Rhodopsin Signaling and Organization in Heterozygote Rhodopsin Knockout Mice

Abstract

Rhodopsin (Rho) resides within internal membrane structures called disc membranes that are found in the rod outer segments (ROS) of photoreceptors in the retina. Rho expression is essential for formation of ROS, which are absent in knockout Rho؊/؊ mice. ROS of mice heterozygous for the Rho gene deletion (Rho؉/؊) may have a lower Rho density than wild type (WT) membranes, or the ROS structure may be reduced in size due to lower Rho expression. Here, we present evidence that the smaller volume of ROS from heterozygous mice is most likely responsible for observed electrophysiological response differences. In Rho؉/؊ mice as compared with age-matched WT mice, the length of ROS was shorter by 30 -40%, and the average diameter of ROS was reduced by ϳ20%, as demonstrated by transmission and scanning electron microscopy. Together, the reduction of the volume of ROS was ϳ60% in Rho؉/؊ mice. Rho content in the eyes was reduced by ϳ43% and 11-cis-retinal content in the eye was reduced by ϳ38%, as determined by UV-visible spectroscopy and retinoid analysis, respectively. Transmission electron microscopy of negatively stained disc membranes from Rho؉/؊ mice indicated a typical morphology apart from the reduced size of disc diameter. Power spectra calculated from disc membrane regions on such electron micrographs displayed a diffuse ring at ϳ4.5 nm ؊1 , indicating paracrystallinity of Rho. Atomic force microscopy of WT and Rho؉/؊ disc membranes revealed, in both cases, Rho organized in paracrystalline and raftlike structures. From these data, we conclude that the differences in physiological responses measured in WT and Rho؉/؊ mice are due to structural changes of the whole ROS and not due to a lower density of Rho.

Two-photon microscopy reveals early rod photoreceptor cell damage in light-exposed mutant mice

“…Importantly, the imaging experiments were performed in a native setting with undisturbed intact eyes, which avoided potential artifacts arising from required tissue-processing. Although ROS sizes are known to be determined by rhodopsin content (75)(76)(77)(78), these light-induced changes were too rapid for de novo protein biosynthesis to account for them. Additionally, it had been shown that rhodopsin mislocalized significantly to rod inner segments only at 48 h after light-induced damage (74).…”

The Function of Guanylate Cyclase 1 and Guanylate Cyclase 2 in Rod and Cone Photoreceptors

“…Recovery from Test Flashes as Determined by Paired Flash Analysis-A paired flash protocol was used to evaluate the ability of GC deletion mutant retinas to recover responsiveness after light stimulation (23,30,35). In this method, short test flashes followed by varying interstimulus intervals, and a probe flash were used to test for a-wave recovery (36,37).…”

“…Leading edges of the ERG responses were fitted with a model of rod photoreceptor activation (29). The double-flash recording followed a previously published protocol (30). The normalized amplitude of the probe flash a-wave versus the time between two flashes was plotted and fit by the linear regression algorithm in the SigmaPlot program (version 9.0).…”

Visual Rhodopsin Sees the Light: Structure and Mechanism of G Protein Signaling

“…The ROS of heterozygous mice for the rhodopsin gene deletion (rhodopsin ϩ/Ϫ ) have a similar density of rhodopsin, but their rhodopsin volume is reduced by ϳ60% when compared with wild-type mice (6). Synthesis of opsin begins in the inner segments of photoreceptors, where it undergoes maturation in the endoplasmic reticulum and Golgi membranes before being vectorially transported to the ROS.…”

“…For example, Asn-55 of TM helix I, Asn-78 and Asp-83 of TM helix II, and Asn-302, Pro-303, and Tyr-306 of TM helix VII (part of the NPXXY(X) 5,6 F motif) are all highly conserved amino acids across the GPCR family that appear to form important contacts for maintaining the inactive state of rhodopsin. FIGURE 1.…”