The general goal of this proposal is elucidation of the mechanism by which light generates the receptor potential in photoreceptors. This process can be divided into early stages involving the light- sensitive molecule rhodopsin, intermediate stages involving chemical amplification, and the final stage in which the chemical message affects ion channels, thereby generating the receptor potential. An aspect of the early steps, the average number of G- proteins activated by a single isomerized rhodopsin, will be investigated in experiments involving agents which block or stimulate G-protein. In addition the possibility of using peptides to inhibit specific reactions involving rhodopsin will be explored by dialyzing such peptides into tiger salamander rods or Limulus photoreceptors. The main questions involving intermediate events have to do with the new hypothesis that a rise in cGMP mediates the excitation of invertebrate photoreceptors. This will be studied in biochemical assays of the effect of light on guanylate cyclase activity, phosphodiesterase activity, and the concentration of cGMP in the living squid eye. Physiological methods will be used to study the effect of putative activators and inhibitors of enzymes involved in cyclic nucleotide metabolism. The general goal is to determine which enzyme is responsible for light-dependent changes in cGMP and how this enzyme might be controlled. Finally, anatomical methods will be used to identify the structural basis for the very inhomogeneous sensitivity to injected cGMP. Ongoing work on the light- activated channels in Limulus using the patch clam method will be continued. Preliminary results indicate that these channel events have two sizes. We will attempt to determine if these are due to two different states of the same channel or to two different channel types. These channels will also be studied in excised patches where it is possible to test whether substances of interest can have direct effects on the channel.
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