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R. L. Brown, Robert M. Duvoisin, Catherine W. Morgans, Neal S. Burke; Regulation Of TRPM1 By The Metabotropic Glutamate Receptor 6. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4126.
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TRPM1 was recently identified as the non-selective cation channel that generates the depolarizing light response in retinal ON-bipolar cells. The purpose of these experiments was to optimize the expression of the mGluR6-TRPM1 signaling pathway in a heterologous system to facilitate further study of the regulatory mechanisms.
We transfected Chinese Hamster Ovary (CHO) tissue culture cells with plasmids encoding different combinations of mGluR6, G-alpha-o, and TRPM1, or G-protein regulated inward rectifier potassium (GIRK) channels. Whole cell patch clamp techniques were used to measure ion channel currents as the cells were exposed to extracellular solutions containing mGluR6 agonists.
In some cells, we observed a constitutively-active linear current that was inhibited by application of glutamate and/or L-AP4. This current was, however, small (~ 50 pA at - 60 mV) and successful expression and regulation by mGluR6 was variable. Confocal fluorescence microscopy confirmed that transfected cells expressed both mGlUR6 and TRPM1. We have tested several factors to investigate this variability, including the functional expression of mGluR6. By using GIRK currents as a readout of mGluR6 function, we determined that most transfected cells did not express functional mGluR6 at the cell surface until 3 days after transfection. In contrast, a CHO cell line that was stably transfected with mGluR6 demonstrated effective regulation of potasium currents within a day after transient transfection with the GIRK channels. The development of this stably-transfected mGluR6-CHO cell line should facilitate successful reconstitution of the mGluR6-TRPM1 pathway.
We have successfully expressed TRPM1 in CHO cells, and demonstrated regulation by mGluR6, similar to the results previously published by Koike et al. (2010). We are currently investigating why successful reconstitution is variable.
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