Substantial changes in CA2+ concentration ([CA2+]) in the fluid surrounding photoreceptors (subretinal space or SRS) follow light/dark transitions. Because Ca2+ affects photoreceptor function, SRS [Ca2+] must be tightly regulated. Though it is known that transport activity in the retinal pigment epithelium (RPE) subserves this regulatory role, the specific mechanisms have not been identified. All cells have only 2 mechanisms that can export Ca2+ across the plasma membrane: Na=:Ca2+ exchangers (NCXs) and plasma membrane Ca2+ ATPases (PMCAs). Except for studies in this application, neither of these proteins, nor the genes that encode them, have been characterized in human RPE. This project targets these two Ca2+ transport proteins. The fundamental hypothesis underlying planned studies is that the combined activities of a PMCA and NCX in RPE regulate light-dependent changes in SRS [Ca2+], and that impaired function of either of these Ca2+ transporters will cause impaired vision. Multiple genes and alternative splicing of primary mRNA transcripts govern the tissue-specific expression and regulation of PMCA and NCX proteins (termed isoforms). Thus, testing these hypotheses requires identifying PMCA and NCX proteins as expressed by RPE, and assaying their roles in RPE physiology. These are goals of this project. Experiments use freshly isolated RPE cells and tissue from human donor eyes and other animal sources. The approach is on 3 levels, molecular (AIM1), structural (AIM2), and biochemical/functional (AIM3). Molecular studies will identify PMCA and NCX isoforms expressed in RPE, and will sequence cDNAs that encode them. Structural studies using immunoEM will identify the membrane location (apical vs. basolateral) of these Ca2+ transport proteins in native RPE tissue. Biochemical studies will identify RPE PMCA and NCX proteins (immunoblotting), and examine their basic properties in freshly isolated RPE cells. Functional studies will examine transepithelial Ca2+ transport (ion flux studies), and regulation of [Ca2+]I (Ca2+ imaging) in freshly isolated RPE tissue. Ion substitution and selective inhibitors will be used to evaluate roles of PMCA and NCX activities in these processes. Proposed studies represent a new initiative in RPE cell biology. The molecular characterization of the PMCA and NCX has been well studied in erythrocytes and excitable cells, respectively. Proposed studies will extend findings to human RPE, and will contribute generally to the field of epithelial Ca2+ transport. Planned studies will identify the physiological significance of PMCA and NCX activities in RPE, and will help to identify ocular pathologies that could be caused by impaired PMCA or NCX function.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY011308-03
Application #
2888496
Study Section
Visual Sciences C Study Section (VISC)
Project Start
1997-08-01
Project End
2000-05-31
Budget Start
1999-08-01
Budget End
2000-05-31
Support Year
3
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
121911077
City
Chicago
State
IL
Country
United States
Zip Code
60612
Kennedy, Brian G; Torabi, Asad J; Kurzawa, Rafal et al. (2010) Expression of transient receptor potential vanilloid channels TRPV5 and TRPV6 in retinal pigment epithelium. Mol Vis 16:665-75
Talarico Jr, Ernest F (2010) Plasma membrane calcium-ATPase isoform four distribution changes during corneal epithelial wound healing. Mol Vis 16:2259-72
Talarico Jr, Ernest F; Mangini, Nancy J (2007) Alternative splice variants of plasma membrane calcium-ATPases in human corneal epithelium. Exp Eye Res 85:869-79
Talarico Jr, Ernest F; Kennedy, Brian G; Marfurt, Carl F et al. (2005) Expression and immunolocalization of plasma membrane calcium ATPase isoforms in human corneal epithelium. Mol Vis 11:169-78
Lamason, Rebecca L; Mohideen, Manzoor-Ali P K; Mest, Jason R et al. (2005) SLC24A5, a putative cation exchanger, affects pigmentation in zebrafish and humans. Science 310:1782-6
Weng, T X; Godley, B F; Jin, G F et al. (2002) Oxidant and antioxidant modulation of chloride channels expressed in human retinal pigment epithelium. Am J Physiol Cell Physiol 283:C839-49
Kennedy, Brian G; Mangini, Nancy J (2002) P-glycoprotein expression in human retinal pigment epithelium. Mol Vis 8:422-30
Kennedy, B G; Haley, B E; Mangini, N J (2000) Creatine kinase in human retinal pigment epithelium. Exp Eye Res 70:183-90
Getz, R K; Kennedy, B G; Mangini, N J (1999) Transthyretin localization in cultured and native human retinal pigment epithelium. Exp Eye Res 68:629-36
Loeffler, K U; Mangini, N J (1998) Immunohistochemical localization of Na+/Ca2+ exchanger in human retina and retinal pigment epithelium. Graefes Arch Clin Exp Ophthalmol 236:929-33

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