Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Nov 22;294(47):17889-17902.
doi: 10.1074/jbc.RA119.008631. Epub 2019 Oct 18.

Neto proteins regulate gating of the kainate-type glutamate receptor GluK2 through two binding sites

Yan-Jun Li  1 , Gui-Fang Duan  1 , Jia-Hui Sun  1 , Dan Wu  1 , Chang Ye  1 , Yan-Yu Zang  1 , Gui-Quan Chen  1   2 , Yong-Yun Shi  3 , Jun Wang  4 , Wei Zhang  5 , Yun Stone Shi  6   2   7
Affiliations

Neto proteins regulate gating of the kainate-type glutamate receptor GluK2 through two binding sites

Yan-Jun Li et al. J Biol Chem. .

Abstract

The neuropilin and tolloid-like (Neto) proteins Neto1 and Neto2 are auxiliary subunits of kainate-type glutamate receptors (KARs) that regulate KAR trafficking and gating. However, how Netos bind and regulate the biophysical functions of KARs remains unclear. Here, we found that the N-terminal domain (NTD) of glutamate receptor ionotropic kainate 2 (GluK2) binds the first complement C1r/C1s-Uegf-BMP (CUB) domain of Neto proteins (i.e. NTD-CUB1 interaction) and that the core of GluK2 (GluK2ΔNTD) binds Netos through domains other than CUB1s (core-Neto interaction). Using electrophysiological analysis in HEK293T cells, we examined the effects of these interactions on GluK2 gating, including deactivation, desensitization, and recovery from desensitization. We found that NTD deletion does not affect GluK2 fast gating kinetics, the desensitization, and the deactivation. We also observed that Neto1 and Neto2 differentially regulate GluK2 fast gating kinetics, which largely rely on the NTD-CUB1 interactions. NTD removal facilitated GluK2 recovery from desensitization, indicating that the NTD stabilizes the GluK2 desensitization state. Co-expression with Neto1 or Neto2 also accelerated GluK2 recovery from desensitization, which fully relied on the NTD-CUB1 interactions. Moreover, we demonstrate that the NTD-CUB1 interaction involves electric attraction between positively charged residues in the GluK2_NTD and negatively charged ones in the CUB1 domains. Neutralization of these charges eliminated the regulatory effects of the NTD-CUB1 interaction on GluK2 gating. We conclude that KARs bind Netos through at least two sites and that the NTD-CUB1 interaction critically regulates Neto-mediated GluK2 gating.

