Cornichon proteins determine the subunit composition of synaptic AMPA receptors

Abstract

Cornichon-2 and cornichon-3 (CNIH-2/-3) are AMPA receptor (AMPAR) binding proteins that promote receptor trafficking and markedly slow AMPAR deactivation in heterologous cells, but their role in neurons is unclear. Using CNIH-2 and CNIH-3 conditional knockout mice, we find a profound reduction of AMPAR synaptic transmission in the hippocampus. This deficit is due to the selective loss of surface GluA1-containing AMPARs (GluA1A2 heteromers), leaving a small residual pool of synaptic GluA2A3 heteromers. The kinetics of AMPARs in neurons lacking CNIH-2/-3 are faster than those in WT neurons due to the fast kinetics of GluA2A3 heteromers. The remarkably selective effect of CNIHs on the GluA1 subunit is probably mediated by TARP γ-8, which prevents a functional association of CNIHs with non-GluA1 subunits. These results point to a sophisticated interplay between CNIHs and γ-8 that dictates subunit-specific AMPAR trafficking and the strength and kinetics of synaptic AMPAR-mediated transmission.

Figure 1. CNIH-2 deletion selectively reduces synaptic AMPAR-mediated transmission

(A–B) Scatter plots show amplitudes of AMPA and NMDA-eEPSCs for single pairs of neurons from Cnih2^fl/fl mice (open circles) and mean ± SEM (filled circles). The scatter plots represent data obtained from acute mouse slices infected with rAAV-CRE-GFP at P0 (red circles) and cultured mouse slices transfected with CRE for 2–3 weeks (black circles). Distributions show a reduction in AMPAR-eEPSC amplitude but no change in NMDAR-eEPSC amplitude. Insets show sample current traces from Control (black) and CRE expressing (green) cells. Bar graphs show mean ± SEM AMPAR and NMDAR-eEPSC amplitudes presented in scatter plots (A, Control [Ctl], 169.2 ± 24.3 pA; CRE, 77.4 ± 10.3 pA; n = 19; *p < 0.001; B, Ctl, 36.5 ± 4.7 pA; CRE, 32.5 ± 5.9 pA; n = 16; p = 0.3). (C) Average AMPAR-eEPSC decay kinetics from pairs of Ctl (black circles) and CRE-infected cells (green circles) (mean Ctl decay ± SEM, 14.4 ± 1.3 ms; mean CRE ± SEM, 11.3 ± 1.4 ms; n = 8; *p < 0.01). Inset shows peak normalized sample traces. (D–E) Bar graphs show mean ± SEM mEPSC amplitude (D, Ctl, 10.0 ± 0.4 pA; n = 8; CRE, 7.6 ± 0.3 pA; n = 8; *p < 0.01) and decay kinetics (E, Ctl, 10.0 ± 0.8 ms; n = 8; CRE, 6.4 ± 0.4 ms; n = 8; *p < 0.01) of Ctl and CRE-infected neurons from Cnih2^fl/fl mice. Average traces are shown to the left, and are peak normalized in (E). (F–G) Bar graphs show mean ± SEM AMPAR deactivation (F, Ctl, 3.6 ± 0.2 ms; n = 12; CRE, 2.7 ± 0.2 ms; n = 18; *p < 0.002) and desensitization (G, Ctl, 13.2 ± 0.8 ms; n = 14; CRE, 8.7 ± 0.4 ms; n = 19; *p < 0.0001) from outside-out patches pulled from Ctl and CRE-infected cells and exposed to 1 and 100 ms applications of 1 mM glutamate, respectively. Peak normalized sample traces are shown to the left. (H) Bar graph shows mean ± SEM 1 mM glutamate-induced current amplitudes from outside-out patches pulled from Ctl and CRE-infected cells (Ctl, 870 ± 148 pA; n = 12; CRE, 458 ± 46 pA; n = 17; *p < 0.01). Sample traces are shown to the left. (I) Bar graph shows mean ± SEM I_KA/I_Glu ratios from outside-out patches pulled from Ctl and CRE-infected cells that were exposed to 1 mM glutamate and 1 mM kainate (Ctl, 0.54 ± 0.03; n = 5; CRE, 0.51 ± 0.03; n = 12; p = 0.56). Sample traces are shown to the left. See also Figure S2.

