As the modest loss of AMPAR protein in the absence of CNIH-2 expression is unlikely to explain the profound effects on physiology, we next examined the effect of deleting CNIH-2 on AMPAR trafficking to the cell surface. AMPARs are glycoproteins, which traffic through the biosynthetic pathway. To determine if CNIH-2 affects AMPAR maturation, we examined receptor glycosylation using endoglycosidase H (Endo H), which digests immature high mannose sugars and PNGase F, which removes all N-linked carbohydrates. Relative to wild-type brains both GluA1 and GluA2 showed increased sensitivity to Endo H in NexCnih2−/− brains (Figure 5F and S6D), as demonstrated by stronger Endo H-sensitive immature bands (red arrows) compared to Endo H-resistant mature bands (blue arrows). These data suggest that a large pool of immature receptors are retained in the ER or cis-Golgi in the absence of CNIH-2. The Endo H-sensitive band co-migrates with completely deglycosylated receptors following treatment with PNGase F.
