Retinal rods and cones signal the presence of light by hydrolyzing cGMP thereby closing cyclic nucleotide gated cation channels in the plasma membrane. An inward Na+ current is interrupted and the cell hyperpolarizes. It has been shown that bicarbonate can increase the flash response amplitude and quicken response kinetics. However, many features of this modulation are not understood including: the full magnitudes of the effects, the dose-response relations, the intra-retinal sources of bicarbonate for photoreceptors, the pathway(s) for access of bicarbonate to the outer segment, differences between rods and cones, and the mechanism of bicarbonate action. Using single cell recording and biochemical assays, we will tackle each of these issues. In elucidating the mechanism of bicarbonate, we will consider three targets: guanylate cyclase, PDE, cyclic nucleotide gated channel. The first two targets will be evaluated in biochemical assays, while the third will be tested in excised membrane patch recordings. Some evidence already suggests that bicarbonate stimulates ROS-GC1 activity. It then becomes important to find out whether ROS-GC2 is similarly affected, to define the bicarbonate binding site of each ROS-GC, and to determine the calcium dependence of bicarbonate stimulation when the calcium sensing subunits GCAPs and S100B are bound to ROS-GCs. The retinal sources of bicarbonate will be localized with in situ hybridization and immunohistochemistry. Pathways for access of bicarbonate into rods and subsequent removal will be elucidated and then manipulated to characterize the impact of bicarbonate on relative sensitivity to flashes, flash response kinetics and the circulating current. Cones operate in brighter light, have quicker flash response kinetics and maintain a higher metabolic rate. It will therefore be important to test whether bicarbonate exerts a greater effect in cones and whether there are differences between various types of cones. Electroretinogram recordings from intact mice will be compared to recordings of single rods to define the physiological bicarbonate levels in situ. Bicarbonate could provide a means of preventing saturation during continuous exposure to bright light, so a contribution to light adaptation will be explored. A role for bicarbonate modulation of guanylate cyclase activity in exacerbating retinal disease will be tested in mutant mice by subjecting them to hypercapnia or by inhibiting endogenous bicarbonate production. A long term goal is to dissect the molecular mechanisms that shape the photon response under light and dark adapted conditions and to understand their roles, be it causative or modulatory, in retinal disease.
Cyclic GMP is the second messenger of visual transduction in retinal photoreceptors;its intracellular levels fall in response to photon absorption, causing ion channels in the plasma membrane to close and the plasma membrane to hyperpolarize. In this proposal, we test and characterize a novel mechanism in which bicarbonate affects the visual transduction mechanism in rods and cones by stimulating cGMP synthesis. The results of this study may lead directly to a therapeutic intervention for blinding retinal diseases that are caused by genetic defects in the cyclic nucleotide gated ion channel or in cGMP metabolism.