The goals of this project are to determine the relationship between rod phosphodiesterase activity and rod electrophysiology, discover the ionic and biochemical consequences of PDE activation and decreased cyclic GMP levels, and to further describe the molecular events responsible for activation and regulation of the rod PDE cascade. Addition of active PDE or GTPase with hydrolysis resistant analog bound (GTPase*) to dark adapted rod outer segment (ROS) membranes in vitro causes cyclic GMP hydrolysis. Pressure injections of purified PDE or GTPase* through micropipettes into isolate rods or rods of superfused retinas will be performed while recording membrane potential or current (from voltage clamped cells). The ability of hydrolysis resistant analogs of GTP to exchange after binding to the GTP binding protein will be measured as a function of both adenine and guanine nucleotide concentration in dark ROS membranes. Such nucleotide dependent exchange may well explain the reversibility of the hyperpolarization induced by intracellular injection of GP(NH)ppG. The influence of buffer composition and concentration on the activity and solubility of GTPase and PDE will be investigated. We will continue to study the relationships between the rod PDE and hormone or neurotransmitter-sensitive adenylate cyclase systems by reconstitution of enzymatic activity using purified components from each system. Since it has been proposed that the rod PDE system may be involved not only in rod physiology, but in cellular maintenance and in the etiology of retinal dystrophies in animal models, a fuller understanding of the relationship between cellular biochemical and electrophysiological processes is needed. The experiments described in this proposal are designed to directly and specifically reveal the functional significance of the rod PDE cascade.
| Clack, J W; Stein, P J (1988) Opsin exhibits cGMP-activated single-channel activity. Proc Natl Acad Sci U S A 85:9806-10 |
| Caretta, A; Stein, P J (1987) Light- and nucleotide-dependent increase in apparent viscosity in a suspension of retinal disks. FEBS Lett 219:97-102 |
| Caretta, A; Stein, P J (1986) Light- and nucleotide-dependent binding of phosphodiesterase to rod disk membranes: correlation with light-scattering changes and vesicle aggregation. Biochemistry 25:2335-41 |
| Caretta, A; Stein, P J (1985) cGMP- and phosphodiesterase-dependent light-scattering changes in rod disk membrane vesicles: relationship to disk vesicle-disk vesicle aggregation. Biochemistry 24:5685-92 |
