G Protein Alpha Transducin
G protein alpha transducin was purified from bovine retinal extracts more than three decades ago. The discovery of transducin was the culmination of a series of findings that started in the mid-1970s (see Bourne 2006 for a recent review), beginning with the identification of the effector molecule cGMP phosphodiesterase (Bitensky et al. 1975). Shortly afterward, the presence of light-induced GTPase activity in rod outer segment extracts was described (Wheeler and Bitensky 1977). In 1978, activation of cGMP phosphodiesterase was shown to require activated rhodopsin and the presence of GTP (Yee and Liebman 1978). In 1979, a soluble guanine nucleotide binding protein with light-activated GTP–GDP exchange was purified (Godchaux and Zimmerman 1979). However, only two components, the a and β subunits, of the heterotrimeric G protein transducin were described. In 1981, the full heterotrimeric G protein was purified in Lubert Stryer’s group, and they named it “transducin.” Using purified transducin and rhodopsin, Fung et al. were able to demonstrate light-dependent binding and hydrolysis of GTP (Fung et al. 1981). These pioneered discoveries were later augmented by human and mouse genetics along with suction-electrode recordings to elucidate the role of transducin in phototransduction. However, many questions remain unresolved.
Role of Transducin in Phototransduction
In the absence of light, photoreceptors maintain a high concentration of intracellular cGMP. The dark cGMP level is sufficient to keep up to 10% of cyclic nucleotide-gated (CNG) channels on the plasma membrane open to allow an inward “dark current” of sodium and calcium ions to depolarize outer segment membranes to a resting voltage of approximately −35 mV. At this depolarized state, photoreceptors tonically release glutamate at their synaptic terminals. When light is introduced, a cascade of events involving transducin and collectively called phototransduction is activated, which leads to photoreceptor hyperpolarization and cessation of the tonic glutamate release.
Distinct phototransduction molecules in mouse rods and cones
Green-sensitive opsin (Opn1mw), blue-sensitive opsin (Opn1sw)
G protein alpha
G protein beta/gamma
Gβ1γ1 (Gnb1, Gngt1)
Gβ3γc (Gnb3, Gngt2)
PDE6A (Pde6a), PDE6B (Pde6b)
The exchange of GTP for GDP on the α subunit of transducin, catalyzed by light-activated rhodopsin, constitutes the first amplification step in the phototransduction cascade. Transducin has a fairly fast guanine nucleotide exchange rate at approximately 1,000/s (Vuong et al. 1984). During its effective lifetime, R* may activate multiple transducin molecules. The estimated rate of transducin activation by a single R* has been reported in the range of 10–3,000/s (Fu and Yau 2007). In mouse rods, the activation rate of transducin by R* is reported to be ~240/s (Fu and Yau 2007). Rods have a large pool of transducin molecules and this characteristic has partly been attributed to the ability of rods to sense a single photon of light. The activation rate of rod transducin is approximately tenfold higher than cone transducin by cone pigment, which also contributes to rod’s higher sensitivity to light (Luo et al. 2008). The effector cGMP phosphodiesterase (PDE) is a tetrameric membrane-associated protein consisting of two active and highly similar catalytic subunits αβ and two identical inhibitory γ subunits. The interaction of T* with PDEγ removes the inhibitory PDEγ from the two catalytic PDEαβ subunits. Since there are two PDEγ subunits in the tetrameric PDEαβγ2, two activated T* molecules are required to achieve full PDE activation.
