The long-term goal of this project is to understand the relation between the structure and the conductance properties of the lens junction connexon. We propose to do this by carrying out parallel electron microscopic and conductance studies on isolated, purified and reconstituted connexon protein from the bovine eye. We expect the information obtained to improve our understanding of ion transport in the lens of the eye and to clarify the roles of calcium and pH in regulating this transport. It is hoped that this knowledge may aid in understanding the role of ion transport in cataract formation. Preliminary studies show that vesicles containing junctional protein induce channels in planar bilayers which are on at zero voltage and turn off at elevated voltages of either sign. But if reconstituted into the planar bilayer directly from detergent, the junctional protein induces an entirely different kind of channel which is off at low voltage and turns on at high voltage. Conductance of these reconstituted connexon channels will be studied under conditions of varied pH and calcium concentration in planar bilayers, in patch-clamped vesicles, and in small bulged bilayers. We will push two patch-clamped vesicles or two small bulged bilayers together to determine how the reconstituted channels behave in single and double bilayers. Results will be analyzed with special emphasis on determining if the channel is controlled by a single gate or two gates in series. Parallel structural studies will be performed on both the reconstituted material and the actual bilayers in which conductance measurements are performed, the object being to obtain as close a correlation between structure and function as possible.
| Fields, James B; Németh-Cahalan, Karin L; Freites, J Alfredo et al. (2017) Calmodulin Gates Aquaporin 0 Permeability through a Positively Charged Cytoplasmic Loop. J Biol Chem 292:185-195 |
| Reichow, Steve L; Clemens, Daniel M; Freites, J Alfredo et al. (2013) Allosteric mechanism of water-channel gating by Ca2+-calmodulin. Nat Struct Mol Biol 20:1085-92 |
| Clemens, Daniel M; Németh-Cahalan, Karin L; Trinh, Lien et al. (2013) In vivo analysis of aquaporin 0 function in zebrafish: permeability regulation is required for lens transparency. Invest Ophthalmol Vis Sci 54:5136-43 |
| Hall, James E (2012) Through a glass darkly. EMBO Mol Med 4:1-2 |
