However, CNIH-2 coimmunoprecipitated with GluA1 from GluA2 KO lysates (Figure S8B), and γ-8 was coimmunoprecipitated with GluA2

from both wild-type and GluA1 KO lysates (Figure S4D). These biochemical studies demonstrate a striking specificity of CNIH-2 binding to GluA1 subunits in the hippocampus. Together, these data indicate that both the physical and functional interactions of CNIH-2 with native AMPARs require the GluA1 subunit. To evaluate the surface expression of GluA1 using immunofluorescence microscopy, we cultured dissociated rat hippocampal neurons transfected with CNIH-2 shRNA and visualized somatic and dendritic surface GluA1 immunoreactivity ∼20 days later. CNIH-2 Selleckchem Paclitaxel shRNA-transfected neurons were compared to adjacent untransfected neurons. CNIH-2 KD dramatically reduced surface GluA1 (Figures 4A and S5A), consistent with our findings showing reduction of synaptic currents. Transfection of a scrambled shRNA or GFP alone had no effect on surface GluA1 staining (Figures 4B, S5B, and S5C). Our data, thus far, demonstrate that synaptic expression of GluA1A2 AMPARs is eliminated in the absence of CNIH-2/-3. What then accounts for the fast kinetics of the remaining AMPARs observed after deleting CNIH-2/-3? Importantly, deletion of GluA1 results in the same fast kinetics, suggesting that the kinetics are a direct result of the specific molecular composition of the remaining receptors,

which are primarily GluA2A3γ-8 complexes (Lu et al., OSI-906 manufacturer 2009). Therefore, we next used heterologous cells to evaluate whether CNIH-2 affects AMPAR kinetics by specifically

regulating GluA1A2 trafficking. We coexpressed GluA2, GluA3, and γ-8 in HEK cells and measured the deactivation of this receptor complex (Figures 4C–4E). For all experiments, flip-type AMPAR subunits were evaluated (see Supplemental Experimental Procedures). GluA2A3γ-8 complex deactivation is twice as fast as GluA1A2γ-8, with GluA2A3γ-8 deactivation being virtually identical to the deactivation of AMPARs in CRE-expressing Cnih2/3fl/fl neurons ( Figure 4D). Furthermore, the difference in deactivation between GluA1A2γ-8 and GluA2A3γ-8 complexes is virtually identical to the magnitude of change in mEPSC decay in both CRE-expressing conditional GluA1 and CNIH-2/-3 (Gria1fl/fl and Cnih2/3fl/fl) KO neurons ( Figure 4E). Thus, these findings indicate that before the kinetic changes caused by the deletion of CNIH-2/-3 in neurons can be fully explained by the selective removal of the GluA1 subunit, leaving GluA2A3γ-8 complexes with faster kinetics. The lack of synaptic GluA1-containing AMPARs in the absence of CNIH-2/-3 expression may be explained by either a selective loss in total GluA1 protein expression or a specific involvement of CNIH proteins in the forward trafficking of GluA1-containing AMPARs to synapses. To examine potential effects of CNIH-2 on synaptic protein expression, Cnih2fl/fl mice were crossed to the Nex-CRE mouse line to create NexCnih2−/− mice.