The avascular lens is coupled electrically and metabolically by communicating junctions, clusters of multimeric membrane intrinsic proteins radially arrayed to form a hydrophilic channel in the fiber plasmamembrane, and which conjoin to multimers in an adjoining fiber excluding the extracellular space. The channel protein, 28.2 kD in size, and the majority membrane protein, has been well-characterized by its immunology, biochemistry, and recently - complete amino acid sequence. Evidence suggests that these and other channels can be closed by H+, Ca++, and activated calmodulin (CaM). Preliminary studies from this laboratory have shown that isolated and purified lens channel proteins insert symmetrically into phospholipid vesicles and self-associate to form hemi-channels. The permeability and gating of these reconstituted channels have been studied using a spectrophotometric isosmotic swelling assay. The channels allow passage of solutes up to 1.5 kD and can be closed by activated CaM, lowered pH, but not by Ca++ alone. Interestingly, the trypsin cleaved 21 kD product, having lost its C-terminus, still forms hemichannels, but can be closed only to large permeants. The general aim of this proposal is to continue these lens channel reconstitution studies.
The specific aims are: (1) to enhance the reliability and efficiency of the isolation, purification, insertion, and self-assembly of lens channel proteins into phospholipid vesicles by varying extraction pardigms, incorporation conditions, and the nature of the lipid matrix, (2) characterize the incorporated vesicles by deep-etch and rotary shadowing and confirm the presence of hemichannels and suspected gating modulators by immunoelectron microscopy. (3) study the interrelationship of pH, Ca++, and Ca++ modulators in carefully prepared Ca++-EGTA buffers. (4) examine the self-assembly, permeability, and gating modulation of modified lens channel proteins which mimic some of the subspecies of this protein found in the lens. These modifications will include proteolysis, phosphorylation, glycosylation, and acylation. (5) determine the channel bore characteristics by kinetic analysis of swelling rates using a variety of permeants: size, shape, charge, sign and polarity (6) use the reconstituted lens junction system as an in vitro model for aging: compare gating in channels reconstituted from nuclear and cortical areas, examine the permeability of channels incorporated using peroxidized lipids, study the gating and dose response curve for lens channels exposed to near-UV in the absence and presence of photosensitizers. (7) study the electrical properties and gating of channels in large vesicles, prepared by Zimmerman cell fusion, using patch clamp techniques, and (8) study the behavior of asymmetrically incorporated lens channels, and the effect of calcium modulators and antagonists in artificial bilayers by voltage clamp dipping experiments. The significance of this proposal is the development and application of a simple reconstituted lens channel assay system to study the gating and permeability regulation of these channels under tightly controlled conditions.
