Effect of accelerated metarhodopsin-II decay on mouse rod responses

Abstract

Purpose : We sought to determine how the rate of metarhodopsin-II (MII) decay affected the sensitivity and time course of light responses of dark-adapted mouse rods. Previous studies have shown that the rhodopsin mutant, E122Q, has a roughly 10 times faster decay of MII compared to wild-type (WT) rhodopsin.

Methods : We utilized ex-vivo electroretinograms (ERGs) to record changes in the photovoltage of the retina in response to flashes covering a wide range of intensities. Mice expressing E122Q rhodopsin were bred into a cone transducin knockout (gnat2^-/-) background to eliminate cone contributions. Retinae were superfused with Ames’ medium containing 1.9 g/L NaHCO₃ and equilibrated with 5% CO₂/95% O_2. Responses from downstream cells were blocked with BaCl₂ and DL-AP4. Brief flashes (10 ms) were delivered from a 505 nm LED, and responses were recorded with a DP-311 differential amplifier and stored on a computer for subsequent analysis.

Results : Plots of a-wave amplitude versus flash intensity were fit by using a Michaelis-Menten equation. Sensitivity was calculated as the light intensity that evoked a half-maximal response. Our results showed a significant decrease (t-test, p = 0.016) between the sensitivity of WT rods (I_1/2 = 39 ± 5.4 photons/µm², n = 3) and rods with E122Q rhodopsin (I_1/2 = 63 ± 4.6 photons/µm², n = 4), with the WT rods being about 1.7 times more sensitive than the E122Q after correction for the E122Q blue-shifted spectrum. However, the integration time of dim-flash responses showed no significant difference (t-test, p = 0.21) between WT mice (180 ± 2.5 ms, n =3) and the E122Q-rhodopsin mice (200 ± 11 ms, n = 4).

Conclusions : We observed a small desensitization in retinae containing E122Q rhodopsin, which may reflect a shorter lifetime of the active state of bleached rhodopsin. The similar integration times suggest that the rate-limiting step for the decay of the dim-flash response is not MII decay but rather the decay of activated phosphodiesterase, as previous work has indicated. Further microspectrophotometric studies will confirm previous biochemical measurements showing faster MII decay in E122Q rods, and time constants for the decay of the photoresponse will be measured with patch or suction electrodes.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.