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Melina A Agosto, Theodore G Wensel; Membrane topology analysis of the ON bipolar cell transduction channel subunit TRPM1. Invest. Ophthalmol. Vis. Sci. 2016;57(12):590.
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© ARVO (1962-2015); The Authors (2016-present)
Neurotransmission between photoreceptors and downstream ON bipolar cells is mediated by a GPCR signaling cascade, for which the TRPM1 channel subunit is required for function. A high-resolution structure of TRPM1 has not yet been determined. Limited homology between the transmembrane (TM) domain of TRPM1 and those of other TRP family channels suggests that TRPM1 has six TM helices. However, no experimental data regarding the topology of the protein has been reported, and different TM prediction algorithms yield varying results. The purpose of this study is to experimentally determine the topology and orientation of TRPM1 in the membrane.
A fluorescence protease protection assay, in conjunction with live cell imaging, was employed to assess the membrane topology of TRPM1 and nyctalopin. Human embryonic kidney (HEK293) cells expressing GFP fusion proteins were treated with digitonin to selectively permeabilize the plasma membrane, followed by proteinase K to digest regions accessible to the cytoplasm.
Mouse nyctalopin (NYX), which is known to have an extracellular or ER-luminal N-terminus and a cytoplasmic C-terminus, was used as a control. As expected, the fluorescence of cells expressing NYX-GFP decreased rapidly upon addition of protease. The fluorescence after 4 min of treatment was greatly reduced compared to control cells treated with digitonin but not protease (p<0.001, Mann-Whitney test). In contrast, GFP-NYX was resistant; cells with and without protease treatment were not significantly different (p=0.176). Both GFP-TRPM1 and TRPM1-GFP were localized to intracellular membranes in HEK293 cells. Unlike nyctalopin, the fluorescence of cells expressing TRPM1 with GFP at either the N- or the C-terminus was sensitive to protease treatment (p<0.001 after 4 min).
The fluorescence protease protection assay revealed that both the N- and C-termini of TRPM1 are cytoplasmic, consistent with the presence of six TM helices. Future studies will further dissect the topology of TRPM1 and the location of the TM helices by testing the accessibility of epitope tags and protease cleavage sites engineered into predicted luminal and cytoplasmic loops.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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