Figure 2. Deletion of CNIH-2/-3 closely resembles GluA1 elimination

(A–B) Scatter plots show amplitudes of AMPA and NMDA-eEPSCs of Ctl and CRE-transfected neurons in cultured slices from Cnih3^fl/fl mice. Distributions show no change in AMPAR-eEPSCs or NMDAR-eEPSCs. Insets show sample current traces. Bar graphs show mean ± SEM AMPAR and NMDAR eEPSC amplitudes presented in scatter plots (A, Ctl, 77.0 ± 11.1 pA; ΔCNIH-3, 83.6 ± 14.2 pA; n = 10; p = 1; B, Ctl, 42.5 ± 5.6 pA; ΔCNIH-3, 35.7 ± 8.3 pA; n = 10; p = 0.5). (C–D) Scatter plots showing mean amplitudes of AMPA and NMDA-eEPSCs ± SEM of Ctl and CRE-transfected neurons in cultured slices from Cnih2/3^fl/fl mice. Distributions show a reduction in AMPAR-eEPSCs (C, Ctl, 242.5 ± 48.4 pA; ΔCNIH-2/-3, 52.6 ± 9.4 pA; n = 10; *p < 0.01) but no reduction of NMDAR-eEPSCs (D, Ctl, 39.8 ± 5.7 pA; ΔCNIH-2/-3, 34.1 ± 4.7 pA; n = 10; p = 0.2). Insets show sample current traces. (E–F) Bar graphs normalized to Ctl ± SEM summarizing eEPSC data from Cnih2^fl/fl, Cnih3^fl/fl and Cnih2/3^fl/fl mice compared to Gria1^fl/fl mice. The light brown bars are published data from the Gria1^fl/fl mouse (Lu et al., 2009). (G–H) Bar graphs show mean ± SEM mEPSC amplitude (G, Ctl, 10.0 ± 0.4 pA; n = 8; ΔCNIH-2/-3, 8.2 ± 0.3 pA; n = 7; *p < 0.01) and decay kinetics (H, Ctl, 10.0 ± 0.7 ms; n = 8; ΔCNIH-2/-3, 5.0 ± 0.5 ms; n = 7; *p < 0.001) of Ctl and CRE-infected neurons from Cnih2/3^fl/fl mice. Average traces are shown to the left and are peak normalized in (H). (I–J) Bar graphs normalized to Ctl ± SEM summarizing mEPSC data from Cnih2^fl/fl, Cnih3^fl/fl and Cnih2/3^fl/fl mice compared to Gria1^fl/fl mice (Lu et al., 2009). (K) Mean ± SEM AMPA-eEPSCs in wild-type (black) and ΔCNIH-2/-3 (green) neurons before and after a whole cell LTP pairing protocol (arrow; V_m = 0 mV, 2 Hz Schaffer collateral stimulation for 90 s normalized to average eEPSC amplitude prior to LTP induction. LTP was severely decreased in ΔCNIH-2/-3 neurons (Ctl, n = 6; ΔCNIH-2/-3, n = 8). Sample traces before and 30–45 min after pairing are shown to the right for Ctl (black) and ΔCNIH-2/-3 (green) neurons. See also Figure S3.

Figure 3. GluA1 is required for CNIH-2’s physical and functional interaction with AMPARs

