Prolonged photoresponses and defective adaptation in rods of Gbeta5-/- mice

Abstract

Timely deactivation of G-protein signaling is essential for the proper function of many cells, particularly neurons. Termination of the light response of retinal rods requires GTP hydrolysis by the G-protein transducin, which is catalyzed by a protein complex that includes regulator of G-protein signaling RGS9-1 and the G-protein beta subunit Gbeta5-L. Disruption of the Gbeta5 gene in mice (Gbeta5-/-) abolishes the expression of Gbeta5-L in the retina and also greatly reduces the expression level of RGS9-1. We examined transduction in dark- and light-adapted rods from wild-type and Gbeta5-/- mice. Responses of Gbeta5-/- rods were indistinguishable in all respects from those of RGS9-/- rods. Loss of Gbeta5-L (and RGS9-1) had no effect on the activation of the G-protein cascade, but profoundly slowed its deactivation and interfered with the speeding of incremental dim flashes during light adaptation. Both RGS9-/- and Gbeta5-/- responses were consistent with another factor weakly regulating GTP hydrolysis by transducin in a manner proportional to the inward current. Our results indicate that a complex containing RGS9-1-Gbeta5-L is essential for normal G-protein deactivation and rod function. In addition, our light adaptation studies support the notion than an additional weak GTPase-accelerating factor in rods is regulated by intracellular calcium and/or cGMP.

Figure 2.

Effect of disrupting Gβ5 on the mean single photon response. a, Population mean single photon responses for +/+ (circles; n = 28), +/- (triangles; n = 13), and -/- (squares; n = 12) rods. Average dark currents (in pA) of the cells used in this determination were 11.3 ± 0.5, 13.0 ± 0.9, and 12.1 ± 1.3, respectively. b, Mean single photon responses from a, shown on an expanded time scale. Error bars represent SEM. c, Initial rate of change of light-evoked PDE activity (dPDE/dt) for four wild-type rods (open symbols) and five Gβ5^-/- rods (filled symbols) (see Materials and Methods).

Figure 1.

Families of responses to increasing flash strengths from representative wild-type, knock-out, and hemizygote mice. Responses have been normalized (r/r_max) by the maximal response amplitudes, which were the following (in pA): 16.3 (+/+), 16.1 (-/-), and 17.7 (+/-). Flash strengths (in photons/μm²) ranged from 11 to 4546 (+/+), 11 to 8563 (+/-), and 19 to 8277 (-/-).

Figure 3.

Comparison of Gβ5^-/- rods (left) and RGS9^-/- rods (right). a, Average dim flash responses of representative Gβ5^-/- and RGS9^-/- rods. Falling phases were fitted with single exponential functions (bold) with time constants (τ) as indicated. Dark currents were 17.1 pA (Gβ5^-/-) and 9.7 pA (RGS9^-/-). Flash strengths (in photons/μm²) were 5.7 (Gβ5^-/-) and 9.4 (RGS9^-/-). b, Time that bright flash responses remained in saturation (T_sat) as a function of the natural log of the flash strength (ln i) (in photons/μm²). Each point in the Gβ5^-/- plot is the average of 12-14 cells, and each point in the RGS9^-/- plot is an average of three to four cells. Error bars reflect SEM. Mean time constants were 8.8 ± 0.3 sec (Gβ5^-/- rods; n = 17) and 9.8 ± 0.6 sec (RGS9^-/- rods; n = 4). c, Saturating flash responses for representative Gβ5^-/- (left) and RGS9^-/- (right) rods. For both cells, the recovery accelerates as the current returns. Dark currents (in pA) and flash strengths (in photons/μm²) for these examples were 9.5 pA and 2255 photons/μm² (Gβ5^-/-) and 14.4 pA and 2095 photons/μm² (RGS9^-/-). Response amplitudes were normalized by the dark current.

Figure 4.

Impaired adaptation of dim flash responses in Gβ5^-/- rods. a, Average dim flash response of representative Gβ5^+/+ (left) and Gβ5^-/- (right) rods before, after, and during steady light exposure. The intensities of the background light were 30 photons/μm²/sec (Gβ5^+/+) and 7.2 photons/μm²/sec (Gβ5^-/-), which turned off 10 and 16% of the original dark current, respectively. The Gβ5^+/+ integration times shortened slightly in the presence of background light, whereas the Gβ5^-/- integration times slowed significantly in the presence of background light. Dim flash responses were normalized (r′) by peak amplitude for comparison of response durations (integration time) (see Materials and Methods). Dark currents (in pA) were 15.2 (Gβ5^+/+) and 16.0 (Gβ5^-/-). In this example, flash strengths were 10 photons/μm² in the presence and absence of background light (Gβ5^+/+), and 10 photons/μm² in darkness and 19 photons/μm² in background light (Gβ5^-/-). b, Integration times from Gβ5^+/+ (left) and Gβ5^-/- (right) dim flash responses before, during, and after exposure to steady light. The background light intensities ranged from 30 to 1340 photons/μm²/sec for the wild-type rods, turning off 10-49% of the original dark current. The background intensities for the Gβ5^-/- rods ranged from 5.9 to 25 photons/μm^-2/sec, turning off 16-40% of the original dark current. c, Average fractional change in integration times during light adaptation for the cells in b. The integration time in the dark is the average of the integration times before and after background light. Error bars represent SEM.

Figure 5.

Adaptation of bright-flash responses in Gβ5^-/- rods. a, Saturating responses of representative Gβ5^+/+ (left) and Gβ5^-/- (right) rods before, after, and during background light exposure. The intensities of the background lights (in photons/μm²/sec) were 340 (Gβ5^+/+) and 17 (Gβ5^-/-), and the flash strengths (in darkness and in the presence of the background light) were 2452 (Gβ5^+/+) and 788 (Gβ5^-/-) photons/μm². The time that both Gβ5^+/+ and Gβ5^-/- responses remained in saturation shortened in the presence of background light. b, Time spent in saturation (T_sat) for Gβ5^+/+ (left) and Gβ5^-/- (right) rods before, during, and after exposure to background light. The background light intensities ranged from 30 to 1340 photons/μm²/sec for wild-type rods, turning off 10-49% of the original dark current. The background intensities for Gβ5^-/- rods ranged from 7.1 to 25 photons/μm²/sec, turning off 16-37% of the original dark current. c, Average fractional change in time spent in saturation during adaptation for the cells in b. The time in saturation in the dark is the average of the saturation times before and after background light. Error bars represent SEM.