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W. Mao, K. Miyagishima, H. Moaven, A. P. Sampath, J. Chen; The Role of Distinct Isoforms of Transducin-Alpha Subunits to the Different Light Sensitivities of Rods and Cones. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5446.
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© ARVO (1962-2015); The Authors (2016-present)
Rods and cones use a similar biochemical cascade to convey photon capture to a change in current at the plasma membrane. However, rod photoreceptors are approximately a hundred times more sensitive to light than cones. The molecular mechanisms that underlie this difference in sensitivity are not well understood. Transducin, a heterotrimeric G-protein involved in phototransduction cascade, consists of different subunits in rods and cones. We hypothesized that cell-specific transducin-alpha may be one underlying factor for the differences in light sensitivity between rods and cones.
The mouse rod opsin promoter was used to direct expression of mouse cone transducin-alpha (cT) in rod cells of rod transducin-alpha knockout (rT-/-) mice. The distribution of cT in both dark-adapted and light-exposed rods was investigated by immunocytochemistry. The ability of cT to heterotrimerize with rod Tβγ was analyzed through pertussis toxin-catalyzed ADP ribosylation of cT. Suction electrode recordings from single rods and ERGs were performed to compare the sensitivity of flash responses between transgenic and wildtype mice.
Western results showed that cT expression level in rods was ~15% of wildtype rT. To further increase cT expression level, we bred the cT transgenic mice to homozygosity. Ectopically expressed cT localizes to the outer segment in the dark and exhibited similar pattern of light-driven translocation as wild-type rT in rods. Whereas rod Tβγ diffuses in the rT-/- rods and does not translocate in response to light, expression of cT in these rods restored the polarized distribution of Tβγ to the outer segment in the dark and to the inner segment in response to light. Similar to wildtype rT, ADP ribosylation of cT was inhibited by light or in the presence of AlF4-. Suction electrode recordings showed that flash responses from cT rods were 10-20 fold less sensitive and recovered faster when compared to wild-type rods.
Our immunocytochemistry data suggest that cT can interact with rod Tβγ and thereby restore their localization to the outer segment in the dark and their ability to translocate to the inner segment in response to light. Biochemical analysis of ADP ribosylation provided direct evidence for the interaction between cT and Tβγ. Exchanging endogenous rT with cT results a decrease of light sensitivity that correlated with the reduced expression level. Therefore, the level of transduction gain exhibited by cell-type specific G-proteins appear to be similar between rods and cones.
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