We next directly tested if the effects of deleting CNIH-2/-3 are specifically related to the regulation of GluA1. To this end we compared the effects of CNIH-2 knock-down on AMPAR-eEPSCs in GluA1 and GluA2 KO mice. The shRNA we generated was highly effective in knocking down CNIH-2 protein levels (Figure S4A) and in wild-type neurons produced a phenotype identical to knocking out CNIH-2 (Figures 1A–B and 3A–B). The knock-down of CNIH-2 in neurons from GluA2 KO mice, which primarily express GluA1 homomers, also resulted in a selective but more pronounced reduction in the AMPAR-eEPSC compared to wild-type mice (Figures 3C–D and G–H). In striking contrast, CNIH-2 knock-down in slices from GluA1 KO mice had no effect on AMPAR-eEPSCs (Figure 3E), AMPAR mEPSC kinetics (Figure S4B) or NMDAR eEPSCs (Figure 3F) demonstrating that CNIH-2 effects on synaptic AMPARs require GluA1. The eEPSC results are summarized in Figures 3G and H. Additionally, residual GluA2A3 receptors in GluA1 KO neurons were found to have a IKA/IGlu ratio of ~0.5 suggesting that all available TARP binding sites on these receptors are occupied (Figure S4C).
