Advances in Photoreception Proceedings of a Symposium on Frontiers of Visual Science (1990) / Chapter Skim
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Phototransduction in Vertebrate Rods: The Electrophysiological Approach to the cGMP Cascade Theory
Phototransduction in Vertebrate Rods: The Electrophysiological Approach to the cGMP Cascade Theory
Pages 59-77
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From page 59... ...
Figure 1 summarizes their finding, overlaying the theoretical interpretation of their data on a scanning electron micrograph of a frog retina. In the dark there is a continually flowing, net outer-segment-inward membrane current, whose spatially integrated magnitude is about 50 to 70 pA in the 25-pm outer-segment rat rod at 37°C (rat rods are essentially identical to human rods)
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From page 60... ...
Of the dark current can be had from considering some numbers: since 1 pA of current corresponds to 6.25 x 106 monovalent charges/second, a 50-pA dark current flowing into a human rod outer segment with a water volume of c. 100 fl demands a complete turnover of the ions carrying the current every 5 seconds (50 pC of monovalent charge into 100 fl corresponds to 5 mM of the charge carrier; we can assume that the internal c[Na]
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From page 61... ...
That the rod should hyperpolarize to light follows immediately from the fact that light suppresses the outer-segment dark current: since that latter current is a depolarizing current, its suppression (in the absence of compensatory reaction) must necessarily drive the membrane potential toward the reversal potential of any K+ conductance.
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From page 62... ...
Internal Messenger Hypothesis The second inference that drove much transduction work was the need for an internal messenger, required because of several different kinds of evidence that the rod disks (which one could presume are the site of most photon adsorptions, based on the action spectrum of night vision) are physically separated from the outer-segment plasma membrane by about the distance of a synaptic cleft.
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From page 63... ...
This experiment demonstrates that substance infused into the inner segment penetrates to the outer segment. The relative differences in intensity of fluorescence of the outer and inner segments probably reflect the differences in the water space—in the outer segment more than half of the volume is occupied by the disks.
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From page 64... ...
Cyclic GMP as Internal Messenger Bill Miller and Grant Nicol (Nicol and Miller, 1978; Miller and Nicol, 1979) were responsible for the beginnings of the electrophysiological testing of the cGMP-transmitter hypothesis: they discovered that injection of cGMP into outer segments depolarizes rods in a magnitude and with a duration that is dose dependent and that light antagonizes the depolarizing effect of cGMP.
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From page 65... ...
SOURCE: Cobbs and Pugh (1985~. a combined suction electrode and intracellular electrode salamander rod preparation like that used by Bavlor and Nunn fl9861, incorporating a ~ ~ ~ ~ ~ ~ .
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From page 66... ...
66 4 o o o ~ E ~ 4o ,` ._ ~ .~ an CL C: Cal +\ ~ X —\ o At _W O\+ O O O — O [(I - ~ )
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From page 67... ...
The cGMP Cascade: A Biochemical Sketch Figure 5 shows a sketch of the so-called cGMP cascade of the outer segment. This diagram summarizes not only the electrophysiological work cursorily reviewed above but also a virtual flood of biochemistry that followed in the wake of Bitensky et al.'s (1971)
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From page 69... ...
FORMAL THEORY The remainder of this paper sketches our recent efforts to develop a formal representation of the reactions that lead to the closure of ghU (reactions represented by the filled arrows in Figure 5) and to test this representation against responses of voltage-clamped rods to intense, brief flashes isomerizing up to 20 percent of the rhodopsin.
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From page 70... ...
The average apparent latency of the velocity- and delay-saturated voltage-clamp photocurrent is about 7 msec (Cobbs and Pugh, 1987~. Theoretical Account of Velocity Saturation Can a formal representation of the cGMP cascade theory account for these phenomena, and is the account quantitatively consistent with the biochemical data on the reactions?
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From page 71... ...
In part A note that the 10-fold increase from 10 4 to 10 3 produces about the same ^~^ aria ~ ~ ~ a^` late rat translation as the 100-fold increase from 10 3 to 10 1 and that in part B the Refold change from 10 1 7 to 10-0 7 produces virtually no change in the lateral position of the photocurrent: this is the phenomenon of delay saturation. Reprinted from Cobbs and Pugh (1987)
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From page 72... ...
Our work on extensions of the theory to incorporate diffusion and cable properties is nearly complete, and we should know shortly which feature of the cascade imposes the photocurrent velocity limitations. Theoretical Account of Delay Saturation and Translation Formal theory also provides interesting insight into the translatory behavior that is seen at fractional isomerizations of about 0.001, and the c.
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From page 73... ...
of a single rod stimulated with 20-psec flashes isomerizing the fraction rhodopsin indicated. The smooth traces are generated by a representation of the cGMP-cascade theory: only one parameter varies between the curves: k20, the pseudo-first-order rate with which rhodopsin activates G-protein.
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From page 74... ...
Since we have independent knowledge of the fraction isomerized, we can thus infer the dependence of the rate constant on isomerization. We are in the process of confronting the theory in this fashion with the individual records of many cells, in effect using the theory as a measuring tool for estimating dependence of the rate on fractional isomerization and thus testing the lateral diffusion hypothesis.
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From page 75... ...
Matthews, and KW. Yau 1980 1\vo components of electrical dark noise in toad retinal rod outer segments.
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From page 76... ...
Torre, and T Lamb 1985 Effects on the photoresponse of calcium buffers and cyclic GMP incorporated into the cytoplasm of retinal rods.
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From page 77... ...
Miller 1978 cGMP injected into retinal rod outer segments increases latency and amplitude of response to illumination. Proceedings of the National Acad~ny of Sciences' USA, 75:5217-5220.
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