Phosducin regulates the expression of transducin betagamma subunits in rod photoreceptors and does not contribute to phototransduction adaptation

Abstract

For over a decade, phosducin's interaction with the betagamma subunits of the G protein, transducin, has been thought to contribute to light adaptation by dynamically controlling the amount of transducin heterotrimer available for activation by photoexcited rhodopsin. In this study we directly tested this hypothesis by characterizing the dark- and light-adapted response properties of phosducin knockout (Pd- / -) rods. Pd- / - rods were notably less sensitive to light than wild-type (WT) rods. The gain of transduction, as measured by the amplification constant using the Lamb-Pugh model of activation, was 32% lower in Pd- / - rods than in WT rods. This reduced amplification correlated with a 36% reduction in the level of transducin betagamma-subunit expression, and thus available heterotrimer in Pd- / - rods. However, commonly studied forms of light adaptation were normal in the absence of phosducin. Thus, phosducin does not appear to contribute to adaptation mechanisms of the outer segment by dynamically controlling heterotrimer availability, but rather is necessary for maintaining normal transducin expression and therefore normal flash sensitivity in rods.

Figure 1.

Families of flash responses from representative rods of WT (A) and phosducin knockout (B) mice. Flash strengths ranged from 8.05 to 6654 photons/μm² by factors of 2. (C) Normalized response amplitudes as a function of flash strength for the rods shown in A and B. Points were fitted by saturating exponential functions, with I_o values of 50.8 (WT) and 80.5 (Pd−/−) photons/μm².

Figure 2.

Saturating flash responses of phosducin knockout rods show normal recovery kinetics. (A) The time that a bright flash response remained in saturation was plotted as a function of the natural log of the normalized flash strength (in photons/μm²). In mouse rods, this relation is linear up to ln i ∼9 (Chen et al., 2000), with the slope equal to the dominant time constant of response recovery (Pepperberg et al., 1992; Nikonov et al., 1998). (B) Normalizing for the difference in sensitivity between Pd−/− and WT rods (dividing the x-axis by the Io values for each individual cell) underscores the similarity of Pd−/− and WT recovery time constants across the entire range of tested flash strengths. The straight line, which has been fitted to the Pd−/− data points, has a slope of 0.198 s.

Figure 3.

Phosducin knockout rods show normal recovery from larger bleaches. Recovery of the dark current (I_d) as a function of time following substantial bleaching exposures in different WT (black, n = 7) and Pd−/− (red, n = 9) rods. The brief (1.4 s) light exposure that bleached an average of 2.6% of the rhodopsin in WT rods suppressed I_d strongly, and it recovered to its dark value slowly, over a period of hundreds of seconds (see Materials and methods). Bleaches that drove transduction to a similar extent in Pd−/− rods (3.7% bleach) yielded responses that recovered along a similar time course.

Figure 4.

Single photon responses of Pd−/− rods are smaller than normal. (A) Population average single photon responses from 19 WT (solid) rods and 18 Pd−/− rods (bold). (B) Same traces as in A on an expanded time scale and normalized by the average dark currents for the population of rods (14.8 pA, WT; 13.9 pA, Pd−/−). Dashed lines are parabolic fits of the Lamb-Pugh model of phototransduction activation (see Materials and methods), resulting in amplification constants of 11.1 s⁻² (WT) and 7.6 s⁻² (Pd−/−). Red trace is the Pd−/− response multiplied by 1.46, compensating for the difference in the amplification constants. The scaled Pd−/− trace reaches a smaller peak amplitude than the WT response, highlighting an effect on the response unexplained by the 32% reduction in amplification constant.

Figure 5.

Quantitative Western blotting of Pd−/− retinal extracts reveals decrease in transducin subunits expression. (A) Whole retinal extracts containing 100 fmol rhodopsin from WT and Pd−/− mice were separated on 10–20% polyacrylamide gels alongside 25, 50, 75, 100, 150 fmol purified transducin standard (1–5), and probed in Western blot analysis with antibodies against rod transducin subunits. (B) Fluorescence of the corresponding transducin standards bands from the top panel were plotted against the amounts of transducin in samples 1–5 (open circles), and fitted with sigmoid curves. Triangles represent the fluorescence of transducin bands from retina extracts. The number of determinations for each subunit is indicated in Table II.

Figure 6.

Adaptation of incremental flash sensitivity is normal in Pd−/− rods. (A) Average dim flash responses (r′; in pA) delivered in darkness (solid) or in the presence of steady light (dash) that decreased the circulating (dark) current by ∼75%. In both WT and Pd−/− rods, steady light decreased the response amplitude and slightly accelerated the response time course as previously described (e.g., Krispel et al., 2003). Dark currents (in pA) were 16.2 (WT) and 16.1 (Pd−/−). (B) Flash sensitivity in background light (S_f) was normalized by the dark-adapted flash sensitivity (S_f ^D), measured before and after each background light intensity. The x-axis was normalized by the average I_o values for WT and Pd−/− rods, which caused the two relations to superimpose, indicating that Pd does not contribute directly to the changes in flash sensitivity that accompany background illumination. Points reflect mean values, with the number of cells varying between 2 and 7 for WT and 2 and 10 for Pd−/−.

Figure 7.

Adaptive acceleration is normal in Pd−/− rods. Prolonged (3 min) exposure to just-saturating light induced a shortening of the time in saturation that persisted for tens of seconds following the adapting light offset (Krispel et al., 2003). (A) Responses of a Pd−/− rod to a test flash (1945 photons/μm²) before, immediately after (dashed), and 100 s after a saturating light that produced a 0.86% cumulative bleach, assuming 7 × 10⁷ rhodopsins per rod (Lyubarsky et al., 2004) and an effective collecting area of 0.36 μm² (Krispel et al., 2006). (B) The shortening of the time in saturation was reversible, decaying exponentially back to the dark-adapted value with a time constant of 45 s.

Figure 8.

Light-dependent dephosphorylation of phosducin in WT mouse retinas. Dark-adapted retinas were exposed to the steady light of indicated intensities, under conditions closely matching those in the experiments described in Figs. 3 and 6. After 2 min of exposure, the retinas were homogenized in SDS-PAGE sample buffer, and the degree of phosducin phosphorylation was determined by Western blotting. (A) Phosducin bands were double labeled with either phosphospecific Pdc54p or Pdc71p antibody (red) and Pdc-pan antibody (green). (B) The fluorescence values of each red band was divided by the fluorescence value of the corresponding green band, and then the amount of phosphorylated phosducin in the light was normalized to the dark-adapted value (SEM, n = 3).