Fraction of the dark current carried by Ca2+ through cGMP-gated ion channels of intact rod and cone photoreceptors

Abstract

The selectivity for Ca2+ over Na+, PCa/PNa, is higher in cGMP-gated (CNG) ion channels of retinal cone photoreceptors than in those of rods. To ascertain the physiological significance of this fact, we determined the fraction of the cyclic nucleotide-gated current specifically carried by Ca2+ in intact rods and cones. We activated CNG channels by suddenly (<5 ms) increasing free 8Br-cGMP in the cytoplasm of rods or cones loaded with a caged ester of the cyclic nucleotide. Simultaneous with the uncaging flash, we measured the cyclic nucleotide-dependent changes in membrane current and fluorescence of the Ca2+-binding dye, Fura-2, also loaded into the cells. The ratio of changes in fura-2 fluorescence and the integral of the membrane current, under a restricted set of experimental conditions, is a direct measure of the fractional Ca2+ flux. Under normal physiological salt concentrations, the fractional Ca2+ flux is higher in CNG channels of cones than in those of rods, but it differs little among cones (or rods) of different species. Under normal physiological conditions and for membrane currents ≤200 pA, the Ca2+ fractional flux in single cones of striped bass was 33 ± 2%, and 34 ± 6% in catfish cones. Under comparable conditions, the Ca2+ fractional flux in rod outer segments of tiger salamander was 21 ± 1%, and 14 ± 1% in catfish rods. Fractional Ca2+ flux increases as extracellular Ca2+ rises, with a dependence well described by the Michaelis-Menten equation. KCa, the concentration at which Ca2+ fractional flux is 50% was 1.98 mM in bass cones and 4.96 mM in tiger salamander rods. Because Ca2+ fractional flux is higher in cones than in rods, light flashes that generate equal photocurrents will cause a larger change in cytoplasmic Ca2+ in cones than in rods.