Light-Dependent Redistribution of Transducin
In darkness, transducin is localized to the outer segment (OS) of rod photoreceptors. After prolonged light exposure, transducin can redistribute throughout the photoreceptor (Slepak and Hurley 2008). Interestingly, the Gβγ subunits also redistribute, but follow a different time course. Upon return to darkness, both Gat and Gβγ return to the rod OS. Two lipid modifications are present in heterotrimeric rod transducin: Gαt is N-acylated and Gγ is S-prenylated on the C-terminus with a farnesyl group. A mutant form of Gαt (Q200L) that lacks the intrinsic GTPase activity was developed. The Q200L Gαt is constitutively bound to GTP and maintained in the active state (T*), and thus cannot reassociate with Gβγ. The Q200L Gαt is unable to stay localized to the rod OS in darkness, suggesting that reassociation with Gβγ was necessary for localization to the rod OS (Artemyev 2008; Slepak and Hurley 2008). Interestingly, cone transducin does not redistribute in response to light. One reason for this difference is the fact that Gβγ in cones has a stronger affinity for cone transducin even in the activated form. When a transgenic mouse was developed replacing rod transducin with cone transducin, the transgenic cone transducin in rods redistributed in a light-dependent manner (Chen et al. 2010). This suggests that it is not the proteins, but rather the cells that are responsible for the lack of transducin redistribution in cones. However, a recent paper by Lobanova et al. demonstrates that intense light exposure for a prolonged period of time can cause cone transducin to redistribute in cones. This entry further shows that increasing cone sensitivity by ectopically expressing rhodopsin also moves cone transducin out of the outer segment in a light-dependent manner (Lobanova et al. 2010).
Role of Transducin in Photoreceptor Sensitivity and Response Decay
Rods are more sensitive to light than cones. Since rods and cones have distinct set of transducin molecules (see Table 1), the distinct proteins found in rods versus cones may have inherent properties that contribute to differences in their responses to light. To study whether transducin has any contribution, a transgenic mouse was generated with full-length mouse cone transducin (Gnat2) expressed in rods (Chen et al. 2010). This transgenic mouse was mated onto the Gnat1–/– mouse background to produce the GNAT2C mouse line. In GNAT2C rods, where cone transducin replaces rod transducin, GNAT2 expression level is similar to that of GNAT1 in normal mouse rods. Using suction-electrode recordings, GNAT2C rods were found to be three times less sensitive than wild-type and required brighter light to produce responses of the same amplitude (Chen et al. 2010). GNAT2C rods also displayed accelerated response decay compared to wild-type. When GNAT2C mice were mated to a mouse line overexpressing transducin GAP, the response recovery was accelerated even further beyond what was achieved from the transducin substitution (Chen et al. 2010). In the RGS9–/– background, GNAT2C rods could still turn off twofold faster than wild-type rods (Chen et al. 2010). These results suggested that the species of Gαt may indeed play an important role in setting the response kinetics and sensitivity of rods and cones.
GNAT1-Independent Phototransduction in Rods
Researchers frequently utilize the Gnat1–/– mouse as a model for loss of rod phototransduction. In 2010, Allen et al. mated Gnat1–/–; Cnga3–/–, Opn4–/– together to generate a triple knockout (TKO) mouse line. The Cnga3–/– mouse has a loss of cone vision, while the Opn4–/– mouse lacks melanopsin, a visual pigment expressed in intrinsically light-sensitive retinal ganglion cells. The TKO mouse theoretically should not have any light response. However, when they were used as “negative” controls in electroretinography (ERG) experiments, they were found to have a reproducible flash ERG response at high stimulus intensities >0.5 log cd/m2 (Allen et al. 2010). Spectral analysis suggests that the light responses of TKO mice are mediated by rhodopsin. Since GNAT2 is known to couple to rhodopsin (Chen et al. 2010), Gnat1–/– was mated to a spontaneous mutant Gnat2cpfl3 mice, which have progressive loss of cone vision. The results from Gnat1–/–, Gnat2cpfl3 mice showed that the ERG response was reduced, but not eliminated. Consistent with their findings: the original characterization of the Gnat1–/– mouse by Calvert et al. showed that one (out of 213 examined) Gnat1–/– rod was found to have robust light responses similarly mediated by rhodopsin (Calvert et al. 2000). Allen et al. speculated that some ERG responses in TKO mice are mediated by GNAT2 (Allen et al. 2010). In an era in which genetics and technology are rampantly expanding the edge of scientific knowledge, the presence of robust light responses in TKO mice exemplifies the opportunities that exist in the vision research field, where scientific dogma can be frequently challenged to reveal underappreciated intricacy and beauty of the visual system that are yet to be fully comprehended.
The critical role of Gαt1 in the phototransduction cascade has been elucidated in rods, while the role of Gαt2 in cone phototransduction is less understood. Custom-made transgenic animals, such as the GNAT2C mouse, should be useful to further dissect the difference of rod and cone phototransduction.
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