(A–F) Scatter plots show amplitudes of AMPA and NMDA-eEPSCs of Ctl and CNIH-2 shRNA transfected neurons in cultured slices from wild-type, GluA2 KO and GluA1 KO mice. Distributions show that the CNIH-2 shRNA reduces AMPAR-eEPSCs in wild-type (A, Ctl, 102.5 ± 16.5 pA; CNIH-2 shRNA, 52.0 ± 8.6 pA; n = 11; *p < 0.05) and GluA2 KO mice (C, Ctl, 128.9 ± 18.2 pA; CNIH-2 shRNA, 40.2 ± 5.1 pA; n = 10; *p < 0.05) but not GluA1 KO mice (E, Ctl, 54.8 ± 13.1 pA; CNIH-2 shRNA, 58.1 ± 12.3 pA; n = 9; p = 0.4). No effects were seen on NMDAR-eEPSCs (B, Ctl, 44.3 ± 7.0 pA; CNIH-2 shRNA, 42.0 ± 4.5 pA; n=10; p = 1; D, Ctl, 39.4 ± 5.1 pA; CNIH-2 shRNA, 34.9 ± 7.4 pA; n = 9; p = 0.4; F, Ctl, 84.9 ± 18.1 pA; CNIH-2 shRNA, 79.5 ± 23.3 pA; n = 8; p = 0.8). Insets show sample current traces. Bar graphs to the right show mean ± SEM AMPAR and NMDAR-eEPSC amplitudes presented in scatter plots. (G–H) Bar graphs normalized to Ctl ± SEM summarizing AMPAR and NMDAR-eEPSC data from CNIH-2 shRNA transfection of wild-type, GluA2 KO and GluA1 KO mice. (I) Immunoprecipitation of GluA2, GluA1 and CNIH-2 from hippocampal lysates of one wild-type mouse and two GluA1 KO mice using antibodies against GluA2 and GluA2/3. See also Figure S4.

Figure 4. Residual GluA2A3 heteromers can account for the effects of CNIH elimination on AMPAR kinetics

(A–B) Immunolabeling of surface GluA1 in untransfected dissociated rat hippocampal neurons (yellow arrows) compared to neurons transfected with either CNIH-2 shRNA (A) or a scrambled shRNA (B) (white arrows). Somatic dark regions are by-products of the confocal image thickness. Dendritic regions of transfected (1) and untransfected (2) neurons are shown at a higher magnification below. (C) Peak normalized sample traces showing AMPAR deactivation in outside-out patches from HEK cells transfected with GluA1A2 and γ-8 or GluA2A3 and γ-8. (D) Bar graph showing mean ± SEM deactivation of GluA1A2γ-8 and GluA2A3γ-8 complexes and the change in AMPAR deactivation kinetics in outside-out patches from ΔCNIH-2 and ΔCNIH-2/-3 CA1 pyramidal neurons (GluA1A2 + γ-8, 3.9 ± 0.4 ms; n = 10; GluA2A3 + γ-8, 1.8 ± 0.2 ms; n = 10; p < 0.001; wild-type. 3.6 ± 0.2 ms; n = 12; ΔCNIH-2, 2.7 ± 0.2 ms; n = 18; *p < 0.002; ΔCNIH-2/-3, 1.6 ± 0.2 ms; n = 6; *p < 0.0001). (E) Bar graph showing mean ± SEM deactivation of GluA2A3γ-8 complexes normalized to GluA1A2γ-8 complexes in outside-out patches from HEK cells (Glu) compared to the percent change in mEPSC decay (mEPSC) in ΔCNIH-2/-3 and ΔGluA1 CA1 pyramidal neurons. See also Figure S5.

Figure 5. CNIH-2 deletion impedes AMPAR trafficking with little effect on other synaptic proteins

