The ultimate purpose of the research proposed in this application is to understand the structural organization and mechanisms of action of gap junctions. These organelles are thought to mediate and regulate the passage of low molecular weight molecules and ions between adjacent cells in an enormous variety of animal tissues. The unraveling of the mechanisms involved in cell-cell communication will be fundamental to further our understanding of normal tissue physiology (i.e., cardiac muscle), development and cell differentiation, and pathological processes such as cancer and lens cataracts. Membranes isolated from lenses of calves and mature bovines contain junctions of different morphology (16-18nm thick, 12-14nm thick, and wavy) and protein composition. They are being studied by electron microscopy, immunocytochemistry, and biochemical and electrical methodology. Specifically, the studies proposed will focus on the localization and electrical characteristics of two putative junction-forming proteins of the lens: MIP26 (the main intrinsic protein of the lens) and MP70 (present in the cortex of lenses from mature animals and in both cortex and nucleus of lenses of calves). The electrical properties of MIP26 and MP70 can be studied independently since these proteins can be isolated separately by using non-ionic detergents at low concentrations. The proteins are then completely solubilized with high concentration of the non-ionic detergent octylglucoside, reconstituted into single-walled liposomes, and fused with planar lipid films. The single and multi-channel properties of these proteins are characterized and compared with each other and those of liver gap junction proteins reconstituted by others. This application also proposes to study the molecular architecture of the gap junction channel at the secondary structural level. Hydrophobicity plots of the primary structure of liver hepatocytes and heart myocite gap junction protein strongly suggest that each monomer is constructed of four intramembrane spanning domains. Similar plots calculated from the primary structure of lens MIP26 suggest six membrane spanning domains. Cryo-electron microscopy and computer image processing will be utilized to experimentally test these predictions.
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