Keywords: CUB domain; N-terminal domain (NTD); allosteric regulation; channel gating; deactivation; desensitization; gating; glutamate ionotropic receptor kainate type subunit 2 (GriK2); glutamate receptor ionotropic kainate 2 (GluK2); ionotropic glutamate receptor; kainate receptor; neuropilin and tolloid-like (Neto); protein-protein interaction; receptor desensitization; recovery from desensitization.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Neto1/2 have two separate interaction sites with GluK2. A and B, immunoblotting of immunoprecipitates from transfected HEK293T cells. The identities of the transfected constructs were indicated above each lane. Full-length GluK2, GluK2NTD, and GluK2ΔNTD were co-immunoprecipitated with Neto1 and Neto2. C, GluK2NTD was co-immunoprecipitated with the CUB1 domains of Neto1/2. D, co-immunoprecipitation of GluK2ΔNTD and Neto proteins without CUB1 domains. E and F, co-immunoprecipitation of GluK2NTD and Neto proteins with or without CUB1 domains. Deletion of CUB1 domains significantly suppressed the interaction between GluK2 and Neto proteins (arrows). G, quantification of immunoprecipitation in E and F. The GluK2NTD pulled down was normalized by the FLAG signal pulled down. Compared with full-length Neto1 (1.00 ± 0.32), the GluK2NTD precipitated by Neto1ΔCUB1 was significantly reduced to 0.38 ± 0.08 (n = 4 pairs; **, p < 0.01, paired t test). Compared with full-length Neto2 (1.00 ± 0.22), the GluK2NTD pulled down by Neto2ΔCUB1 was significantly reduced to 0.24 ± 0.03 (n = 4 pairs; ***, p < 0.001, paired t test). H, a schematic model shows the two interaction sites between GluK2 and Neto proteins. Error bars, S.D.
Figure 2.
Figure 2.
Neto regulation on GluK2 desensitization. A, deletion of NTD has no effect on GluK2 desensitization. Top, superimposed average desensitization traces of GluK2 and GluK2ΔNTD. Bottom left, statistical comparison between the τdes of GluK2 and GluK2ΔNTD. Bottom right, a schematic model depicts deletion of NTD. B, modulatory effects of Neto1 and Neto2 on GluK2 desensitization. The top panel shows superimposed average desensitization traces. Bottom left, statistical comparison among the τdes of GluK2 with and without Netos. Whereas Neto1 has no effect on GluK2 desensitization, Neto2 slows GluK2 desensitization by about 4 times (***, p < 0.001). Neto1 and Neto2 exhibited differential modulation on GluK2 desensitization (GluK2 + Neto1 versus GluK2 + Neto2; ***, p < 0.001). Bottom right, a schematic model depicts the NTD-CUB1 and core-Neto interactions. C, the modulatory effects of Neto1 and Neto2 on GluK2ΔNTD desensitization. The top panel shows superimposed average desensitization traces. Bottom left, statistical comparison among the τdes of GluK2ΔNTD with and without Netos. Both Neto1 and Neto2 have modest slowing effects on GluK2ΔNTD (*, p < 0.05); no difference was found between GluK2ΔNTD + Neto1 and GluK2ΔNTD + Neto2. Bottom right, a schematic model depicts the core-Neto interaction under these conditions. D, CUB1-deleted Netos have no apparent effects on GluK2 desensitization. E, modulatory effects of CUB1-deleted Netos on GluK2ΔNTD. Neto1ΔCUB1 slowed the desensitization of GluK2ΔNTD (**, p < 0.01). Neot2ΔCUB1 slowed the desensitization of GluK2ΔNTD (*, p < 0.05). No significant difference was found between GluK2ΔNTD + Neto1ΔCUB1 and GluK2ΔNTD + Neto2ΔCUB1. The raw desensitization traces are depicted and the average desensitization traces are calculated in Fig. S2A. The data were analyzed using two-way ANOVA with post hoc Tukey's multiple-comparison tests (Netos or mutants, F(4, 151) = 22.80, p < 0.001; NTD, F(1, 151) = 0.16, p = 0.69; interaction, F(4, 151) = 25.69, p < 0.001). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. Error bars, S.D.
Figure 3.
Figure 3.
Neto regulation on GluK2 deactivation. A, deletion of NTD has no effect on GluK2 deactivation. B, modulatory effects of Neto1 and Neto2 on GluK2 deactivation. Top, superimposed average deactivation traces. Bottom, whereas Neto1 has no effect on GluK2 desensitization, Neto2 slows GluK2 deactivation (*, p < 0.05). Neto1 and Neto2 exhibited differential modulation on GluK2 deactivation (GluK2 + Neto1 versus GluK2 + Neto2; ***, p < 0.001). C, modulatory effects of Neto1 and Neto2 on GluK2ΔNTD deactivation. Top, superimposed average desensitization traces. Bottom, both Neto1 and Neto2 have modest slowing effects on GluK2ΔNTD (*, p < 0.05). No difference was found between GluK2ΔNTD + Neto1 and GluK2ΔNTD + Neto2. D, Neto1ΔCUB1 slightly