The electrical response of cone photoreceptors to light is generated by changes in the activity of ion channels located in the plasma membrane of the cell. the activity of some of these channels is directly modulated by light, while that of others responds to membrane voltage and neurotransmitter molecules. The overall objective of this grant is to understand the mechanisms of the electrical photoresponse of cones through studies of the biophysical properties and the regulation of some of the ion channels in the membrane. The activity of the light -sensitive channels in the cone outer segment appears to be under indirect regulation by cytoplasmic Calcium. the cytoplasmic Calcium concentration may be controlled by the kinetic balance between its influx and its efflux across the plasma membrane. We will quantitatively test this hypothesis by measuring the kinetics of Calcium efflux and influx in single cones isolated from a fish retina. Calcium influx will be measured as a membrane current with suction electrodes and Calcium efflux will be studied by measuring, with ion selective electrodes, the Calcium concentration in a small volume outside the outer segment. We also will measure directly the cytoplasmic Calcium concentration in the outer segment and will investigate the quantitative relationship between this concentration and the photocurrent and the state of light- or dark-adaptation of the photoreceptor. Calcium concentration will be followed by measuring the space resolved fluorescence of single cells loaded with the Calcium indicator dye fura2. The cone inner segment contains five different types of ion channels that help shape its voltage response to light and mediate coupling between photoreceptors. We will study the features of two of these channels: Voltage-activated Calcium channels and Calcium-dependent Chloride channels, because of their particular relevance to understanding the synaptic function of cones. The biophysical properties of the channels will be investigated with voltage-clamp studies of solitary cone inner segments isolated from a lizard retina. Their spatial distribution will be determined both with electrophysiologic and microphotometric protocols.
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