Mechanistic basis for the failure of cone transducin to translocate: why cones are never blinded by light

Abstract

The remarkable ability of our vision to function under ever-changing conditions of ambient illumination is mediated by multiple molecular mechanisms regulating the light sensitivity of rods and cones. One such mechanism involves massive translocation of signaling proteins, including the G-protein transducin, into and out of the light-sensitive photoreceptor outer segment compartment. Transducin translocation extends the operating range of rods, but in cones transducin never translocates, which is puzzling because cones typically function in much brighter light than rods. Using genetically manipulated mice in which the rates of transducin activation and inactivation were altered, we demonstrate that, like in rods, transducin translocation in cones can be triggered when transducin activation exceeds a critical level, essentially saturating the photoresponse. However, this level is never achieved in wild-type cones: their superior ability to tightly control the rates of transducin activation and inactivation, responsible for avoiding saturation by light, also accounts for the prevention of transducin translocation at any light intensity.

Figure 1.

Diagram illustrating the light intensity threshold-dependent mechanism of transducin translocation. A, At light intensities below the threshold, activated transducin binds to PDE and is rapidly inactivated by RGS9, providing little time for dissociation from the membrane. B, At light intensities above threshold, more transducin is activated than can bind to PDE and be readily inactivated by RGS9. This led to a fraction of transducin staying activated sufficiently long enough to dissociate from the membrane to the cytosol. The amount of activated transducin in each case is represented by the thickness of the arrows. The basic elements of this diagram are reproduced with permission from Lobanova et al. (2007).

Figure 2.

A–D, Determination of the molar ratio among transducin subunits (A, Gα_tc; B, Gγ₈), PDEγ_c (C), and phosducin (Pdc; D) in cones of Nrl ^−/− mice. Retina lysate aliquots of indicated volumes were separated by SDS-PAGE along with 0.1, 0.2, 0.3, and 0.4 pmol of each protein standard and immunoblotted using the corresponding antibodies. The examples of calibration curves for each protein are shown below the blots. All data were obtained with retina extract obtained from a single animal; the results obtained in multiple experiments are summarized in the text.

Figure 3.

Transducin translocation in cones of R9AP knock-out mice. Wild-type C57BL/6 or R9AP ^−/− mice were either dark adapted or exposed to 25 min of illumination from a calibrated light source producing a light intensity of 100,000 lux at the cornea surface. Animals were killed, eyes fixed, and retina cross sections stained with antibodies against Gα_tc (top panels) or Gγ₈ (bottom panels). Note that weak Gγ₈ immunostaining was observed in cone synaptic terminals of wild-type and dark-adapted R9AP ^−/− mice, perhaps due to its engagement into Gβγ complexes distinct from transducin. However, this signal was further enhanced in light-adapted R9AP ^−/− cones. The subcellular compartments of the cone are labeled as follows: OS, Outer segment; IS, inner segment; N, nucleus; ST, synaptic terminal.

Figure 4.

The light-dependency of cone transducin translocation in R9AP knock-out mice. The animals were treated as described in the Figure 3 legend at the light intensities indicated above each panel. Retina cross sections were immunostained with antibodies against Gα_tc (top panels) or Gα_t (bottom panels).

Figure 5.

Transducin translocation in rods and cones of GRK1 knock-out mice. A, B, GRK1 ^−/− (A) and wild-type mice (B) were either kept in the dark or exposed to 25 min illumination from a calibrated light source producing the light intensities indicated above the panels. Animals were killed and retina cross sections were stained with antibodies against Gα_t. C, A similar protocol using brighter illumination levels was applied to GRK1 ^−/− mice, and the sections were stained with antibodies against Gα_tc.

Figure 6.

Cone transducin translocation in cones expressing rod visual pigment. Representative Gα_tc immunostaining in retina sections obtained from wild-type mice coinfected with AAV5-PR2.1-mRhodopsin and AAV5-PR2.1-GFP. A total of 13 animals were tested in the dark or at a range of light intensities from 10 to 100,000 lux. Animals were killed and their eyes fixed. Retina cross sections were obtained from portions of the retina displaying the brightest GFP fluorescence and stained with antibodies against Gα_tc.

Figure 7.

Evaluation of cone light responses at background light below and above transducin translocation threshold in R9AP knock-out mice. ERGs evoked by test flashes producing 1000 cd · s/m² were recorded from wild-type C57BL/6 or R9AP ^−/− mice according to the protocol illustrated in A: first after subjecting animals to 3 min illumination at 40 lux, which is sufficient to completely suppress rod-driven responses, next after applying bright light of the various intensities indicated for 25 min, and finally after returning to 40 lux illumination for a period of 6 min (first flash applied 1 min after cessation of bright light). The period between all flashes was 1 min, which was sufficient for complete response recovery from the preceding flash. Animals were killed immediately after completing ERG recordings, and their retinas were processed for Gα_tc immunostaining. B, ERG recordings averaged from 6 (40 lux) or 25 (other conditions) individual trials are shown in the middle. Representative immunostaining images of retina cross sections from selected illumination conditions (marked by arrows) are shown on the sides. C, The averaged amplitudes of ERG b-waves recorded under the initial 40 lux condition and all conditions of bright illumination are plotted as a function of background illuminance. Data points are connected by smooth dashed lines.