It is the overall aim of this proposal to use the techniques of patch voltage clamping and whole cell voltage clamping to localize and characterize the ionic channels which exist in lens membranes and in corneal epithelial and endothelial membranes and to find blockers for the channels. The work proposed is intended to begin the detailed characterization of channels and blockers that is required if future channel blocker therapy of anterior segment diseases is to become a reality.
The specific aims are as follows: 1. Identify the various channel types from lens membranes (epithelial and fiber), corneal epithelial membranes from all epithelial layers and from corneal endothelial membranes. We hope to be able to record from both the apical and basal surfaces of all of the cells of interest. This will require the development of techniques to isolate all of the cell surfaces of interest and to assess the quality of the cells following the isolation procedures. 2. Characterize each of the frequently occurring channels with respect to: a. Selectivity for important physiological ions b. Voltage dependence of gating c. Channel """"""""density"""""""". d. Apparent number of conformational states e. Specific and non-specific blockers 3. Compare the channel population in several species to determine if the localization of channel types is consistent from one species to another. Also do a detailed comparison of the properties of similar appearing channels which occur in more than one tissue or more than one species to assess if the channels have important differences in different locations. 4. Determine the macroscopic conductance properties of each of the cell types following isolation from their respective tissues. The measurements will be made using the technique of whole cell voltage clamping with several different ionic gradients and drugs present.
| 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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