Reliable and precise signaling at the first visual synapse, where ON bipolar cells are located, is crucial for the proper functioning of the retina. Visual disorders such as congenital stationary night blindness are the result of aberrant information transmission from photoreceptors to bipolar cells. Recently, the transient receptor potential channel TRPM1 has been implicated as the transduction channel responsible for the light response of ON bipolar cells. TRPM1 is a nonselective cation channel, kept closed in darkness by a G-protein signaling cascade coupled to the glutamate receptor mGluR6. At least 10 splice variants of the Trpm1 gene have been identified in the retina and purification of a major TRPM1 isoform indicates a dimer. However, the composition of the TRPM1-dependent channel is still unknown and the functional role of splice variants is as yet undetermined. Furthermore, the identity of the channel gating G-protein subunit remains unresolved. The goal of this project is to test the hypotheses that 1) the functional transduction channel in ON-bipolar cells is a homo- or hetero-oligomer of one or more TRPM1 splice variants and possibly other proteins; and that 2) TRPM1 splice variants interact differentially with the G-protein subunits Galpha-0 and Gbeta-gamma. These will be tested in the following specific aims: 1) to identify new TRPM1 splice variants and quantify their relative abundance in the mouse retina, using TRPM1-targeted mRNA deep sequencing; 2) to characterize channel function of TRPM1 variants in their homomeric or heteromeric state, and determine channel interactions with G-protein subunits, using patch clamp electrophysiology, live cell Ca2+ imaging and co-immunoprecipitation and 3) to identify TRPM1 binding partners in the retina using mass spectrometry, and determine their co-localization with epitope-tagged splice variants in vitro, using super resolution microscopy and proximity ligation. This study will be the first to address the functional role of TRPM1 splice variants in the retina and investigate the interactions that form the functional TRPM1-dependent channel. Improved understanding of the signal transduction mechanisms at the photoreceptor-bipolar cell synapse will help identify novel targets for therapeutic intervention in the treatmentof diseases associated with impaired transmission of visual information.
Information processing in the retina is dependent on faithful signal transmission between pre- and post-synaptic neurons and is fundamental for proper vision. ON bipolar cells are retinal post-synaptic neurons, which rely on the ion channel TRPM1 to convey visual information, but the composition of the channel is still undetermined. The goal of the proposed research is the investigation of differentially processed products of the Trpm1 gene and their importance in forming the functional channel.
