Although it is well established that CNIH-2 binds to AMPARs (Kato et al., 2010a; Schwenk et al., 2009; Shi et al., 2010), the relative binding to GluA subunits has not been reported. Because CNIH-2 knock-down has a profound and selective effect on GluA1-containing AMPARs, we compared GluA1 and GluA2 binding to CNIH-2. We first immunoprecipitated GluA2 from wild-type hippocampal lysates using two different antibodies (anti-GluA2 or anti-GluA2/3). We found that CNIH-2 coimmunoprecipitated with GluA2 from wild-type hippocampal lysates, as expected (Figure 3I). In sharp contrast, we observed no coimmunoprecipitation of CNIH-2 with GluA2 when using GluA1 KO lysates. 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 hippocampal lysates. Together these data indicate that both the physical and functional interaction of CNIH-2 with native AMPARs requires the GluA1 subunit.
