Abstract
Purpose: :
The light-induced decrease in glutamate release from photoreceptors results in the opening of a nonselective cation channel in ON-bipolar cells. This channel is known to be negatively coupled to the metabotropic glutamate receptor, mGluR6, via the heterotrimeric G-protein, Go. Despite intensive investigation over the past decade, the molecular identity of the cation channel has remained elusive. Recently, we identified several cation channels belonging to the TRP family that were enriched in ON-bipolar cells (see poster by Duvoisin et al., ARVO 2009). We aimed to determine the cellular distribution of these channels in the retina by immunohistochemistry.
Methods: :
The expression and subcellular localization of TRP channels, including TRPM1, was assessed in the mouse and macaque retina by immunofluorescence confocal microscopy. Human TRPM1 and mouse TRPM1-GFP cDNAs were expressed in HEK293 and tsa-201 cells by transient transfection.
Results: :
Immunohistochemical analysis demonstrates that TRPM1 is found in the retina where it is localized exclusively to the dendrites and cell bodies of ON-bipolar cells. Double labeling for TRPM1 and ON-bipolar cell markers indicates that the channel is not in the tips of the dendrites with mGluR6, but is located further down the dendrites toward the cell body. The specificity of the TRPM1 antibody was confirmed by staining HEK293 cells transfected with TrpM1 cDNA.
Conclusions: :
Recently, it has been shown that reduced TRPM1 expression in the retina of Appaloosa horses results in congenital stationary night blindness (CSNB) and reduced ON-bipolar cell depolarization in ERG recordings (Bellone et al., 2008). Our results demonstrate that TRPM1 is localized in the dendritic region of ON-bipolar cells, but it remains to be determined if TRPM1 is, in fact, the cation channel coupled to mGluR6. Future directions will include a pharmacological comparison between expressed TRPM1 channels and the mGluR6-modulated current, as well as an evaluation of ON-bipolar cell responses in the TRPM1-/- mouse.
Keywords: bipolar cells • ion channels • immunohistochemistry