexes, but little is known about KAR complex proteins. We previously identified the CUB (complement C1r/C1s, Uegf, Bmpl) domain protein, Neto1, as an NMDA receptor-associated polypeptide. Here, we show that Neto1 is also an auxiliary subunit for endogenous synaptic KARs. We found that Neto1 and KARs coimmunoprecipitated from brain lysates, from postsynaptic densities (PSDs) and, in a manner dependent on Neto1 CUB domains, when coexpressed in heterologous cells. In Neto1-null mice, there was an ∼50% reduction in the abundance of GluK2-KARs in hippocampal PSDs. Neto1 strongly localized to CA3 stratum lucidum, and loss of Neto1 resulted in a selective deficit in KAR-mediated neurotransmission at mossy fiber-CA3 pyramidal cell (MF-CA3) synapses: KAR-mediated EPSCs in Neto1-null mice were reduced in amplitude and decayed more rapidly than did those in wild-type mice. In contrast, the loss of Neto2, which also localizes to stratum lucidum and interacts with KARs, had no effect on KAR synaptic abundance or MF-CA3 transmission. Indeed, MF-CA3 KAR deficits in Neto1/Neto2-double-null mutant mice were indistinguishable from Neto1 single-null mice. Thus, our findings establish Neto1 as an auxiliary protein required for synaptic function of KARs. The ability of Neto1 to regulate both NMDARs and KARs reveals a unique dual role in controlling synaptic transmission by serving as an auxiliary protein for these two classes of ionotropic glutamate receptors in a synapse-specific fashion.

Figure 1.

Neto1 and Neto2 associate with KARs in vivo. A–D, Immunoblots of immunoprecipitates from adult wild-type (+/+) and Neto1-null (Neto1^tlz/tlz) or Neto2-null (Neto2^−/−) crude synaptosomes. Both the input and the unbound fraction for the immunoblot were 2% of the volume of the sample used in the immunoprecipitation experiment. E, F, Neto1 and Neto2 are part of PSD KAR protein complexes. Immunoblot of immunoprecipitates from adult wild-type (+/+) and Neto1-null (Neto1^tlz/tlz) or Neto2-null (Neto2^−/−) PSD. The input for the immunoblot analysis is 1% of the volume of sample used in the immunoprecipitation experiment. G, The purity of PSD preparations of wild type (+/+), Neto1-null (Neto1^tlz/tlz), and Neto2-null (Neto2^−/−) mice was tested by immunoblotting for the presynaptic protein VAMP-2. Blot, Antibody used for immunoblot analysis; I, input, IP, immunoprecipitate; U, unbound fraction, to show that the Neto1 antibody can efficiently immunoprecipitate Neto1 protein from the synaptosomal samples.

Figure 2.

Generation of Neto2-null mice. Neto2 gene-targeting strategy. A, Top, A portion of the Neto2 gene showing exons (Ex). SS (signal sequence), CUB1, and CUB2 are encoded motifs; open box, noncoding sequences; solid boxes, coding sequences; P, Pstl restriction enzyme site. Middle, Neto2 targeting construct. neo^r, Neomycin resistance gene; dta, Diphtheria toxin A gene-negative selection cassette. Bottom, Targeted Neto2^−/− allele after homologous recombination. Arrows indicate the direction of transcription. The 5′ external probe is shown by a gray rectangle. B, Genomic Southern blot from ES cell clones digested with Pstl and hybridized with the 5′ probe. C, Immunoblot of brain lysates from Neto2^+/+, and Neto2-null mice using anti-Neto2 antibodies raised to the C-terminal 70 aa of Neto2. The top arrowhead indicates the specific Neto2-immunoreactive band.

Figure 3.

