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A. V. Kolesnikov, O. G. Kisselev, V. J. Kefalov; Role of Transducin -subunit in the Activation and Inactivation of Mouse Rod Transducin. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3004.
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Light-activated visual pigment triggers phototransduction by activating a heterotrimeric G-protein, transducin (Gt). Domains on both Gt and Gtβγ subunits can interact with photoactivated (Meta II) rhodopsin indicating that Gtβγ might participate in allosteric regulation of nucleotide exchange on Gt subunit upon binging to Meta II rhodopsin. To investigate the role of Gtβγ in the activation and inactivation of transducin, we compared flash responses from wild type (WT) and Gtγ knockout (Gtγ-/-) mouse rods.
Using a suction electrode, we recorded membrane current from single mouse rod photoreceptors. We compared flash sensitivity, time-to-peak, integration time and recovery time constant (τrec) of dim flash responses from rods of 2-3 months old WT and Gtγ-/- mice. We also used saturating flash responses to compare WT and Gtγ-/- dominant time constants of recovery (τD). In mouse rods, τrec and τD are believed to be identical and to represent the rate of transducin inactivation.
We found that rods lacking Gtγ-subunit were ca. 77 times less sensitive to light than WT rods. As Gt content in rod outer segments of our KO mice was reduced to 10% of that in WT rods without significant outer segment degeneration, we attributed the remaining 7.7 fold difference in sensitivity to the lack of Gtγ. Thus, Gtγ appears to be required for the effective activation of transducin. The inactivation phase of the dim flash response was 2 times faster in Gtγ-/- rods compared to WT rods (τrec was 87 ± 6 ms (n = 27) and 172 ± 14 ms (n = 18), respectively). The faster response inactivation correlated with the substantially faster time-to-peak (95 ± 2 ms (n = 27) vs. 140 ± 4 ms (n = 18)) and integration time (128 ± 6 ms (n = 27) vs. 229 ± 16 ms (n = 18)) in Gtγ-/- rods compared to WT rods. Surprisingly, in contrast to the large reduction in τrec derived from dim flash responses, τD derived from saturating flash responses was only slightly decreased in Gtγ-/- rods compared to WT rods (177 ± 11 ms (n = 36) vs. 228 ± 13 ms (n = 22)).
Our results indicate that Gtγ is required for the effective activation of Gt heterotrimer by Meta II rhodopsin. Deletion of Gtγ also results in significant acceleration of the dim flash response inactivation, possibly due to the 10-fold decrease in the Gt to regulator of G-protein signaling (RGS9) complex ratio. Finally, we find that τrec and τD can be uncoupled by deletion of Gtγ indicating that they might be rate-limited by separate processes.
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