Investigating the Role of Rhodopsin F45L Mutation in Mouse Rod Photoreceptor Signaling and Survival

Abstract

Rhodopsin is the critical receptor molecule which enables vertebrate rod photoreceptor cells to detect a single photon of light and initiate a cascade of molecular events leading to visual perception. Recently, it has been suggested that the F45L mutation in the transmembrane helix of rhodopsin disrupts its dimerization in vitro To determine whether this mutation of rhodopsin affects its signaling properties in vivo, we generated knock-in mice expressing the rhodopsin F45L mutant. We then examined the function of rods in the mutant mice versus wild-type controls, using in vivo electroretinography and transretinal and single cell suction recordings, combined with morphologic analysis and spectrophotometry. Although we did not evaluate the effect of the F45L mutation on the state of dimerization of the rhodopsin in vivo, our results revealed that F45L-mutant mice exhibit normal retinal morphology, normal rod responses as measured both in vivo and ex vivo, and normal rod dark adaptation. We conclude that the F45L mutation does not affect the signaling properties of rhodopsin in its natural setting.

Keywords: electroretinogram; phototransduction; retinal degeneration; rhodopsin; rods.

Figure 1.

The Rho^F45L knock-in mutation. A 2D cartoon of mouse rhodopsin showing the mutation site at amino acid position 45 in the transmembrane helix I, where phenylalanine is replaced by leucine in the Rho^F45L KI mice (A). Side (B) and axial (C) views of the 3D structure of bovine rhodopsin’s dimer as determined by cryo-electron microscopy (PDB ID 6OFJ), highlighting the position of F45.

Figure 2.

Effect of the Rho^F45L knock-in mutation on photoreceptor morphology. Representative images from retinas of three-, six-, and nine-month-old wild-type mice (A–C, respectively), and age-matched Rho^F45L KI mice (D–F, respectively). Quantitative spider plots of ONL thickness as a function of distance from the optic nerve disk from wild-type mice (black; n = 4 each), and from Rho^F45L KI mice (red; n = 5, 4, 5) measured at three-, six-, and nine-month time points (G–I, respectively). Quantitative spider plots of the number of photoreceptor nuclei per column in the ONL as a function of distance from the optic nerve disk from wild-type mice (black; n = 4 each), and from Rho^F45L KI mice (red; n = 5, 4, 5) measured at three-, six-, and nine-month time points (J–L, respectively). Error bars indicate SEM; *p < 0.05.

Figure 3.

Effect of the Rho^F45L knock-in mutation on rhodopsin expression. Representative difference absorbance spectra of rhodopsin (before vs after bleaching) from wild-type mice (A) and Rho^F45L KI mice (B). Averaged spectra from wild-type mice (black; n = 2 eyes) and Rho^F45L KI mice (red; n = 2 eyes; C). All measurements were done from three-month-old animals.

Figure 4.

Effect of the Rho^F45L knock-in mutation on in vivo scotopic ERG responses. Representative families of ERG responses to flashes of increasing intensity (Cd · s m⁻²: 2.5 × 10⁻⁵, 2.5 × 10⁻⁴, 2.5 × 10⁻³, 2.5 × 10⁻², 0.25, 2.5, 20, and 250) recorded in scotopic conditions from wild-type mice (A) and Rho^F45L KI mice (B). For comparison, the responses to a flash of 2.5 × 10⁻⁴ Cd · s m⁻² are highlighted in red in the two panels. Population-averaged a-wave response amplitudes (C) and b-wave response amplitudes (D) from groups of wild-type mice (black; n = 5) and Rho^F45L KI mice (red; n = 5) are plotted together as a function of flash intensity. Error bars indicate SEM. Differences in C and D were not significant (p > 0.05) for all data points. All measurements were done from three-month-old mice.

Figure 5.

Effect of the Rho^F45L knock-in mutation on ex vivo ERG rod responses. Representative families of responses to flashes of increasing intensity (photons μm⁻²: 0.3, 1, 3, 10.7, 35, 117, 386, and 1271) for retinas from wild-type mice (A) and Rho^F45L knock-in mutant mice (B). For comparison, the responses to a flash of 35 photons μm⁻² are highlighted in red in both panels. Average flash response amplitudes (C) and average normalized flash response amplitudes (D) for rods from wild-type mice (black; n = 5 mice, 8 retinas) and Rho^F45L knock-in mutant mice (red; n = 6 mice, 10 retinas) are plotted together as a function of flash intensity. Error bars indicate SEM. The lines represent curves fitted to the data using a hyperbolic Naka–Rushton function. All measurements were done from three-month-old mice.

Figure 6.

Effect of the Rho^F45L knock-in mutation on individual rod responses. Representative families of responses of individual rods to flashes of increasing intensity (photons μm⁻²: 1, 3, 10.7, 35, 117, 386, and 1271); (A) rods from wild-type mice, and (B) rods from Rho^F45L knock-in mutant mice. For comparison, the responses to a flash of 35 photons μm⁻² are highlighted in red in the two panels. Population-averaged flash response amplitudes (C) and averaged normalized flash response amplitudes (C, inset) for rods from wild-type mice (black; n = 14 cells), and rods from Rho^F45L knock-in mutant mice (red; n = 13 cells) plotted together as a function of flash intensity. Error bars indicate SEM. The lines represent curves fitted to the data points using a Naka–Rushton function. D, Normalized averaged dim flash responses for rods from wild-type mice (black; n = 10 cells), and from Rho^F45L knock-in mutant mice (red; n = 13 cells) plotted together for comparison of response kinetics. All measurements were done from three-month-old mice.

Figure 7.

Effect of the Rho^F45L knock-in mutation on dark adaptation of rods. Recovery of absolute (A) and normalized (B) scotopic ERG maximal a-wave amplitudes (R_max; mean ± SEM) after bleaching >90% of rhodopsin in eyes of wild-type mice (black, n = 12) and Rho^F45L knock-in mutant mice (red, n = 12). Bleaching was achieved by a 35-s illumination with bright 520 nm LED light at time 0. R_max^DA refers to the prebleach maximal response in the dark (DA). Averaged data points were fitted with single exponential functions yielding time constants of 18.7 ± 0.9 and 16.2 ± 0.6 min for wild-type and Rho^F45L knock-in mice, respectively. Recovery of absolute (C) and normalized (D) scotopic ERG a-wave flash sensitivity (S_f; mean ± SEM) after bleaching >90% of rhodopsin in the same wild-type mice (black, n = 12) or Rho^F45L knock-in mice (red, n = 12). S_f^DA designates the sensitivity of dark-adapted (DA) rods. All measurements were done from three-month-old mice.