Abstract
Abstract: :
Purpose: Retinal pigment epithelium (RPE) regulates bi–directional ion transport between the retina and the choroid, facilitated in part by plasmalemmal ion channels. Several ion channels have been identified and characterised in the RPE, but this work is far from complete. The aim of the current study was to provide a more comprehensive analysis of the ion channels expressed in RPE using DNA microarray technology. Methods: A database comprising genbank accession and unigene numbers for human ion channel genes (alpha subunits) was compiled following a review of the literature and homology searches with known ion channel protein sequences. Total RNA was isolated from ARPE–19 cells after four days or one month in culture. RNA was reverse transcribed and hybridised to a cDNA microarray containing 12,000 expressed human sequences (Agilent, Human 1). Fluorescent labelling was performed with a 3DNA Submicro Kit (Genisphere) and spot intensities were measured using ScanAlyze. Ion channel data was extracted from microarrays using VectorXpression (Informax) in conjunction with our database, and key results validated by RT–PCR. Results: A database of 252 ion channels was created, of which 137 were represented on the human microarray. The same six ion channels were the most highly expressed in both proliferating (4–d) and quiescent (1–mo) RPE cells. Of these, an inward rectifier potassium ion channel (KCNJ13) and a chloride channel (CLCN5) have been previously characterised in RPE. The remaining four channels, a gap junction membrane channel protein (GJA1), a gamma–aminobutyric acid receptor (GABRA1), a polycystic kidney disease cation channel (PKD2) and a voltage dependent anion channel (VDAC1) have not previously been identified. Conclusions: This study has demonstrated the use of a focussed database to extract specific information from RPE cDNA microarrays. Evidence has been obtained for the presence of known and previously uncharacterised ion channels in RPE. The most highly expressed channels appear characteristic of both proliferating and mature RPE.
Keywords: retinal pigment epithelium • gene/expression • ion channels