Abstract
cGMP is the second messenger for visual excitation in vertebrate rod photoreceptors. However, no direct correlation has been observed between the measured total cGMP concentration in the rod outer segment and the electrical response of these cells to photic stimulation. To address this discrepancy, we have quantitated the number and affinities of cGMP binding sites in the rod outer segment to determine the cytoplasmic free cGMP concentration that is involved in visual transduction. We identified two distinct classes of cGMP binding sites in amphibian rod outer segments: 1) high affinity binding sites with a KD1 = 60 nM and a site density of 30 microM, and 2) moderate affinity binding sites with a KD2 = 6.6 microM and a site density of 78 microM. These two classes of binding sites are calculated to bind 94% of the total cellular cGMP, thereby lowering the cytoplasmic cGMP concentration to 3.5 microM in dark-adapted rod outer segments. This value is consistent with predictions of the cytoplasmic cGMP concentration based on activation of the cGMP-gated ion channel of rod photoreceptors. The kinetics of cGMP dissociation from high affinity binding sites indicate that this class of sites would dissociate its bound cGMP too slowly to participate in visual excitation and recovery to flash illumination. This binding of cGMP to intracellular binding sites provides a non-enzymatic mechanism by which photoreceptor cells regulate the concentration and restrict the diffusion of this second messenger during visual transduction.
Highlights
30 WM, and 2) moderate affinity binding sites with a Despite extensive evidence implicating cGMP as the sole
In this report we demonstrate the existence of high and moderate affinity cGMP binding sites in the amphibian rod outer segments (ROS) that serve to sequester greater than 90% of the total cellular cGMP
Averaging the binding parameters from the two different equilibrium methods (KO,= 60 nM, €Irnarl = 0.005 cGMP per Rho, K D=~6.6 PM, Bmer=:! 0.013 cGMP per Rho), we estimate that 98% of the high affinity cGMP binding sites and 35% of the moderate affinity binding sites are occupied under equilibrium conditions inthe outer segmentof rod photoreceptors (Fig. 7)
Summary
The abbreviations used are: Rho, rhodopsin; PDE, rod photore- proposes that rod outer segments (ROS) contain cGMP bindceptorcGMPphosphodiesterase holoenzyme; ROS,rodouter seg- ingsites that reduce the free cGMP concentration in the ments; KD,dissociation constant B,., maximum binding site density; [CGMP]~,,, total acid-extractable cGMP concentration; [cGMP]~,, free cytoplasmic cGMP concentration; BP, incremental cytoplasmic cytoplasm. content of. Prior to measuring the extent of binding of exogenous cGMP, permeabilized photoreceptor preparations were depleted of endogenous cGMP by incubation at 22 "C for 225 min (see Fig. 1). Determination of Nucleotide Concentration and Purity-The cGMP contentof photoreceptor preparations was measured by radioimmunoassay as follows. Filter Binding Assay of cGMP Binding-Nucleotide-depleted permeabilized ROS (Rho concentration 10-30 pM) were mixed with an equal volume of intracellular medium containing various concentrations of ['HIcGMP along with 200 p~ zaprinast (M&B 22,948;see Gillespie and Beavo (1989a)). mM EDTA was added when the [Rho] was greater than 10 p~ to furtherinhibit PDE activity. TOmeasure nonspecific binding to the filters, 3 mM cold cGMP was added to ROS along with [3H]cGMP, andthe radioactivity retained by the filter subtracted from samples lacking the excess cGMP. P D E Assay-Measurements of the extent of cGMP hydrolysis were carried outusing anion-exchange chromatography (Kincaid and Manganiello, 1988)
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