Rod and cone photoreceptors in the vertebrate retina provide the organism with two alternative and complementary signaling systems that support the full behavioral range of the visual system. In general, the photoresponse in rods is slower in time course, more sensitive to light and adapts over a more restricted range of intensities than that of cones. Within a given species, cones sensitive to different wavelengths of light may also differ in their photosensitivity and kinetics. The long term objective in this research is to understand the mechanisms of phototransduction in retinal photoreceptors, and to explain the difference in transduction between the two receptor types. Ca ions in the cytoplasm of the outer segment in cone photoreceptors play a critical role in phototransduction. Yet, the Ca concentration in darkness and the magnitude and time course of changes expected to follow illumination are unknown. Also, the mechanisms that regulate Ca concentration and the mechanisms through which Ca achieves its function in transduction are generally not known from direct investigation, rather, they are inferred by analogy to the processes discovered in rod photoreceptors.
The specific aims of this proposal are to investigate the concentration, function and homeostasis of Ca in cone outer segments. Recent discoveries suggest that rods and cones differ in the rate and extent of voltage- and light-dependent changes in Ca concentration in their outer segments, in the Ca permeability of their cGMP-dependent ion channels as well as in the attributes of Ca-dependent biochemical processes in the outer segment. Using an indicator dye, we propose to measure the Ca concentration in the outer segment of isolated cone photoreceptors and to determine the magnitude and time course of light-dependent changes in this concentration. We will investigate the role of Ca in cone outer segments as modulator of three different biochemical processes: the activity of the enzyme guanylate cyclase, the photoactivation of the enzyme phosphodiesterase and the binding of cGMP to cGMP-gated ion channels. We will conduct these studies both in intact cones and in truncated outer segments using electrophysiological and photometric techniques. Using electrophysiological methods we will study the Ca permeability of the cGMP-gated channels of cones as well as their macroscopic and microscopic kinetics. We will also investigate the structural determinants of ion flow through these channels by combining electrophysiological methods with techniques of molecular biology.
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