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ERGs were obtained from dark-adapted mice that were anesthetized with intraperitoneal urethane (40 mg/ml), ketamine (1 mg/ml), and xylazine (0.4 mg/ml) at 15 μl/g body weight. The corneal ERG was detected with a cotton wick electrode. Flashes were obtained from a modified strobe lamp with a 3-cm aperture that was positioned 9 cm from the dilated pupil of the mouse. The ERG response was measured with a Nicolet Instruments CA-1000 computer, which averaged 2 to 100 responses at a flash frequency of 0.05 to 1 Hz. The maximum flash intensity photoisomerized ∼2000 rhodopsin molecules per rod [A. L. Lyubarsky and E. N. Pugh, J. Neurosci. 16, 563 (1996)].
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2, 10 mM Hepes (pH 7.5), 1 mM dithiothreitol, 10 μM leupeptin, and 100 kallikrein units per 1 ml of aprotinin (final concentrations).
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2) was measured by a photomultiplier that was connected to a digital pulse counter. The mean percent of absorbed light (expressed as minimum, maximum, and n, number of determinations) was 27.6%, (23.6, 34.7, n = 3) in 129/SvJ retinas and 27.5% (21.3, 31.8, n = 4) in W70A retinas. (ii) The amount of rhodopsin in the retinas of four mice was determined through difference spectroscopy [M. D. Bownds, A. Gordon-Walker, A.-C. Gaide-Huguenin, W. Robinson, J. Gen. Physiol. 58, 225 (1971)] after solubilization in 30 mM cetyltrimethylammonium chloride. The rhodopsin content of both control and W70A retinas was 0.3 nmol per retina.
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The absolute rates of GTP hydrolysis in control ROS in vitro were slower than the rate of recovery of the photoresponse. This is consistent with many previous reports that show that dilution of cellular components, most likely RGS9, slows the rate of GTP hydrolysis (6) [E. A. Dratz, J. W. Lewis, L. E. Schaechter, K. R. Parker, D. S. Kliger, Biochem. Biophys. Res. Commun. 146, 379 (1987); V. Y. Arshavsky, M. P. Antoch, K. A. Lukjanov, P. P. Philippov, FEBS Lett. 250, 353 (1989)]. Similarly, the 2.7-fold difference in GTPase rate between control and W70A ROS in Fig. 2E should be considered only as the lowest estimate for the difference in physiologically intact photoreceptors.
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The absolute rates of GTP hydrolysis in control ROS in vitro were slower than the rate of recovery of the photoresponse. This is consistent with many previous reports that show that dilution of cellular components, most likely RGS9, slows the rate of GTP hydrolysis (6) [E. A. Dratz, J. W. Lewis, L. E. Schaechter, K. R. Parker, D. S. Kliger, Biochem. Biophys. Res. Commun. 146, 379 (1987); V. Y. Arshavsky, M. P. Antoch, K. A. Lukjanov, P. P. Philippov, FEBS Lett. 250, 353 (1989)]. Similarly, the 2.7-fold difference in GTPase rate between control and W70A ROS in Fig. 2E should be considered only as the lowest estimate for the difference in physiologically intact photoreceptors.
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2, 3 mM Hepes (pH 7.4), 0.02 mM EDTA, 10 mM glucose, and 0.1% vitamin and amino acid supplement (Sigma). The responses were low-pass filtered at 20 Hz with an eight-pole Bessel filter and digitized at 100 Hz with an acquisition program written by F. Rieke for IgorPro (Wave Metrics, Lake Oswego, OR). Brief flashes (10 ms) of 500-nm light were used for stimulation. The intensity of the light source was calibrated with a silicon detector (UDT350, Graseby Optronics, Orlando, FL), and the flash strength was controlled with calibrated neutral density filters. When white light was needed to evoke the maximal response from a W70A rod, its intensity was expressed as the equivalent intensity at 500 nm, using the relative ability of white and 500-nm light to stimulate the rod.
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2, 3 mM Hepes (pH 7.4), 0.02 mM EDTA, 10 mM glucose, and 0.1% vitamin and amino acid supplement (Sigma). The responses were low-pass filtered at 20 Hz with an eight-pole Bessel filter and digitized at 100 Hz with an acquisition program written by F. Rieke for IgorPro (Wave Metrics, Lake Oswego, OR). Brief flashes (10 ms) of 500-nm light were used for stimulation. The intensity of the light source was calibrated with a silicon detector (UDT350, Graseby Optronics, Orlando, FL), and the flash strength was controlled with calibrated neutral density filters. When white light was needed to evoke the maximal response from a W70A rod, its intensity was expressed as the equivalent intensity at 500 nm, using the relative ability of white and 500-nm light to stimulate the rod.
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act value of ∼4 ms is obtained. Allowing for the finite flash duration of 10 ms and assuming an effective delay of 3 ms (24), one would expect activation of the PDE in a normal mouse rod to be completed within a few milliseconds after the end of the flash.
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We thank E. R. Makino and J. W. Handy for help with biochemical experiments; D. B. Farber for providing Pdeg cDNA; J. Xu and M. I. Simon for providing the opsin promoters; T. G. Wensel for providing antibodies against RGS9; F. Costantini, C. Liu, and members of their laboratories for sharing ideas and equipment; V. I. Govardovskii, R. Axel, and E. Kandel for critically reading the manuscript; and M. Mendelsohn, K. Doi, H. Kjeldbye, J. Ma, and D. Wiener for discussion. Supported by NIH grants T32 EY07105, EY05750, EY10336, and EY11510; the Ruth and Milton Steinbach Fund; the McKnight Foundation; and Research to Prevent Blindness (RPB). S.P.G. is an investigator of the Howard Hughes Medical Institute. V.Y.A. is a recipient of a Jules and Doris Stein professorship from RPB.
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