The overall goal of this research is to elucidate the molecular basis of visual excitation and adaptation in retinal rod cells. We plan to carry out the following enzymatic, spectroscopic, structural, and electrophysiological studies of rod outer segment proteins: (1) The light-triggered amplification cycle in rod outer segments involving photoexcited rhodopsin, transducin, and the cyclic GMP phosphodiesterase (PDE) will be investigated in molecular detail. Fluorescence energy transfer studies will be carried out to establish the location of the subunits of transducin relative to each other, R*, and the disk membrane. (2) The structural differences between membrane-bound and soluble GTP- transducin will be determined and the functional significance of the two forms will be ascertained by reconstitution experiments. (2) Energy transfer studies will also be carried out to determine how activated transducin reverses the inhibition of PDE imposed by its gamma subunit. Specifically, does transducin displace the gamma subunit from the inhibited holoenzyme or does it carry it away? (3) A synthetic gene for the gamma subunit has been prepared for site-specific mutagenesis studies.
The aim i s to pinpoint the region of gamma that binds to the catalytic subunits and blocks their activity It will also be interesting to engineer mutants that irreversibly inhibit PDE. (4) Nonhydrolyzable analogs of cGMP will be used to determine where most of the cGMP in rod outer segments is bound and how its uptake and release are controlled. Defects of cGMP buffer sites may be important in the pathogenesis of some degenerative diseases of the retina. (5) We have recently found that guanylate cyclase is activated by small decreases in the concentration of Ca2+ in the vicinity of 10-7 M. The control of guanylate cyclase by Ca2+ is likely to be important for recovery and adaptation. The calcium-binding protein mediating this highly cooperative effect will be purified and its mechanism of regulation will be investigated. (6) The molecular architecture of the cGMP- activated channel in the plasma membrane will be probed by fluorescent analogs of cGMP. Energy transfer will be used as a spectroscopic ruler to map the channel. Fluorescence and conductance studies of reconstituted membranes containing purified channel protein will define the allosteric mechanism by which cGMP cooperatively opens the conductance pathway. The conductance properties of channels containing a defined number of covalently attached cGMP analog molecules should be highly informative in revealing how cGMP allosterically opens the channel. A detailed understanding of the cyclic GMP cascade of vision will be highly rewarding in understanding signal transduction processes generally and molecular diseases arising from defective coupling.
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