It is the long term goal of this research to identify and characterize at a molecular level, transport proteins and receptors in corneal cells which contribute to maintenance of corneal deturgescence and transparency. This includes determining their primary amino acid sequence, prevalence, regulation, specific mechanisms of function and relative role in the maintenance of the health of corneal cells.
Four specific aims are proposed: l) Using microdissection, molecular biology, and patch voltage clamping, determine the location of ion channels in both corneal epithelium and endothelium. 2) For corneal epithelium, determine the relative role of its major ion channels in controlling the transmembrane voltage using molecular biology, ratio imaging, cell culture, and patch voltage clamping. This includes determining the primary amino acid sequence of the potassium channels and non-selective cation channels and how each channel is regulated. 3) For corneal endothelium, determine the relative role of its major ion channels in controlling the transmembrane voltage using molecular biology, ratio imaging, cell culture, and patch voltage clamping. This includes determining the primary amino acid sequence of the potassium channels and non-selective cation channels and how each channel is regulated. 4) Compare channel sequences in corneal cells to those in ciliary epithelium and conjunctival epithelium. 5) Characterize the channel and receptor proteins in corneal keratocytes using molecular biology, cell culture patch voltage clamping, and imaging. Determine whether or not the cells contain muscarinic receptors and if so what types. Sequence the channel proteins and determine their sensitivity to toxins and Prozac. Quantify dye and electrical coupling between the cells. Polymerase chain reaction and DNA sequencing techniques will be used to determine the primary amino acid sequence of each transporter or receptor. Site directed mutagenesis and transfections into expression systems will be used along with patch voltage clamp to measure at a whole cell and single channel level the current flow through these transporters to assess structure-function relationships for each as well as to determine how each is regulated. The roles of selected drugs and/or antibodies in regulating transporter function will be investigated.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY006005-13
Application #
2444289
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1987-07-01
Project End
2000-06-30
Budget Start
1997-07-01
Budget End
1998-06-30
Support Year
13
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
City
Rochester
State
MN
Country
United States
Zip Code
55905
Rae, J L; Levis, R A (2004) Fabrication of patch pipets. Curr Protoc Neurosci Chapter 6:Unit 6.3
Mathias, Richard T; Rae, James L (2004) The lens: local transport and global transparency. Exp Eye Res 78:689-98
Ou, Yijun; Strege, Peter; Miller, Steven M et al. (2003) Syntrophin gamma 2 regulates SCN5A gating by a PDZ domain-mediated interaction. J Biol Chem 278:1915-23
Rae, James L; Levis, Richard A (2002) Single-cell electroporation. Pflugers Arch 443:664-70
Rae, J L; Shepard, A R (2000) Kir2.1 Potassium channels and corneal epithelia. Curr Eye Res 20:144-52
Rae, J L; Shepard, A R (2000) Kv3.3 potassium channels in lens epithelium and corneal endothelium. Exp Eye Res 70:339-48
Rich, A; Farrugia, G; Rae, J L (1999) Effects of melatonin on ionic currents in cultured ocular tissues. Am J Physiol 276:C923-9
Shepard, A R; Rae, J L (1999) Electrically silent potassium channel subunits from human lens epithelium. Am J Physiol 277:C412-24
Shepard, A R; Rae, J L (1999) ""Microprep"" method for rapidly isolating plasmid DNAs for restriction enzyme analysis. Biotechniques 26:868-70
Shepard, A R; Rae, J L (1998) Ion transporters and receptors in cDNA libraries from lens and cornea epithelia. Curr Eye Res 17:708-19

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