Transfection of CNIH-2 alone did not rescue synaptic AMPA receptors whereas transfection with γ-8 produced mEPSCs that decayed with a τ of ∼2.5 ms (Figure 7D). Importantly, coexpression of CNIH-2 with γ-8 slowed mEPSCs (τ∼4 ms) and did not have significant effects on amplitude relative to wild-type or γ-8-transfected stargazer granule cells (Figure 7D). Taken together, these results show that CNIH-2 can modulate decay kinetics of synaptic AMPA receptors through synergic actions with γ-8-containing receptors. We next evaluated

for CNIH-2 modulation BKM120 mouse of cyclothiazide (CTZ) actions on kainate-evoked currents (IKA) from AMPA receptors, for which the hippocampal neuronal phenotype has yet to be recapitulated with coexpression of GluA and TARP subunits. Previous studies CX-5461 mouse found that CTZ potentiates kainate-evoked currents ∼2-fold in hippocampal neurons (Patneau et al., 1993), whereas in oocytes injected with GluA1 + γ-8, CTZ augments kainate-evoked currents by only ∼40% (Tomita et al., 2007a). In the present studies, CTZ minimally potentiated kainate-evoked currents from GluA1o/2 + γ-8 (Figures 8A5 and 8B). By contrast, CTZ potentiation of kainate-evoked currents for GluA1o/2 alone was ∼12-fold (Figures 8A1 and 8B), which was not significantly different from

CTZ-potentiated kainate-evoked currents from GluA1o/2 + CNIH-2 (∼7-fold). Importantly, coexpression of CNIH-2 with γ-8 modulated GluA1o/2 receptors to yield CTZ potentiation of kainate currents of ∼2-fold, which was quantitatively similar to that observed in acutely isolated hippocampal neurons (Figures 8A3, 8A6, and 8B). The effect of CNIH-2 on CTZ-mediated potentiation of kainate-evoked currents was sensitive to a 50% reduction in the amount of CNIH-2

transfected, which minimized the potentiation of kainate currents to near γ-8 alone levels (Figure 8A4). These data suggest that CNIH-2 stoichiometry in AMPA receptors may modulate CTZ pharmacology (Figure 8B). Furthermore, this requirement for both γ-8 and CNIH-2 to produce hippocampal AMPA receptor-like kainate/CTZ pharmacology was also observed for transfections with GluA1i/GluA2 heteromeric receptors (Figure S7). Cultured hippocampal neurons transfected with CNIH-2 all shRNA exhibited reduced CTZ potentiation of IKA (Figure 8B). CNIH-2 knockdown also produced resensitization in only one out of nine hippocampal neurons (data not shown), supporting the hypothesis that complete elimination of CNIH-2 expression is necessary to reveal γ-8-mediated resensitization, whereas a graded stoichiometric mechanism likely explains the effect of CNIH-2 on kainate/CTZ pharmacology. Collectively, these results indicate that γ-8 and CNIH-2 are required to recapitulate native hippocampal AMPA receptor complexes.