MOLECULAR AND CELLULAR CONSEQUENCES OF DISEASE ASSOCIATED PROCESSING DEFECTS IN CONE PHOTORECEPTOR CYCLIC NUCLEOTIDE-GATED CHANNELS

Abstract

Cyclic nucleotide-gated (CNG) channels are heterotetrameric (2A3:2B3) cation channels critical to the molecular cascade that couples light stimulus to membrane potential changes in photoreceptor cells. CNG channels are mostly confined to the continuously shed outer segment (OS) of these highly specialized cells. The function and viability of photoreceptors depend on the structure, localization, and performance of CNG channels. Many loss-of-function mutations associated with human retinal-degenerative disease result in intracellular accumulation of CNG channels. Misfolded and trafficking-defective membrane proteins typically are degraded through the ubiquitin-proteasome system (UPS). Accumulation of proteins in the endoplasmic reticulum (ER) causes ER stress and triggers the unfolded protein response (UPR), a cellular program that restores homeostasis or induces apoptosis. Yet the cues and mechanisms of degradation and the involvement of UPR-mediated cell death in the pathophysiology of these disorders remain unexamined. We show that cone CNG channels expressed in a cone photoreceptor-derived cell line are degraded by the proteasome after successive ubiquitin conjugation. Overexpression of ubiquitin accelerated degradation of CNGB3 subunits. In addition, CNGB3 degradation was increased by coexpression with wild-type CNGA3 or by a deletion promoting CNGA3-independent forward trafficking. Disease-associated mutations that cause CNGA3 folding and trafficking defects led to increased ubiquitination and degradation of both CNGA3 and coexpressed CNGB3 subunits. Ubiquitination of native CNG channels was confirmed in chicken retinal lysates, further implicating the UPS in the disposal of CNG channels. Taken together, these results indicate that CNG channels are subject to the UPS in accordance with quality control directives, localization cues, and intersubunit interactions. In addition, we show that trafficking defective CNGA3 subunits cause ER stress and invoke the UPR. ER retention was associated with increased expression of UPR-related markers of ER stress and with decreased cell viability. Chemical and pharmacological chaperones (TUDCA, 4PBA, and the cGMP analog CPT-cGMP) differentially reduced degradation and/or promoted plasma-membrane localization of defective subunits. Improved subunit maturation was concordant with reduced expression of ER-stress markers and improved viability of cells expressing localization-defective channels. These results indicate that ER stress can arise from expression of localization defective CNG channels, and may represent a contributing factor in photoreceptor degeneration.