Rhodopsin Kinase Expression Level and Its Effect on Recovery Phase

Abstract

Purpose: : Rhodopsin kinase (GRK1) phosphorylates light-activated rhodopsin. Previous studies indicate that in dark-adapted rods rhodopsin inactivation by GRK1 is required but not rate-limiting for proper flash response termination. However it is not known whether GRK1 phosphorylation affects rod function during dark- or light adaptation. To address this question, we investigated the function of rods from Grk1 transgenic mice (Grk1-ox), with 3-fold higher expression of GRK1 compared to wild type (WT).

Methods: : Using a suction electrode, we recorded membrane currents from singe rod photoreceptors of Grk1-ox mice and WT littermates. We compared their dim flash sensitivity, time-to-peak, integration time (T_int), and recovery time constant (τ_rec) as well as their dominant time constant of recovery (τ_D). We also investigated how GRK1 affects the recovery of dark current following a bleach and the PDE adaptation in background light of various intensities to lower rod [Ca²⁺]_i.

Results: : Rising phase and time-to-peak of dim flash response of Grk1-ox rods were comparable with WT rods. However, τ_rec and T_int were shortened significantly by Grk1 overexpression (τ_rec: 275 ± 17 ms (n = 36) for WT rod and 200 ± 16 ms (n = 21) for Grk1-ox rod; T_int: 356 ± 21 ms (n = 36) for WT rod and 254 ± 15 ms (n = 21) for Grk1-ox rod). Notably, unlike τ_rec, τ_D was not affected by GRK1 overexpression (269 ± 13 ms (n = 26) for WT and 274 ± 17 ms (n = 14) for Grk1-ox). After a 3% rhodopsin bleach, the recovery of dark current was accelerated significantly from 5.5 ± 0.8 min (n = 10) in WT to 2.9 ± 0.6 min (n = 9) in Grk1-ox rods. The [Ca²⁺]_i-dependent acceleration of response termination observed in WT rods was essentially absent in Grk1-ox rods indicating no PDE adaptation in background light.

Conclusions: : Surprisingly, GRK1 overexpression accelerated rod dim flash response shutoff indicating that rhodopsin inactivation could be rate-limiting. Enhancing rhodopsin inactivation also affected dark adaptation and background adaptation. The lack of PDE adaptation in Grk1-ox rods could explain the uncoupling of τ_rec and τ_D, believed until now to both reflect transducin inactivation.

Keywords: electroretinography: non-clinical • photoreceptors • phosphorylation