EARLY RECEPTOR CURRENT OF RHODOPSIN ACTIVATION

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During the initial event in vision light triggers conformational changes in rhodopsin that lead to photoreceptor excitation. Spectroscopic assays of mutant rhodopsins suggest roles for specific amino acids in the rapid conformational changes leading to the active Metarhodopsin-II state that binds and activates transducin. The Early Receptor Current (ERC), the conformation-dependent charge flow in rhodopsin during activation, can be studied using whole-cell patch clamp techniques and is the fundamental process underlying the familiar early receptor potential (ERP). The ERC provides sufficient microsecond time resolution for studying fast conformational changes in rhodopsin and permits tests of the role of specific amino acids in activation in both the chromophore and remote protein environments. The ERC technique should also be useful in studying how mutations of human rhodopsin, such as those caused by autosomal dominant retinitis pigmentosa (adRP), disrupt phototransduction and lead to photoreceptor degeneration. The ERC technique is suitable because over half of the fifty mutations in adRP are in the intramembrane region of rhodopsin. To better understand the role of specific amino acids in the activation of wild type and mutant (adRP) rhodopsins, I propose to: (1) Characterize the Early Receptor Current (ERC) produced during the activation of human rhodopsin expressed in a cell line. (2) Investigate the contribution of specific intramembrane amino acids to the ERC. (3) Use the ERC to test the hypothesis that adRP mutations decrease thermal stability of rhodopsin and create alternative paths to Metarhodopsin-II. The proposed experiments should lead to a better understanding of the fundamental biophysical processes underlying the ERC, the roles of specific amino acids in activation of rhodopsin, and how adRP leads to rhodopsin dysfunction. I propose to carry out these studies in a cellular expression system in which ERCs from normal human rhodopsin have been measured and in which rhodopsin mutants can be expressed.