frequency (Figure S2A). To quantitatively determine the effects of CNIH-2 on AMPAR kinetics we pulled somatic outside-out patches and used ultra-fast glutamate application to measure AMPAR deactivation (Figure 1F) and desensitization (Figure 1G). Both desensitization and deactivation time constants were faster in the absence of CNIH-2. We also examined AMPAR currents generated from somatic extrasynaptic outside-out patches in the presence of cyclothiazide to block desensitization. Similar to AMPAR-eEPSCs, extrasynaptic currents were reduced by 47 ± 5% in CRE-infected neurons (Figure 1H). Furthermore, if CNIH-2 reduces the stoichiometry of TARP γ-8 binding to AMPARs as previously proposed (Gill et al., 2011; Kato et al., 2010a) then in the absence of CNIH-2 the γ-8/AMPAR stoichiometry should increase and thus, the kainate/glutamate (IKA/IGlu) ratio, a sensitive assay for γ-8/AMPAR stoichiometry (Shi et al., 2009), should also increase. However, no change in IKA/IGlu was seen in neurons lacking CNIH-2 (Figure 1I). We also observed no change in AMPAR-eEPSC rectification in the absence of CNIH-2, indicating no change in GluA2 content (Figure S2B). CNIH-2 deletion also failed to influence paired-pulse ratio indicating an exclusively post-synaptic role for CNIH-2 (Figure S2C).
