Functional interchangeability of rod and cone transducin alpha-subunits

Abstract

Rod and cone photoreceptors use similar but distinct sets of phototransduction proteins to achieve different functional properties, suitable for their role as dim and bright light receptors, respectively. For example, rod and cone visual pigments couple to distinct variants of the heterotrimeric G protein transducin. However, the role of the structural differences between rod and cone transducin alpha subunits (Talpha) in determining the functional differences between rods and cones is unknown. To address this question, we studied the translocation and signaling properties of rod Talpha expressed in cones and cone Talpha expressed in rods in three mouse strains: rod Talpha knockout, cone Talpha GNAT2(cpfl3) mutant, and rod and cone Talpha double mutant rd17 mouse. Surprisingly, although the rod/cone Talpha are only 79% identical, exogenously expressed rod or cone Talpha localized and translocated identically to endogenous Talpha in each photoreceptor type. Moreover, exogenously expressed rod or cone Talpha rescued electroretinogram responses (ERGs) in mice lacking functional cone or rod Talpha, respectively. Ex vivo transretinal ERG and single-cell recordings from rd17 retinas treated with rod or cone Talpha showed comparable rod sensitivity and response kinetics. These results demonstrate that cone Talpha forms a functional heterotrimeric G protein complex in rods and that rod and cone Talpha couple equally well to the rod phototransduction cascade. Thus, rod and cone transducin alpha-subunits are functionally interchangeable and their signaling properties do not contribute to the intrinsic light sensitivity differences between rods and cones. Additionally, the technology used here could be adapted for any such homologue swap desired.

Fig. 1.

Translocation of Tα in rd17 retinas. AAV-expressed rod Tα and cone Tα was observed by immunostaining in both rods (arrows) and cones (arrow heads) in rd17 retinas after treatment with AAV5-CBA-rod Tα (A) or AAV5-CBA-cone Tα (B) vectors. Cones were labeled with PNA (green); rod or cone Tα was labeled red by rod or cone-specific antibodies. (A) Localization of vector-expressed rod Tα in cones paralleled that of endogenous cone Tα. In the dark, AAV-expressed rod Tα was observed only in the OS in both rods and cones. Under light conditions, rod Tα was observed in both rod OS and IS, whereas rod Tα remained only in cone OS. (B) Localization of vector-expressed cone Tα in rods was similar to that of endogenous rod Tα. In the dark, cone Tα was observed only in OS of both rods and cones. Under light conditions, exogenously expressed cone Tα was redistributed into rod IS, whereas the majority of cone Tα remained localized to cone OS. (Scale bar, 20 μm.)

Fig. 2.

Electrophysiological analysis of Trα−/−, GNAT2^cpfl3, and rd17 mice after treatment with rod or cone Tα vectors. Each data point represents the mean ± SD of b-wave amplitudes recorded for each group at the indicated input flash intensity. (A) Comparison of dark-adapted ERG responses from wild-type, Trα−/−, and contralateral Trα−/− eyes treated with AAV5-mOP-cone Tα. The rod-driven b-wave (flash intensity at 0.01 cd.s.m⁻²) missing in the untreated Trα−/− eye was partially restored after treatment with AAV5-mOP-cone Tα. Paired t test analysis showed that the b-wave amplitude at this intensity was significantly different between Trα−/− untreated eyes and fellow AAV5-mOP-cone Tα-treated eyes (P < 0.001). (B) Comparison of light-adapted cone-mediated ERGs in wild-type, GNAT2^cpfl3 untreated and contralateral AAV5-PR2.1-rod Tα-treated GNAT2^cpfl3eyes. ERG responses were recorded after adaptation to a rod-saturating background light. Statistical analysis showed significant differences between untreated and fellow treated GNAT2^cpfl3 eyes at flash intensities of 5 and 10 cd.s.m⁻² (P < 0.001). (C and D) Both dark-adapted (C) and light-adapted (D) ERG b-waves were partially restored in rd17 mice after treatment with either AAV5-CBA-rod Tα or AAV5-CBA-cone Tα vectors. Statistical analysis demonstrated significant differences between untreated and fellow vector-treated eyes for dark-adapted b-waves at 0.01, 0.1, 1, and 5 cd.s.m⁻² (P < 0.001) and for light-adapted b-waves at 5 and 10 cd.s.m⁻² (P < 0.05). No statistical difference in recovered b-wave amplitudes was found between AAV5-CBA-rod Tα and AAV5-CBA-cone Tα treated rd17 eyes under either dark-adapted or light-adapted conditions. B-wave amplitudes at indicated flash intensities were compared by repeated-measures ANOVA, with the Bonferroni post hoc test for ANOVA (P < 0.05) used to compare means at individual flash intensities.

Fig. 3.

Response families from ex vivo transretinal ERG recordings obtained from wild-type (A), rod Tα-treated (B), and cone Tα-treated (C) rd17 retinas. Test flashes of incremental intensities, in 0.5 log steps, were delivered at time 0. The dimmest flashes delivered 7.6 (A) and 25 (B and C) photons μm⁻². (D) Intensity-response relations of individual retinas normalized for maximal response (R_max) and half-saturating flash intensity (I_o). Whereas the intensity-response relations in wild-type retinas (black symbols, n = 4) were well fit by Eq. 1 with k = 1 (solid line), those for rod Tα-treated (blue symbols, n = 4) and cone Tα-treated (red symbols, n = 5) retina were less steep than Eq. 1. (Inset) Cumulative results of normalized sensitivity (S_f) from individual wild-type (black, n = 4), rod Tα-treated (blue, n = 4), and cone Tα-treated (red, n = 5) rd17 retinas. Statistical analysis was carried out by the one-way ANOVA with the post hoc Bonferroni test.

Fig. 4.

Response families from single-cell recordings obtained from individual wild-type (A), rod Tα-treated (B), and cone Tα-treated (C) rd17 rods. Test flashes of incremental intensities, in 0.5 log steps, were delivered at time 0. The dimmest flashes delivered 1.0 (A) and 4.4 (B and C) photons μm⁻². (D) Intensity-response relations of individual cells normalized for maximal response (R_max) and half-saturating flash intensity (I_o). Data from wild-type rods (black symbols, n = 9), rod Tα-treated rd17 rods (blue symbols, n = 9), and cone Tα-treated rods rd17 (red; n = 12) were all well fit by Eq. 1 with k = 1. (Inset) Cumulative results of normalized sensitivity (S_f) from individual wild-type (black, n = 9), rod Tα-treated (blue, n = 9), and cone Tα-treated (red, n = 12) rods. Statistical analysis was carried out by the one-way ANOVA with the post hoc Bonferroni test.

Fig. 5.

Transretinal ERG response families from rd17 retinas treated with AAV5-PR2.1-rod or cone Tα vector. (A) Although ERG responses could be restored in rd17 retina by treating with AAV5-PR2.1-cone Tα, the resulting responses had rod-like kinetics and sensitivity, indicating rescue of rod function without detectable rescue of cone function. (B) Both rod (slow) and cone (fast) ERG responses could be restored in rd17 retina by treating with AAV5-PR2.1-rod Tα, indicating rescue of both rod and cone function. Test flashes of incremental intensities, in 0.5 log steps, were delivered at time 0. In both A and B, the dimmest flash delivered 25 photons μm⁻². The largest response in each panel was generated by unattenuated white light. (Inset) Normalized cone dim flash responses from the retina in B (red trace) and from wild-type retina (blue trace) extracted by double-flash stimulation. For comparison, wild-type rod response (black trace) is also shown.