The Journal of Neuroscience, April 26, 2006, ():
GAP-Independent Termination of Photoreceptor Light Response by Excess 
Subunit of the cGMP-Phosphodiesterase
J. Neurosci. Tsang et al.
26: 4472
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1. Levels of additional transduction proteins in WT and wt6C mice (OX). The relative levels of retinal proteins were determined by immunoblotting and enhanced chemiluminescence (ECL) with primary antibodies against RGS9-1 (CT317, 1:3,000), Gβ5 (CT215, 1:3,000), PDE6γ (PA1-723, 1:1,000, Affinity Bioreagents (ABR)), guanylyl cyclase-E (K285, 1:5,000, from David Garbers), guanylyl cyclase-F (A670, 1:5,000, David Garbers), PDE6α (PA1-720, 1:500, ABR), rhodopsin kinase (MA1-721, 1:5,000, ABR), arrestin (Marr, 1:20,000), transducin α subunit (UUTA1, 1:5,000), phosducin (Gerti, 1:5,000, from Rehwa Lee), GCAP1 (α-GCAP1, 1:1,000, from Alexander Dizhoor), and GCAP2 (α-GCAP2, 1:1,000, from Alexander Dizhoor). This was followed by treatment with peroxidase conjugated secondary goat-antirabbit or goat-antimouse IgG (Santa Cruz Biotechnology, 1:5,000).
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Supplemental Figure 2. Morphology of wt6C retinas is normal and indicates no loss of photoreceptor number by comparison to WT animals. WT and wt6C mice were both 3 months old at time of sacrifice. Scale bar, 25 μm.
- supplemental material
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Supplemental Figure 3. One conceivable interpretation of our data would be that excess PDE6γ produces a decrease in rise time and an acceleration of decay by the same mechanism, that is by decreased activation of the PDE. On this view, a decrease in activation, presumably by binding of excess PDE6γ to Tα-GTP, would cause the current to rise with a slower time course, decrease sensitivity, and also speed inactivation, since there would be less activated PDE available to be turned off. The data in Fig. 4C of our paper indicate that this interpretation is unlikely, since the responses at these bright intensities rise along nearly the same time course, indicating a similar PDE activation, but the wt6C response decays much more rapidly. In the supplemental figure shown above, we illustrate a similar phenomenon but for responses at dimmer light intensities, which for the WT and wt6C rods produce a similar suppression of about a third of the current. Wave forms are from the same cells as in Fig. 4 and have been averaged after normalizing to the maximum photocurrent for each rod, from 35 WT rods to an intensity of 17 photons μm-2 s-1 and 28 wt6C rods to an intensity of 43 photons μm-2 s-1. Parts A and B show the same responses but at a different temporal resolution. The wt6C response actually rises somewhat more rapidly and reaches peak amplitude sooner (see part B) but then crosses the WT response and decays markedly more rapidly. Thus even when responses are chosen that rise along a similar time course and reach a similar peak suppression of circulating current, indicating a similar PDE activation, the excess PDE6γ causes a more rapid decay of the response. This shows that the acceleration of decay caused by the PDE6γ cannot be produced simply by a decrease in activation of the PDE and supports our view, also supported by a large body of biochemical evidence (Wensel and Stryer, 1990; Erickson et al., 1992; Angleson and Wensel, 1993; Antonny et al., 1993; Angleson and Wensel, 1994; Bondarenko et al., 1999;Yamazaki, Moskvin &Yamazaki, 2002), that PDE6γ can turn off PDE independently of its effect on activation.
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Supplemental Figure 4. Excess PDE6γ alters both the rise time and rate of turn off of rod responses. Schema illustrating possible molecular mechanisms. Numbers indicate steps that occur normally in WT rods, and the letters are additional steps that we postulate to be responsible for the changes in wave form of the rod responses in the presence of excess PDE6γ. RGS 9 is illustrated for simplicity as if it were cytoplasmic, though it is normally part of a membrane-associated complex. The other members of this complex have been left out of the figure.