(A) Bar graphs show mean ± SEM AMPA/NMDA ratios of primary neurons in CA1, dentate gryrus and barrel cortex from wild-type, NexCnih2^+/− and NexCnih2^−/− mice. (CA1, Ctl, 3.6 ± 0.5; n = 8; NexCnih2^+/−, 3.7 ± 0.5; n = 5; NexCnih2^−/−, 1.8 ± 0.2; n = 8, *p < 0.001), (DG, Ctl, 3.6 ± 0.3; n = 8; NexCnih2^−/−, 1.7 ± 0.2; n = 8) and (BC, Ctl, 2.9 ± 0.5; n = 5; NexCnih2^−/−, 1.6 ± 0.2; n = 6; *p < 0.05). AMPA and NMDA sample current traces from CA1 of wild-type and NexCnih2^−/− mice normalized to NMDAR current at 150 ms are shown to the left. (B–C) Scatter plots showing that transfection of NexCnih2^−/− neurons with CNIH-2 restores the AMPAR-eEPSC amplitude to wild-type levels. Bar graphs to the right of scatter plots show corresponding mean ± SEM eEPSC amplitudes (B, NexCnih2^−/−, 53.3 ± 16.9 pA; NexCnih2^−/− + CNIH-2, 109.1 ± 29.6 pA; n = 7; *p < 0.05; C, NexCnih2^−/−, 60.2 ± 8.7 pA; NexCnih2^−/− + CNIH-2, 55.5 ± 7.7 pA; n = 7; p = 0.8). Insets show corresponding sample traces. (D) Immunoblots from hippocampal lysates of wild-type and NexCnih2^−/− mice comparing expression levels of synaptic proteins. Bar graph to the right shows average synaptic protein levels in NexCnih2^−/− mice normalized to wild-type mice ± SEM (CNIH-2, 0.04 ± 0.008; GluA1, 0.84 ± 0.033; GluA2, 0.82 ± 0.057; γ-8, 0.97 ± 0.062; PSD-95, 0.97 ± 0.039; NR2A, 1.01 ± 0.081; n = 3–5; *p < 0.05). (E) Immunoblots from hippocampal lysates of wild-type NexCnih2^−/− and γ-8 KO mice comparing total GluA1, GluA2, γ-8 and CNIH-2 expression levels. Bar graph to the right shows average GluA1, GluA2, γ-8 and CNIH-2 expression levels in NexCnih2^−/− and γ-8 KO mice normalized to wild-type mice ± SEM (NexCnih2^−/− mice: GluA1, 0.83 ± 0.03; GluA2, 0.89 ± 0.02; γ-8, 0.99 ± 0.05; CNIH-2, 0.05 ± 0.02; n = 3; γ-8 KO mice: GluA1, 0.49 ± 0.05; GluA2, 0.50 ± 0.04; γ-8, 0.03 ± 0.01; CNIH-2, 0.28 ± 0.02; n = 3; *p < 0.05). (F) Glycosylation analysis of GluA1 and GluA2 in wild-type and NexCnih2^−/− mice. The representative blot to the left shows the relative amount of mature GluA1 receptor subunits (blue arrows) to immature GluA1 subunits (red arrows) in hippocampal lysates from wild-type and NexCnih2^−/− mice. Bar graph to the right shows the average ratio of immature to mature GluA1 and GluA2 subunits in NexCnih2^−/− mice normalized to wild-type mice ± SEM (GluA1, 1.99 ± 0.28; GluA2, 1.70 ± 0.13; n = 3–5; *p < 0.05). (G) Biotinylation analysis of GluA1, GluA2, γ-8 and CNIH-2 in dissociated hippocampal neurons. See also Figure S6.

Figure 6. γ γγ-8 blocks CNIH-2’s functional interaction with GluA2 but not GluA1

(A) Bar graph shows mean ± SEM deactivation kinetics of GluA1 homomers expressed in HEK cells alone, with γ-8, with CNIH-2 and with γ-8 and CNIH-2 (Ai, GluA1, 1.8 ± 0.2 ms, n = 10; GluA1 + γ-8, 4.9 ± 0.3 ms, n = 8; GluA1 + CNIH-2, 8.7 ± 0.6 ms, n = 11; GluA1 + γ-8 + CNIH-2, 9.4 ± 0.7 ms, n = 12). Mean ± SEM I_KA/I_Glu ratios for GluA1 + γ-8 and GluA1 + γ-8 + CNIH-2 were also compared (Aii, GluA1 + γ-8, 0.57 ± 0.03, n = 8; GluA1 + γ-8 + CNIH-2, 0.54 ± 0.05, n = 5). (B–C) Bar graphs show mean ± SEM deactivation kinetics of GluA2(Q) homomers and GluA1A2(R) heteromers expressed in HEK cells alone, with γ-8, with CNIH-2 and with γ-8 and CNIH-2 (B, GluA2(Q), 1.2 ± 0.2 ms, n = 8; GluA2(Q) + γ-8, 4.5 ± 1.0 ms, n = 4; GluA2(Q) + CNIH-2, 10.0 ± 1.5 ms, n = 7; GluA2(Q) + γ-8 + CNIH-2, 6.0 ± 0.7 ms, n = 6) (C, GluA1A2(R), 1.7 ± 0.4 ms, n = 6; GluA1A2(R) + γ-8, 3.9 ± 0.4 ms, n = 10; GluA1A2(R) + CNIH-2, 11.7 ± 1.0 ms, n = 6; GluA1A2(R) + γ-8 + CNIH-2, 5.7 ± 0.5 ms, n = 9). Corresponding peak normalized sample traces are shown to the left of bar graphs. See also Figure S7.