slowed (*, p < 0.05), whereas Neto2ΔCUB1 has no effect on GluK2 deactivation. However, the effects were not significantly different between Neto1ΔCUB1 and Neto2ΔCUB1. E, modulatory effects of CUB1-deleted Netos on GluK2ΔNTD deactivation. Both Neto1ΔCUB1 and Neto2ΔCUB1 slowed the deactivation of GluK2ΔNTD (***, p < 0.001). No significant difference was found between GluK2ΔNTD + Neto1ΔCUB1 and GluK2ΔNTD + Neto2ΔCUB1. The raw deactivation traces are depicted and the average deactivation traces are calculated in Fig. S2B. The data were analyzed using two-way ANOVA with post hoc Tukey's multiple-comparison tests (Netos or mutants, F(4, 129) = 14.68, p < 0.001; NTD, F(1, 129) = 47.39, p < 0.001; interaction, F(4, 129) = 10.00, p < 0.001). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. Error bars, S.D.
Figure 4.
Figure 4.
Neto regulation on GluK2 recovery from desensitization. A, representative recording traces of GluK2 and GluK2ΔNTD evoked by pairs of 50-ms applications of 10 mm glutamate. The interval time between two applications ranged from 5 to 8000 ms. The amplitude was normalized to the first peak. B, analysis of the recovery rates of GluK2 and GluK2ΔNTD. Left, the recovery was calculated by the peak amplitude of the second response divided by that of the first response. Data points represent mean ± S.D. The average data were fitted with a single-exponential equation (black, GluK2; red, GluK2ΔNTD). Right, the time constants (τrec) were compared between GluK2 and GluK2ΔNTD. Deletion of NTD significantly sped the receptor recovery from desensitization (***, p < 0.001). C, Netos regulate GluK2 recovery from desensitization. Both Neto1 and Neto2 sped GluK2 recovery from desensitization (p < 0.05 (*) and p < 0.01 (**), respectively). D, Netos differentially regulate GluK2ΔNTD recovery from desensitization. Neto1 has no effect on GluK2ΔNTD recovery from desensitization. Neto2 slowed GluK2ΔNTD recovery from desensitization (*, p < 0.05). E, CUB1-deleted Netos differentially regulate GluK2 recovery from desensitization. Neto1ΔCUB1 has no effect on GluK2 recovery from desensitization. Neto2ΔCUB1 slowed GluK2 recovery from desensitization (***, p < 0.001). F, CUB1-deleted Netos differentially regulate GluK2ΔNTD recovery from desensitization. Neto1ΔCUB1 has no effect on GluK2ΔNTD recovery from desensitization. Neto2ΔCUB1 slowed GluK2ΔNTD recovery from desensitization (*, p < 0.05). The representative traces for recovery from desensitization are depicted in Fig. S3. The data were analyzed using two-way ANOVA with post hoc Tukey's multiple-comparison tests (Netos or mutants, F(4, 93) = 29.92, p < 0.001; NTD, F(1, 93) = 109.80, p < 0.001; interaction, F(4, 93) = 20.22, p < 0.001). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. Error bars, S.D.
Figure 5.
Figure 5.
Critical residues on CUB1 domains for NTD-CUB1 interactions. A, homology model for Neto2CUB1 domain. Left, distribution of the charges on the Neto2CUB1 surface. Negatively charged residues are shown in red, and positively charged residues are shown in blue. The molecule is polarized according to its charge distribution. Middle, the model is rotated for better view of the negatively charged pole. Right, sequence alignment around the negatively charged residues between Neto1 and Neto2. B, co-immunoprecipitation of Neto2CUB1 mutants with GluK2NTD. The interaction between GluK2NTD and Neto2CUB1 was significantly diminished when 4 negatively charged residues were mutated to alanine residues (arrow). The mutation of 5 residues on the positively charged pole did not affect CUB1 interaction with GluK2NTD. Right, bar graph shows the relative pulldown efficiency from three experiments. The GluK2NTD pulled down was normalized by the FLAG signal pulled down. C, mutation of the 3 negatively charged residues in Neto1CUB1 domain diminished its binding to GluK2NTD (arrow). Right, bar graph shows the relative pulldown efficiency from three experiments. The GluK2NTD pulled down was normalized by the FLAG signal pulled down. D, neutralization of the negative charges affects Neto regulation on GluK2 desensitization. Left, superimposed average desensitization traces of GluK2 with or without mutated Netos recorded in a parallel experiment. Right, bar graph shows the weighted τdes. GluK2, 2.69 ± 0.35 ms, n = 8; GluK2 + Neto1(DE3A), 4.10 ± 0.91 ms, n = 10; GluK2 + Neto2(DE4A), 3.75 ± 0.83 ms, n = 8. One-way ANOVA with post hoc Tukey's multiple comparisons tests was used: F(2, 23) = 7.24, p < 0.01. *, p < 0.05; **, p < 0.01; ns, not significant. E, neutralization of the negative charges affects Neto regulation on GluK2 recovery from desensitization. Left, recovery of GluK2 with or without mutant Netos recorded in a parallel experiment. Right, analysis of the recovery rates. GluK2 + Neto1(DE3A) (2.36 ± 0.77 s, n = 13) was not different from GluK2 (2.45 ± 0.92 s, n = 12). Neto2(DE4A) slowed GluK2 recovery (4.24 ± 1.40 s, n = 8; **, p < 0.01, one-way ANOVA with post hoc Tukey's multiple comparisons, F(2, 30) = 9.17, p < 0.001). Error bars, S.D.
