Abstract
Purpose: :
Retinal ON-bipolar cells (ON-BPCs) hyperpolarize in darkness in response to glutamate released from photoreceptors, and depolarize in response to the light-induced decrease in the synaptic glutamate level. These changes in membrane potential are mediated by the metabotropic glutamate receptor, mGluR6, which signals via a G-protein (Go) to control the gating of a cation channel. Apart from Go, the identity of the other components of the mGluR6-based signaling pathway, including the cation channel remain elusive. To facilitate identification of additional components of the pathway, we have generated a transgenic mouse line with GFP-tagged ON-BPCs.
Methods: :
A transgenic mouse was generated with the mGluR6 promoter (Ueda et al., 1997) driving the expression of the bacterial Cre recombinase in ON-BPCs. In double transgenic mice carrying the mGluR6-Cre gene and the ACTB-βgeo floxed GFP transgene (Novak et al., 2000), ON-BPCs are fluorescent. Retinas from these mice were dissociated with papain, and the GFP-expressing cells were purified by fluorescence-activated cell sorting (FACS). Total RNA was prepared from the FACS-purified cells, as well as from total retina. mRNA was amplified, applied to an Illumina Expression Array, and data was analyzed using the Genesifter program.
Results: :
Of the 46,642 markers analyzed, the expression of 4,097 markers was detected in ON-BPCs (p<0.05). Of these, 2,937 markers were enriched at least 7.3 fold in ON-BPCs, the level of enrichment obtained for mGluR6. As expected these include transcription factors (BLHB8, LHX6), G protein signaling proteins (Gβ2) and ion channels. Among the ion channels, TrpM1 was shown to be enriched 11-fold.
Conclusions: :
Reduced expression of TrpM1 has been shown to lead to congenital stationary night blindness in the Appaloosa Horse (Bellone et al., 2008). Together, with our data indicating that TrpM1 is expressed in mouse ON-BPCs, this raises the possibility that TrpM1 could be the ion channel mediating the depolarizing response of ON-BPCs to light.
Keywords: bipolar cells • gene microarray • ion channels