Abstract
This study examines whether changes in cGMP concentration initiated by illumination of frog rod photoreceptors occur rapidly enough to implicate cGMP as an intermediate between rhodopsin activation in the disc membrane and permeability changes in the plasma membrane. Previous studies using whole retinas or isolated outer segments have provided conflicting evidence on the role of cGMP in the initial events of phototransduction. The rod photoreceptor preparation employed in this work consists of purified suspensions of outer segments still attached to the mitochondria-rich ellipsoid portion of the inner segment. These photoreceptors are known to retain normal electrophysiological responses to illumination and have cGMP levels comparable to those measured in the intact retina. When examined under several different conditions, changes in cGMP concentrations were found to occur as rapidly or more rapidly than the suppression of the membrane dark current. Subsecond changes in cGMP concentration were analyzed with a rapid quench apparatus and confirmed by comparison with a rapid freezing technique. In a 1 mM Ca2+ Ringer's solution, cGMP levels decrease to 65% of their final extent within 200 ms after bright illumination; changes in membrane dark current follow a similar time course. When the light intensity is decreased to 8000 rhodopsins bleached per rod per s, the light-induced cGMP decrease is completed within 50 ms, with 7 X 10(5) cGMP molecules hydrolyzed per rhodopsin bleached. During this time the dark current has not yet begun to change. Thus, under physiological conditions it is clear that changes in cGMP concentration precede permeability changes at the plasma membrane. The correlation of rapid changes in cGMP levels with changes in membrane current leave open the possibility that changes in cGMP concentration may be an obligatory step in the reaction sequence linking rhodopsin activation by light and the resultant decrease in sodium permeability of the plasma membrane.
Highlights
This study examines whether changescGinMP con- membrane andpermeability changes int,heplasma membrane centration initiated by illumination of frog rodphoto- has been inferred from both the anatomyof the rod photorereceptors occur rapidly enough to implicate cGMP as ceptor and theamplification of the electrical response of the an intermediate between rhodopsin activation in the rod when one rhodopsin molecule is activated (see Bownds disc membrane and permeability changes in the plas(m19a81) and Korenbrot (1984) for reviews)
The cGMP concentrationof purified 0s.-is. prepared in a 1mM Ca2+ bufferwas found to be 0.010 mol of cGMP permol of rhodopsin (Table I).Assuming that therhodopsin concentration in the rod is 3 mM, the cGMP concentration in the extradiscal space is estimatedto be 60 p ~0s..-is. have a 30% higher cGMP concentration thanO.S. when they are comparably prepared (Table I)
This 30% greater cGMP concentration in 0s.-i.s. was observed withinindividualexperiments where density gradient centrifugation allowed a purification of o s . and 0.s.-is. suspensionsbased on their different densities
Summary
Light-induced Decreases in cGMP Concentration Precede Changes in Membrane Permeability in FrogRod Photoreceptors*. The signal generator activates a shutter placed by cGMP is that the light-induced changes in cGMP must in front of a continuous lightsource, synchronizing acid ejection precede the suppressionof the dark current In this studwye into the fifth sample with the onset of illumination. The number of have correlated the kineticsof light-induced cGMP changes to the kinetics of dark current suppression.A preparation of rod outer segments with the ellipsoid portion of the inner segment still attached (0.s.-is.) offers the advantage of consamples automatically quenched can be varied, allowing for manual quenching of the remaining samples a t later times.
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