Although speculative, given the likelihood that γ-8 inhibits the interaction of CNIH on GluA2 subunits, we believe that γ-8 may similarly inhibit CNIH interaction with GluA3. Previous studies, including our own, report little effect of CNIH overexpression on endogenous AMPARs. However, CNIHs clearly interact with AMPARs in heterologous cells and in neurons (Harmel et al., 2012; Shi et al., 2009; Schwenk et al., Idelalisib in vitro 2009; Kato et al., 2010a; Gill et al.,
2011, 2012). To test whether CNIHs have an important role in neurons but are expressed at saturating levels, we performed extensive analyses using genetic deletion and KD of CNIHs. Indeed, we found that deletion of CNIH-2/-3 causes a profound and selective reduction in AMPAR-eEPSC amplitude. selleck This is accompanied by faster decay of mEPSCs, faster deactivation and desensitization of glutamate-evoked currents from somatic patches, and compromised LTP induction. These results demonstrate a critical role for CNIHs in neuronal AMPAR regulation and are particularly fascinating given that the profound synaptic changes seen with the deletion of CNIH-2/-3 match those seen with the selective deletion of GluA1
(Lu et al., 2009). Because neurons lacking CNIH proteins look physiologically similar to neurons lacking GluA1, we hypothesized that removal of CNIH-2/-3 might have different effects in various AMPAR KO mice and therefore used these tools to probe CNIH-2 function. Knocking down CNIH-2 in hippocampal slices from GluA2 KO mice causes a profound reduction of AMPAR-eEPSCs, whereas knocking down CNIH-2 in slices from GluA1 KO mice has no effect, either on the amplitude or kinetics of AMPAR EPSCs. These physiological results support a selective action of CNIH-2/-3
on GluA1-containing receptors. We also found that CNIH-2 and GluA1 coimmunoprecipitate with GluA2 when using wild-type hippocampal no homogenates. However, in striking contrast, when using homogenates from GluA1 KO mice, CNIH-2 does not coimmunoprecipitate with GluA2. Furthermore, GluA2A3/γ-8 receptors, the most likely composition of the receptors remaining in neurons lacking GluA1 or CNIH-2/-3, are twice as fast as GluA1A2/γ-8 receptors. Thus, the 50% reduction in mEPSC decay observed in neurons lacking GluA1 and CNIH-2/-3 can be explained by the selective loss of synaptic GluA1-containing AMPARs. Why is the action of CNIH-2/-3 confined to the GluA1 subunit? Previous studies in heterologous systems have shown that CNIH-2 has significant effects on AMPARs containing and lacking GluA1 subunits (Schwenk et al., 2009). To address this seeming contradiction, we examined the interactions between CNIH-2 and γ-8, the most prevalent TARP in the hippocampus (Rouach et al., 2005), on the kinetics of AMPARs of defined subunit composition.