Neto1 and Neto2 bind to GluK2 KARs through the extracellular CUB domains. A–D, Immunoblot of immunoprecipitates from transfected HEK293 cell lysates. The identities of the transfected cDNAs are indicated above each lane. Blot, Antibody used for immunoblot analysis; IP, antibody used for immunoprecipitation; HA, hemagglutinin tag. In the diagram of deletion proteins, the dashed-line boxes represent the domain that has been deleted from the full-length protein. A, B, The anti-Neto1 antibody used for immunoblotting in A is raised against the C-terminal cytoplasmic domain of Neto1. C, D, The anti-Neto2 antibody used for immunoblotting in C is raised against the C-terminal 70 aa of Neto2. The anti-Neto2 antibody used for immunoprecipitation in D is raised against the extracellular domain of Neto2. Similar results were observed in each of three experiments.

Figure 4.

KAR-mediated EPSCs are reduced at hippocampal mossy fiber-CA3 pyramidal cell synapses in Neto1-null, but not in Neto2-null, mice. A, Confocal micrographs of immunostained hippocampal slices showing the CA3 and dentate gyrus region. Antibodies used are indicated in each box. Pyr, Pyramidal cell layer; SL, stratum lucidum; SR, stratum radiatum. Scale bars: top two panels, 100 μm; lower four panels, 20 μm. B, Representative traces of AMPAR, NMDAR, and KAR MF-CA3 EPSCs from individual wild-type (left), Neto1-null (Neto1^tlz/tlz, lower right), Neto2-null (Neto2^−/−, lower left), or Neto1/Neto2-null (Neto1^tlz/tlz/ Neto2^−/−, right) CA3 pyramidal neurons. Initially, the dual AMPAR/KAR-mediated synaptic responses (bottom two traces) were monitored at a V_h of −70 mV, then the KAR-mediated component was pharmacologically isolated by applying the AMPAR-specific antagonist GYKI 53655. The GYKI-resistant component at V_h = −70 mV was confirmed to be KAR mediated by subsequent application of DNQX in the continued presence of GYKI 53655. The holding potential is then moved to +40 mV to obtain the NMDAR-mediated component of EPSCs (top trace). Insets for the Neto1-null and Neto2-null sets of traces display the fourth KAR-mediated event at higher gain. C–E, Histograms summarizing group data for EPSC_KA/EPSC_AMPA, and EPSC_NMDA/EPSC_AMPA ratios (C), mean EPSC_KA decay time constants (D), and ratios of fourth and first AMPAR- or NMDAR-mediated EPSCs (P4/P1) (E), for wild-type (n = 10), Neto1-null (n = 14), Neto2-null (n = 12), and Neto1/Neto2-double-null (n = 12) neurons. **p < 0.01.

Figure 5.

Reduced NMDAR-mediated transmission at A/C-CA3 synapses. A, Representative traces of AMPAR-, and NMDAR-mediated EPSCs from individual wild-type (left) and Neto1-null (right) CA3 pyramidal cell recordings evoked by stimulating MF (top) or A/C (bottom) inputs. B, C, Histograms summarizing data for EPSC_NMDA/EPSC_AMPA ratios for wild-type or Neto1-null mice at MF-CA3 and A/C-CA3 synapses (B), and ratios of second to first AMPAR or NMDAR-mediated EPSCs (P2/P1) for wild-type or Neto1-null mice at MF-CA3 synapses (C) (n = 9 and 6 for MF-CA3 synapses in wild-type and Neto1-null mice respectively; n = 12 and 12 for A/C-CA3 synapses in wild-type and Neto1-null mice, respectively).

Figure 6.

KAR subunits are reduced in the hippocampal PSD of Neto1-null mice. A–E, Immunoblots and histogram of synaptic proteins from whole hippocampal homogenates and hippocampal PSD fractions from +/+ and Neto1-null (Neto1^tlz/tlz) mice (A, B); +/+ and Neto2-null (Neto2^−/−) mice (C, D); and +/+ and Neto1/Neto2-null (Neto1^tlz/tlz Neto2^−/−) mice (E, F). Antibodies used for detection are indicated on the left. Blots shown are representative of three experiments. Histogram shows normalized levels of different synaptic proteins in null-mice hippocampal homogenate relative to that of +/+ (white bars), and in null-mice hippocampal PSD relative to that of +/+ (black bars). **p < 0.01, paired t test, n = 3.