Figure 7. CNIH-2 slows synaptic AMPAR currents in the absence of γγ γ-8

(A–B) Bar graphs show mean ± SEM mEPSC amplitude (A) and decay (B) of wild-type, NexCnih2^−/−, CNIH-2 overexpressing (OE), γ-8 KO and γ-8 KO + CNIH-2 CA1 neurons in slice culture (A, wild-type, 17.4 ± 1.6 pA; n = 8; NexCnih2^−/−, 9.5 ± 0.5 pA; n = 9; CNIH-2 OE, 17.3 ± 1.6 pA; n = 5; γ-8 KO, 10.7 ± 0.9 pA; n = 9; γ-8 KO + CNIH-2, 19.1 ± 3.5 pA; n = 7; *p < 0.05) (B, wild-type, 6.3 ± 0.4 ms; n = 8; NexCnih2^−/−, 4.4 ± 0.3 ms; n = 9; CNIH-2 OE, 6.4 ± 0.4 ms; n = 5; γ-8 KO, 7.8 ± 0.6 ms; n = 9; γ-8 KO + CNIH-2, 14.2 ± 0.63 ms; n = 7; *p < 0.05). Select color-matched sample traces are shown above bar graphs. Sample traces in (B) are peak normalized. Note that compared to acute slices baseline mEPSC amplitude is larger and mEPSC kinetics are faster in slice culture (see Supplemental Experimental Procedures).

Figure 8. Model of CNIH and γ γγ-8 interactions with AMPARs

(A) GluA1 AMPAR subunits simultaneously associate with CNIH proteins and TARP γ-8. Therefore, we propose surface tetrameric GluA1 homomers associate with 4 γ-8 molecules and 1–4 CNIH molecules. (B) CNIH protein association with GluA2 AMPAR subunits appears to be prevented by γ-8. Therefore, in the presence of γ-8, we propose surface GluA2 homomers associate with 4 γ-8 molecules and 0 CNIH molecules. (C) Because of GluA1 and GluA2’s respective relationships with γ-8 and CNIH proteins, we propose surface GluA1A2 heteromers associate with 4 γ-8 molecules and 1–2 CNIH molecules. (D) Because GluA1 is required for the physical association of CNIH proteins but not γ-8 with AMPARs in neurons, we propose surface GluA2/3 heteromers associate with 4 γ-8 molecules and 0 CNIH molecules. (E) In neurons CNIH proteins selectively promote the trafficking of GluA1A2 heteromers but not GluA2A3 heteromers to the neuronal surface. γ-8 prevents CNIH interaction with non-GluA1 subunits and provides a mechanism for the subunit specific action of CNIH on GluA1A2 receptor trafficking. Overexpression of CNIH in wild-type neurons does not slow AMPAR gating kinetics indicating CNIH cannot displace γ-8 on non-GluA1 subunits. Together these data suggest a model whereby in the ER/Golgi γ-8 associates with AMPARs prior to CNIH (1) thus limiting subsequent CNIH interactions to only GluA1 subunits, which uniquely associate with both γ-8 and CNIH (2). CNIH proteins would then selectively enable the forward trafficking of GluA1A2 heteromers to the neuronal surface (3). CNIH deletion prevents GluA1A2 receptors from leaving the ER/Golgi.