The tetraspanin disc proteins, peripherin-2 and ROM1, facilitate CNG channel localization to the rod outer segment.

The light-responsive outer segment of rod photoreceptors is composed of two distinct membrane subdomains: discs and the plasma membrane that surrounds them. To understand how proteins are delivered to these subdomains, we focused on the plasma membrane-specific cyclic nucleotide-gated (CNG) channel. It was previously reported that the β1 subunit of the CNG channel is absent from rods in peripherin-2 knockout ( Rds -/- ) mice. It is unclear how the disc-resident protein peripherin-2 would impact the localization of CNGβ1 in the plasma membrane. In this study, we investigate how peripherin-2 is engaged in CNG channel delivery to the outer segment. We overexpressed a MYC-tagged CNGβ1 in Rds -/- rods and found that it is trapped in intracellular membranes. CNGβ1 localization was restored to the outer segment when full-length, FLAG-tagged peripherin-2 was expressed. This led us to investigate the region of peripherin-2 that is required for CNGβ1 delivery by expressing peripherin-2 chimeras containing either the N-terminus, tetraspanin core, or C-terminus. We show that the peripherin-2 tetraspanin domain is sufficient to localize CNGβ1 to the outer segment. Recent studies have shown that peripherin-2 and ROM1 act redundantly in outer segment disc formation and enclosure. Consistent with this, we found that expression of ROM1 chimeras containing its tetraspanin region could also restore CNGβ1 localization. We postulated that the structural properties of these tetraspanin proteins could facilitate CNG channel delivery to the outer segment. Interestingly, this notion is supported by endogenous staining of CNGβ1, which reappears in aged Rds -/- rods that have produced ciliary membrane protrusions.