Abstract
Purpose:
Recently, the ion channel TRPM1 has been identified as the protein responsible for the sign-inverting response of retinal ON-bipolar cells. In the mouse retina there are at least five TRPM1 isoforms containing predicted transmembrane domains that arise from alternative splicing, but their distribution, or roles in the formation of the functional channel, are still unknown. The purpose of this study was to improve the resolution of TRPM1 subcellular localization and to determine whether there are differences in distribution among isoforms.
Methods:
Monoclonal antibodies generated against full-length TRPM1 and recognizing either a C-terminal epitope, found in all transmembrane-containing isoforms, or an N-terminal epitope, found only in isoforms containing exon 19, were used for protein localization. Mouse retinas were examined by fluorescent immunohistochemistry and imaged by super-resolution and confocal microscopy.
Results:
TRPM1 co-localizes with markers of ON-bipolar cell dendritic tips (RGS7, Gβ5 and mGluR6), localizes to the tips of rod bipolar cells (PKCα) and is adjacent to photoreceptor ribbons (Ribeye). Localization of TRPM1 in the dendritic arms and somas of ON-bipolar cells appears to be mostly in intracellular compartments, as it does not co-localize with the plasma membrane marker Na/K ATPase. TRPM1 expression is robust at the dendritic tips and a clear dyad of bipolar cell dendrites can be observed with confocal and structured illumination microscopy (SIM), a morphological feature typically only seen with electron microscopy. In contrast, the signal intensity from staining with N-term antibodies is much lower, especially at the dendritic tips. The distribution of TRPM1 isoforms lacking exon 19 in the dendritic arms and somas appears similar to what is observed when staining the C-term antibody, but the signal intensity is lower. Staining is absent in TRPM1 knockout mice.
Conclusions:
Long isoform TRPM1 expression at the site of action - the dendritic tips of ON-bipolar cells - is robust as detected by the C-term antibody, but most of the TRPM1 in the cells is intracellular. The extensive intracellular localization of TRPM1 isoforms in the dendrites and somas, as well as the possibility of alternative distribution in the tips of variants lacking exon 19, suggest unknown functions for the large intracellular pool and for some of the differentially processed products of the Trpm1 gene.