Purpose: Hydration and ionic composition of the subretinal space (SRS) is modulated by the retinal pigment epithelium (RPE). In particular calcium concentration (Ca2+) in the SRS varies with light exposure, and although this change is regulated by RPE transport activity, the specific transport proteins involved have yet to be defined. Two members of the transient receptor potential vanilloid family, TRPV5 and TRPV6, are calcium selective ion channels and are known to be expressed in calcium-transporting epithelial tissues. The present work characterizes of TRPV5 and TRPV6 in RPE. Methods: Reverse transcriptase PCR was used to examine the presence of TRPV5 and TRPV6 mRNA in cultured human RPE. Protein expression was assessed by western blotting using TRPV5- and TRPV6-specific antibodies. Immunocytochemistry was employed to examine subcellular localization of TRPV5 and TRPV6 in frozen, formaldehyde-fixed sections of native RPE-choroid tissue and in cultured human RPE monolayers. Finally, TRPV5/TRPV6 activity was assessed in cultured RPE, using Ca2+ indicator dyes to follow [Ca2+]i as a function of changes in [Ca2+]o with and without addition of the TRPV5/TRPV6 inhibitor ruthenium red. Results: Direct sequencing of PCR DNAs documented the presence of TRPV5 and TRPV6 transcripts in human RPE. Immunocytochemistry showed that TRPV5 and TRPV6 are expressed in native RPE-choroid tissue with strong immunoreactivity for both channels on the apical as well as the basal plasma membranes. Immunostaining for both channels was also positive in monolayers of cultured RPE cells. In cultured cells subcellular localization was variable with immunoreactivity present in the cytoplasmic domain as well as on the plasma membrane. Plasma membrane staining was increased with phagocytosis. The reported molecular weight of the core protein for both TRPV5 and TRPV6 is about 75 kDa, with the expected size of the glycosylated proteins in the range of 85-100 kDa. Western blot analysis of TRPV6 in RPE detected a distinct band at approximately 85 kDa, with another strong band at approximately 60 kDa. A similar pattern was seen for TRPV5, with strong bands at 82 kDa and 71 kDa. In live-cell imaging experiments, [Ca2+]i was lower in the presence of the TRPV5/TRPV6 inhibitor ruthenium red. Conclusions: RPE expresses the epithelial calcium channels TRPV5 and TRPV6, the most calcium-selective channels of the TRP superfamily. Present findings suggest that these channels could function in RPE to mediate calcium influx from SRS and thus regulate changes in SRS calcium composition that accompany light/dark transitions.
|Original language||English (US)|
|Number of pages||11|
|State||Published - Aug 20 2010|
ASJC Scopus subject areas