In this study, we report that KAR slow kinetics and high affinity binding in the brain, presumably the two most distinct properties of native KARs, are determined by the auxiliary subunit Neto1. This KAR auxiliary subunit augments the agonist binding affinity and off-kinetics of KARs. Additionally, Neto1 controls the decay kinetics and amplitude of KAR-EPSCs by modulating the channel properties, rather than KAR synaptic expression. As a result of this modification, the postsynaptic KAR/Neto1 complex can regulate KAR-EPSC spike generation, indicating a potential role for this complex in modulating neural circuit function.
A perennial question in the field has been how the distinct distribution of high-affinity KAR in the brain is defined. Strong [3
H]kainate binding signals can be observed in the hippocampal s. lucidum6
. However, this experimental approach alone cannot distinguish between binding to pre or postsynaptic KARs. Here, we found that disruption of Neto1 in CA3 pyramidal cells, as indicated by our anatomical, biochemical and electrophysiological evidence, diminished most of the [3
H]kainate binding (), consistent with functional evidence that the KARs in the s. lucidum
are predominantly localized postsynaptically41
. However, we still detected 50% residual high-affinity KARs in biochemical binding experiments. The residual binding could be the result of KARs associated with the Neto1 homologue Neto2, which is also expressed in CA3 pyramidal cells albeit at lower levels ()38
. Alternatively, this binding could reflect presynaptic KARs that lack Netos. To distinguish between these possibilities, an analysis of the Neto1/ Neto2 double knockout will be required.
Several KAR binding proteins have been reported to support synaptic localization of KARs, and most are PDZ binding proteins that recognize the GluK2 PDZ binding motif31, 32, 34, 35, 42
. Neto1 possesses a typical class 1 PDZ binding domain (-TTRV) at its C-terminus. Indeed, a PDZ dependent interaction of Neto1 with PSD-95 was previously reported36
, although we could not confirm this interaction in vivo
(). Our findings show that loss of Neto1 impaired KAR-mediated synaptic transmission without obvious changes in the distribution of KARs. We therefore conclude that the KAR itself, but not Neto1, possesses the signal for its synaptic localization. Consistent with this possibility, a reduction in synaptic KAR distribution has recently been reported in GluK4/5 double knockouts, indicating that the GluK4/5 subunits partly determine the synaptic localization of the KAR complex43
. We also found that GluK2/3 binds preferentially to Neto2 in the cerebral cortex, suggesting a robust interaction between GluK2/3 and Neto2 (). It is worth noting that the GluK2/3 antibody we used likely recognizes one of three GluK2/3 splicing isoforms44
. An alternative scenario is that Neto1 and Neto2 might interact preferentially with specific KAR splicing isoforms.
Another query of longstanding contention has been that the slow decay kinetics of native KAR-EPSCs are significantly different from the fast kinetics of recombinant KARs expressed in heterologous cells. The underlying explanation for this discrepancy has been elusive, while various mechanisms have been proposed1, 2, 44, 45
. For example, KARs could be located extrasynaptically and be activated by glutamate spillover. However, this possibility was discarded early on given that reducing glutamate diffusion and/or antagonizing glutamate reuptake failed to alter the kinetics and amplitude of KAR-EPSCs7, 10, 14
. Another possibility is that the presence of GluK5 could confer slow gating properties to native KARs. However, as GluK5 slows down glutamate-induced currents of recombinant GluK2/5 in heterologous cells46
, KAR-EPSCs are slightly faster in GluK5 knockout mice47
. Importantly, our results reveal that the decay kinetics of KAR-EPSCs at the mf-CA3 synapse is significantly faster in Neto1-knockout mice () strongly suggesting that Neto1 is a major determinant in generating the characteristically slow decay kinetics of native KAR-EPSCs.
A recent study has reported that Neto1 is an NMDAR-interacting protein36
. According to this study, Neto1 co-immunoprecipitates with NMDARs and also regulates NMDAR-mediated transmission and NMDAR-dependent LTP in the CA1 region of the hippocampus. In contrast to these findings, we were unable to demonstrate any biochemical interaction between Neto1 and the NMDAR, or change in NMDAR-mediated transmission at synapses impinging on CA3 pyramidal cells where Neto1 is strongly expressed36, 38, 48
and the current study. This apparent discrepancy might be caused by differences in the genetic background of the two Neto1 KO lines. The Neto1 KO line in the present study was generated using VGB6 ES cell lines derived from C57BL/6NTAC
and backcrossed with C57BL/6J at least 6 times, whereas the other Neto1 KO line36
was generated using the R1 ES cells (129X1/Sv
J) and backcrossed with C57BL/6J, presumably rendering a 129/BL6 hybrid genetic background. Notably, Neto1 mRNA is strongly expressed in hippocampal CA3 pyramidal cells and CA1 interneurons36, 38
, a pattern more consistent with the functional expression of KARs, and our findings that Neto1 is an auxiliary subunit specific to KARs.
In addition to mediating synaptic transmission (e.g. KAR-EPSCs), KARs can also regulate transmitter release4
. However, we found no functional or anatomical evidence that Neto1 regulates presynaptic KAR function. We have shown that the capability of KAR-EPSCs to elicit action potentials from CA3 pyramidal cells in a robust and temporally precise manner is diminished in the Neto1-knockout. These findings, when viewed in the context of the function of the hippocampal tri-synaptic circuit, could be relevant to specific types of hippocampal memory function.