title: Expression of transient receptor potential vanilloid channels TRPV5 and TRPV6 in retinal pigment epithelium.
abstract: OBJECTIVE
  Hydration and ionic composition of the subretinal space (SRS) is modulated by the retinal pigment epithelium (RPE). In particular calcium concentration (Ca(2+)) 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 Ca(2+) indicator dyes to follow [Ca(2+)](i) as a function of changes in [Ca(2+)](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, [Ca(2+)](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.
authors.affiliation: Department of Cellular & Integrative Physiology, Indiana University School of Medicine-Northwest, Gary, IN 46408, USA. brkenned@iun.edu
authors.forename: Brian G
authors.initials: BG
authors.lastname: Kennedy
authors.forename: Asad J
authors.initials: AJ
authors.lastname: Torabi
authors.forename: Rafal
authors.initials: R
authors.lastname: Kurzawa
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Echtenkamp
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Mangini
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000814:Aniline Compounds / Q000378:metabolism; D015153:Blotting, Western; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002648:Child; D005260:Female; D005453:Fluorescence; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D021381:Protein Transport; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050916:TRPV Cation Channels / Q000235:genetics / Q000378:metabolism*; D014966:Xanthenes / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; D015220:Calcium Channels; D050916:TRPV Cation Channels; C492239:TRPV5 protein, human; C422827:TRPV6 protein, human; D014966:Xanthenes; C059715:Fluo-3
references.citation: Exp Eye Res. 1977 Sep;25(3):235-48
references.pmid: 304010
references.citation: Adv Exp Med Biol. 2002;514:225-35
references.pmid: 12596924
references.citation: Invest Ophthalmol Vis Sci. 2001 Jul;42(8):1921-9
references.pmid: 11431462
references.citation: J Pharmacol Exp Ther. 1999 Jun;289(3):1447-53
references.pmid: 10336538
references.citation: Exp Eye Res. 1991 May;52(5):591-8
references.pmid: 2065727
references.citation: Graefes Arch Clin Exp Ophthalmol. 2008 May;246(5):685-92
references.pmid: 18270725
references.citation: Mol Vis. 2008;14:2340-8
references.pmid: 19096717
references.citation: Virology. 1974 Dec;62(2):483-99
references.pmid: 4432376
references.citation: Exp Eye Res. 1999 May;68(5):629-36
references.pmid: 10328977
references.citation: J Physiol. 2001 Dec 15;537(Pt 3):747-61
references.pmid: 11744752
references.citation: Vis Neurosci. 1999 Nov-Dec;16(6):1157-67
references.pmid: 10614595
references.citation: J Neurosci. 1997 Apr 1;17(7):2324-37
references.pmid: 9065493
references.citation: Invest Ophthalmol Vis Sci. 1989 Oct;30(10):2114-7
references.pmid: 2551835
references.citation: Invest Ophthalmol Vis Sci. 2007 Dec;48(12):5767-72
references.pmid: 18055830
references.citation: J Biol Chem. 2008 Feb 15;283(7):4077-86
references.pmid: 18077461
references.citation: Invest Ophthalmol Vis Sci. 1993 Nov;34(12):3408-18
references.pmid: 8225876
references.citation: J Physiol. 1999 Nov 1;520 Pt 3:745-59
references.pmid: 10545141
references.citation: Pigment Cell Res. 1990 Sep;3(3):141-5
references.pmid: 2290785
references.citation: Exp Eye Res. 1996 Nov;63(5):547-56
references.pmid: 8994358
references.citation: Cell Signal. 2008 Jun;20(6):1159-68
references.pmid: 18395422
references.citation: Biochem Biophys Res Commun. 2005 Nov 11;337(1):241-7
references.pmid: 16188231
references.citation: Cell Calcium. 2007 Aug;42(2):225-32
references.pmid: 17368756
references.citation: Mol Cell Biol. 2007 Feb;27(4):1486-94
references.pmid: 17178838
references.citation: Mol Vis. 2007 Mar 27;13:443-56
references.pmid: 17417605
references.citation: J Gen Physiol. 1992 Feb;99(2):263-90
references.pmid: 1319462
references.citation: Cell Calcium. 2003 May-Jun;33(5-6):497-507
references.pmid: 12765695
references.citation: Cell Calcium. 2009 Apr;45(4):331-9
references.pmid: 19157541
references.citation: Am J Physiol Renal Physiol. 2008 May;294(5):F1212-21
references.pmid: 18305097
references.citation: Am J Physiol. 1990 Jul;259(1 Pt 1):C29-34
references.pmid: 2372049
references.citation: Graefes Arch Clin Exp Ophthalmol. 1998 Dec;236(12):929-33
references.pmid: 9865624
references.citation: Curr Eye Res. 1996 Jan;15(1):9-15
references.pmid: 8631209
references.citation: Curr Eye Res. 1993 Mar;12(3):261-70
references.pmid: 8482115
references.citation: Mol Vis. 2002 Nov 11;8:422-30
references.pmid: 12432340
references.citation: Arch Ophthalmol. 1972 Jul;88(1):63-9
references.pmid: 5049457
references.citation: Br J Pharmacol. 2001 Oct;134(3):453-62
references.pmid: 11588099
references.citation: Am J Physiol. 1994 Mar;266(3 Pt 1):C676-83
references.pmid: 8166231
references.citation: Exp Eye Res. 1997 Dec;65(6):821-34
references.pmid: 9441706
references.citation: J Biol Chem. 2007 Aug 3;282(31):22582-91
references.pmid: 17550901
references.citation: Biochem Soc Trans. 2007 Feb;35(Pt 1):115-9
references.pmid: 17233615
references.citation: Physiol Rev. 2005 Jul;85(3):845-81
references.pmid: 15987797
references.citation: Exp Eye Res. 1994 Mar;58(3):331-42
references.pmid: 7513648
references.citation: Prog Retin Eye Res. 2007 May;26(3):263-301
references.pmid: 17258931
references.citation: Exp Eye Res. 2000 Feb;70(2):183-90
references.pmid: 10655143
references.citation: Curr Eye Res. 1992 Mar;11(3):195-201
references.pmid: 1375137
references.citation: Vis Neurosci. 1994 Jul-Aug;11(4):753-61
references.pmid: 7918225
references.citation: Vision Res. 1990;30(6):853-61
references.pmid: 2385926
references.citation: Vis Neurosci. 2005 Mar-Apr;22(2):163-70
references.pmid: 15935109
references.citation: Invest Ophthalmol Vis Sci. 1995 Feb;36(2):373-80
references.pmid: 7843908