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P. Bojang, Jr., J. N. Pearring, R. G. Gregg; Nyctalopin Interacts with Transient Receptor Potential Channels in Yeast. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5176.
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The novel protein nyctalopin is expressed in depolarizing bipolar cells (DBC) of the retina and is required for signaling from the metabotropic glutamate receptor 6 (Grm6) to a non-specific cation channel, most likely of the transient receptor potential (Trp) family. The absence of nyctalopin results in either the loss of the cation channel from the membrane or complete closure of the channel and its inability to be gated. Nyctalopin contains a leucine rich repeat (LRR) domain that is predicted to interact with other proteins and also to be located in the extracellular space. In Appaloosa horses with CSNB the expression level of Trpm1 is dramatically decreased (Bellone et al. 2008. Genetics 179:1861). Therefore, we wished to determine if nyctalopin interacted with either Trpm1 or other closely related Trp channels.
We used yeast two hybrid analyses to determine if nyctalopin interacted with Trp channels. The membrane split-ubiquitin yeast two-hybrid (MYTH) system was used. A full length cDNA that encoded nyctalopin was inserted into the bait vector pCCW-SUC vector (Dualsystems Biotech), such that the ubiquitin moiety and a LexA-VP16 transcription factor were fused to the C-terminus of nyctalopin. As a control we also cloned a mutant version of nyctalopin that was predicted to disrupt the LRR domain. To analyze interactions, full length TrpM1 and TrpV1 were cloned into the pDL2Nx prey vectors. The two vectors were co-transformed into the NYM32 yeast strain. Interaction was assessed by growth on selective media and colony lift assays.
Full length nyctalopin was expressed in yeast and the bait vectors did not show any self-activation. Co-transformation with prey vectors expressing either Trpm1 or TrpV1 showed that the two proteins interacted genetically. Control experiments utilizing the mutated version of nyctalopin did not show evidence for interaction, supporting the conclusion that nyctalopin does interact with TrpM1 and TrpV1.
Our yeast two hybrid analyses indicate that nyctalopin interacts directly with Trp channels. These data in conjunction with currently published data suggest that nyctalopin may be critical to either the expression, localization or function of the Trp channels. Reports that suggest TrpM1 mice have abnormal ERG b-waves (Koike, C. Neurosc. Res. 2007. 58S: S1-S244. Abstract O1P-E11) would suggest the interaction between nyctalopin and Trpm1 is critical for fucntion of eth DBCs.
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