One long term objective is to contribute to the prevention of cataracts. However, a cataract is the ultimate outcome of most alterations in normal physiological properties of the lens. We must therefore first understand how a normal lens maintains transparency. We are addressing this goal by studying biophysical properties of the intact lens, transport properties of isolated lens epithelial cells and functional properties of isolated lens proteins. We are using the techniques of molecular biology to identify, clone and express lens membrane proteins. The most prevalent protein in lens fiber cell membranes is MIP. It has been hypothesized to be a gap junction, a volume regulator and a neutral solute transporter, but we have no direct data on its function. We have cloned MIP from the frog lens and are expressing it in frog oocytes. Membrane properties, volume regulation and cell coupling will be studied in oocytes with and without MIP. We will also do the """"""""inverse"""""""" experiment of injecting total lens mRNA with and without MIP to determine if MIP works synergistically with other protein(s) to perform its function. This line of study will be extended to other membrane proteins. One of the more important transport proteins in the lens is the Na/K pump, which is localized to cells near the surface of the lens. This protein consists of alpha and beta subunits of which there are 3 isoforms of the alpha-subunit. Our recent data suggest that functional properties as well as hormonal regulation of the different isoforms are very different. We will use dihydro-ouabain to specifically and reversibly inhibit the Na/K pump and thus measure its rate of transport and changes in transport induced by regulatory factors. These studies will mostly be done on isolated lens epithelial cells using the whole cell patch damp technique. In the intact normal lens there is a complicated steady-state circulation of ionic current, which we believe is followed by fluid flow. The regional localization within the lens of specific transport properties drives these fluxes. We will use linear frequency domain impedance techniques to determine membrane conductance and selectivity in different regions of the lens. These techniques also determine the pattern of gap junctional coupling between cells. Fluorescent dyes will be injected into cells at various locations within the lens and the pattern of dye movement will provide data on local cell coupling as well as fluid movement. Moreover, calcium and hydrogen indicator dyes will be used to determine the local concentration of these ions. From these data, we will create a model of fluxes in the intact lens and relate the pattern of flow to particular transport properties of the constituent cells.
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