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Yuan Pan, Joseph Laird, Sheila Baker; Multiple isoforms of TRIP8b, a regulator of HCN channels, are expressed in retina. Invest. Ophthalmol. Vis. Sci. 2013;54(15):705.
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Hyperpolarization-activated cyclic nucleotide-gated channels (HCN) are cation-selective channels present in retina, brain and heart. The activity of HCN channels contributes to signal integration, cell excitability and pacemaker activity. Rods express homomeric channels composed of HCN1; these channels participate in keeping light responses transient. The subcellular localization of HCN1 varies among cell types. In rods, HCN1 is found in the inner segments, to a lesser extent in the synaptic terminals, but excluded from the outer segments. In contrast, HCN1 in the hippocampus is concentrated in a subset of dendrites. A key regulator of HCN1 trafficking and activity is TPR-containing Rab8b interacting protein (TRIP8b). Up to nine splice isoforms of TRIP8b are expressed throughout the brain and can differentially regulate surface expression and activity of HCN1. The purpose of the present study was to determine which isoforms of TRIP8b are expressed in photoreceptors and to test if the C-terminus of HCN1, containing TRIP8b binding sites, could confer targeting in rods.
RT-PCR using RNA isolated from total mouse retina or photoreceptor inner segments collected by laser capture micro-dissection was used to test for the presence of specific TRIP8b splice isoforms. Immunohistochemistry was used to test for the expression of TRIP8b protein in mouse retina. To assess targeting, the C-terminus of HCN1 and deletions of this region, were fused to a membrane reporter and expressed in transgenic Xenopus rods. Localization of the transgenically expressed proteins was visualized with confocal microscopy.
We found that multiple splice isoforms of TRIP8b are expressed in photoreceptors, including those with opposing effects on HCN1 trafficking. TRIP8b protein was found in the same retina compartments as HCN1. In transgenic Xenopus rods, the membrane reporter localized primarily to outer segments but addition of HCN1’s C-terminus changed its localization to the inner segment. Deletions of HCN1’s C-terminus uncovered a strong synaptic targeting signal located in a stretch of amino acids in between the two TRIP8b binding sites. In order to achieve the normal localization pattern of HCN1, this synaptic targeting signal needs to be suppressed.
HCN1 trafficking involves interactions among multiple signals within its C-terminus, and TRIP8b is a possible regulator of this process in photoreceptors.
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