Figure 6.
Figure 6.
Critical residues in GluK2NTD for NTD-CUB1 interaction. A, the NTD dimer of GluK2 is adapted from the cryo-EM structure of GluK2 (PDB entry 5KUF). Highly positive patches on the surface of subunit A containing at least two positively charged residues were identified (in blue). Positively charged residues scattered on the NTD surface are shown in pink. Right, residues composed of the six highly positive patches on the NTD surface. B, co-immunoprecipitation of Neto2CUB1 domain coexpressed with GluK2NTD with or without mutations. Lanes 1–6, GluK2NTD mutations carrying alanine replacement of positively charged residues identified in A. The interaction between GluK2NTD and the CUB1 domain was significantly disrupted when the 4 positively charged residues in group 1 were mutated to alanine residues (arrow). Bottom, the bar graph shows the pulldown efficiency from three experiments. C, co-immunoprecipitation of Neto2CUB1 domain with GluK2NTD mutations. 4A, the same mutant as lane 1 in B. 2A, R58A_K82A. The interaction between GluK2NTD and CUB1 domain was significantly disrupted in the 4A mutation of the GluK2NTD but not in single or double mutations. Bottom, the bar graph quantifies the pulldown efficiency from three experiments. D, Neto1 and Neto2 regulation on the desensitization of GluK2(RK4A). Both Neto1 (5.89 ± 3.01 ms, n = 19) and Neto2 (5.78 ± 1.72, n = 12) slowed the desensitization of GluK2(RK4A) (2.47 ± 0.23 ms, n = 10). **, p < 0.01, one-way ANOVA with post hoc Tukey's multiple comparisons, F(2, 30) = 9.17, p < 0.001. E, GluK2(RK4A) recovery from desensitization with or without Netos. Neto1 (1.27 ± 0.22 s, n = 13) had no effect on GluK2(RK4A) recovery (1.19 ± 0.38 s, n = 18). Neto2 (1.95 ± 0.69 s, n = 15) slowed GluK2(RK4A) recovery. ***, p < 0.001; ns, not significant, one-way ANOVA with post hoc Tukey's multiple comparisons, F(2, 43) = 11.08, p < 0.001. Error bars, S.D.
Figure 7.
Figure 7.
Schematic models summarizing NTD and Neto modulation of GluK2 gating. A, effects of the three factors, NTD, core-Neto interaction, and NTD-CUB1 interaction, on GluK2 desensitization. The model starts from the smallest functional receptor, GluK2ΔNTD. NTD has no effect on desensitization (comparison 1). Core-Neto interaction with either Neto1 or Neto2 slows desensitization (comparison 2). NTD-Neto1 speeds up desensitization, whereas NTD-Neto2 slows desensitization (comparison 3). These models can be applied for GluK2 deactivation. B, the three factors on the recovery from desensitization. Among the three factors, NTD appears to have most dramatic effects and stabilizes the receptor in the desensitization state (comparison 1). Core-Neto1 has little effect, whereas core-Neto2 slows the recovery speed either on NTD-truncated (comparison 2) or full-length GluK2 (comparison 2′). The NTD-CUB1 interaction speeds GluK2 recovery (comparison 3).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Traynelis S. F., Wollmuth L. P., McBain C. J., Menniti F. S., Vance K. M., Ogden K. K., Hansen K. B., Yuan H., Myers S. J., and Dingledine R. (2010) Glutamate receptor ion channels: structure, regulation, and function. Pharmacol. Rev. 62, 405–496 10.1124/pr.109.002451 - DOI - PMC - PubMed
    1. Lerma J., and Marques J. M. (2013) Kainate receptors in health and disease. Neuron 80, 292–311 10.1016/j.neuron.2013.09.045 - DOI - PubMed
    1. Schmitz D., Mellor J., and Nicoll R. A. (2001) Presynaptic kainate receptor mediation of frequency facilitation at hippocampal mossy fiber synapses. Science 291, 1972–1976 10.1126/science.1057105 - DOI - PubMed
    1. Volk L., Chiu S. L., Sharma K., and Huganir R. L. (2015) Glutamate synapses in human cognitive disorders. Annu. Rev. Neurosci. 38, 127–149 10.1146/annurev-neuro-071714-033821 - DOI - PubMed
    1. Zhu S., and Paoletti P. (2015) Allosteric modulators of NMDA receptors: multiple sites and mechanisms. Curr. Opin. Pharmacol. 20, 14–23 10.1016/j.coph.2014.10.009 - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources