Ion channels have a major role in maintaimng the transmembrane voltage in epithelial cells in addition to their participation in many different cell signaling mechanisms. Through their action on transmembrane voltage and via their ion permeation pathways, they influence the regulation of intracellular Na+ and intracellular Ca++ as well as all transport processes that are voltage dependent. It is the overall goal of this research to identify at a molecular level, all of the channels in the corneal epithelium and endothelium involved in regulating Na+ and Ca++ movements and promoting cellular homeostasis and ion transport in these important epithelia. We propose three specific aims: 1) Identify, characterize, and localize the K+ channels in the epithelium and endothelium. 2) Sequence the Na+ and Ca+ channels in the epithelium and endothelium. 3) Identify, characterize, and localize selected C1- channels in the epithelium and endothelium. The following approaches will be utilized for studying each channel identified: 1) Determine both the nucleotide and amino acid sequence. 2) Clone the channel cDNA into an expression plasmid, express the protein in a mammalian cell line, and determine biophysical functions of the channel. 3) Make a fusion protein with green fluorescence protein (GFP) analog and transfect the DNA into a mammalian expression system. Verify that the fusion protein functions like the channel protein alone. Use the GFP fusion protein to study targeting of the channel protein in natural ocular preparations. Our goal is to end the application period with sequences, clones in expression vectors, and channel-GFP fusion protein clones in expression vectors for each important channel in corneal epithelium and endothelium. We also hope to have done sufficient characterization of each to have unequivocally identified it, localized it and quantified its general properties. Once the channel amino acid sequences are known, drugs aimed at modulating cell signaling mechanisms and enhancing ion transport in these cells can be developed.
| 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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