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, 96 (7), 3718-22

Abnormal Photoresponses and Light-Induced Apoptosis in Rods Lacking Rhodopsin Kinase

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Abnormal Photoresponses and Light-Induced Apoptosis in Rods Lacking Rhodopsin Kinase

C K Chen et al. Proc Natl Acad Sci U S A.

Abstract

Phosphorylation is thought to be an essential first step in the prompt deactivation of photoexcited rhodopsin. In vitro, the phosphorylation can be catalyzed either by rhodopsin kinase (RK) or by protein kinase C (PKC). To investigate the specific role of RK, we inactivated both alleles of the RK gene in mice. This eliminated the light-dependent phosphorylation of rhodopsin and caused the single-photon response to become larger and longer lasting than normal. These results demonstrate that RK is required for normal rhodopsin deactivation. When the photon responses of RK-/- rods did finally turn off, they did so abruptly and stochastically, revealing a first-order backup mechanism for rhodopsin deactivation. The rod outer segments of RK-/- mice raised in 12-hr cyclic illumination were 50% shorter than those of normal (RK+/+) rods or rods from RK-/- mice raised in constant darkness. One day of constant light caused the rods in the RK-/- mouse retina to undergo apoptotic degeneration. Mice lacking RK provide a valuable model for the study of Oguchi disease, a human RK deficiency that causes congenital stationary night blindness.

Figures

Figure 1
Figure 1
Inactivation of the RK gene. (A) Genomic structure of RK gene, targeting vector, and altered RK locus. The targeting vector contained 10 kb (long arm) of the 3′ downstream and 0.8 kb (short arm) of the 5′ upstream homologous sequence. The MC1neopA cassette (hatched bars) replaced the 2.7-kb BamHI fragment that contained the exon bearing the translation initiating codon (filled box). Restriction sites: B, BamHI; A, ApaI; K, KpnI. (B) Immunoblot analysis of RK from mice at 1 month of age; the analysis used a polyclonal antibody (8585) raised against the first 50 amino acids of bovine RK. The RK content of the hemizygous (+/−) retina was approximately 50% of that of the RK+/+. No detectable RK was found in the RK−/− retina.
Figure 2
Figure 2
Absence of light-dependent phosphorylation of rhodopsin in RK−/− retina. Light caused about 43 phosphates to be incorporated per 100 rhodopsins in RK+/+ retinas; no light-dependent phosphorylation was observed in RK−/− retinas. The number of phosphates incorporated per 100 rhodopsins was calculated as follows: 1 × (% monophosphorylated rhodopsin) + 2 × (% di-phosphorylated rhodopsin) + 3 × (% triphosphorylated rhodopsin). Bars equal SD, n = 2 except for RK+/+ dark, where n = 3.
Figure 3
Figure 3
Flash responses from rods lacking rhodopsin kinase. (A) Saturating responses from RK+/+ and RK+/− rods (Inset), and an RK−/− rod to a flash that elicited about 300 photoisomerizations in each case. Unlike the +/+ and +/− responses (see Inset), the RK−/− response was greatly prolonged, and it recovered in a series of stepped transitions that represent the deactivation and occasional reactivation of individual rhodopsin molecules. The dark currents of the three cells were 15.0 pA (+/+), 16.7 pA (+/−), and 16.6 pA (−/−). Flashes were delivered at t = 0. (B) Form of the single photon response from rods of each of the three lines. The RK−/− trace is the average of 14 responses; the RK+/− trace is the average of 80 responses; and the RK+/+ trace is the average of 57 responses. The dark current of the three cells was 13.8, 18.9 and 11.3 pA, respectively. (C) Distribution of the durations of 198 RK−/− quantal events measured at half-maximal amplitude from three RK−/− cells. The smooth curve is an exponential function with a time constant of 3.3 s. The Inset is an example of a quantal event from an RK−/− rod whose dark current was 7.0 pA. (D) Dependence of the rate of change of PDE activity on the number of flash-induced photoisomerizations in RK+/+ and −/− rods. Slope of the linear regression is 0.99.
Figure 4
Figure 4
Altered morphology of RK−/− retina. (A) Morphology of the central retina of cyclic-light reared and dark-reared mouse retinas at 6 weeks of age. (1) Control (+/+), raised in cyclic illumination (50 lux). (2) Hemizygous knockout (+/−), raised in cyclic illumination (50 lux). (3) Homozygous knockout (−/−), born and raised in the dark. (4) Homozygous knockout (−/−), reared in cyclic illumination (50 lux). The outer segments in 4 lacked the characteristic orderly arrangement seen in 1, 2, and 3 and were approximately 50% shorter. (Scale bar = 16 μm.) (B) Light-induced photoreceptor apoptosis of dark-reared RK−/− mice after 24 hr in continuous room light (450 lux). (Upper) Results of TUNEL staining photographed in the presence of dim background light. Positive nuclei were in the outer nuclear layer. (Scale bar = 100 μm.) (Lower) Corresponding morphological changes shown in Upper. (Scale bar = 16 μm.) (C) DNA degradation. Lane 1, control (+/+); lane 2, RK−/− after 24 hr of continuous illumination; lane 3, RK−/− after 48 hr of continuous illumination. DNA size markers are shown in base pairs as indicated at right. OS, outer segments; IS, inner segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

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