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Y. Xu, N. Vardi; Modulation of the Light-Activated Cation Channel in Retinal ON Bipolar Cells by G-Protein Subunits. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4797.
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© ARVO (1962-2015); The Authors (2016-present)
In darkness, glutamate released from photoreceptors hyperpolarizes retinal ON bipolar cells by activating the heterotrimeric G-protein Go and closing non-selective cation channels thought to be TRPM1. Here we asked which subunit, Gαo1 or Gβγ, mediates the channel closure.
Whole-cell patch recordings were made from mouse rod bipolar cells clamped at -60 mV. Retina was perfused with AMES solution including strychnine and picrotoxin (to block Cl- channels). Control pipette solution included cesium (to block K+ channels), BAPTA (to buffer Ca2+) and ATP. Test solutions included control solution and either Gαo1, GTP-γ-s, GDP-β-s or phosducin. The retina was either dark or light adapted. A light pulse (ON or OFF) was given every 35 seconds. The holding current and light responses were compared over time between different dialyzed solutions.
Under light adaptation, dialyzing GTP-γ-S (25 µM to 50 µM) quickly decreased the basal current and diminished the light OFF response, confirming that activated G protein cascade closes the channel. But when Gαo1-GTP (100 nM) was dialyzed into bipolar cells (n=18), there was no change in either basal current or light OFF responses, indicating Gαo1 does NOT close the channel. Dialyzing GDP-β-s (500 µM, n=3) to de-activate Gαo1 didn’t cause any change either.Under dark adaptation, dialyzing Gαo1-GTP (n=25) increased the holding current significantly while dialyzing the control solution caused no change. De-activating Gαo1 with GDP-β-S (n=15) removed the effect leaving the holding current as stable as in control cells (n=20). These data suggest that Gαo1 opens the channel instead of closing it.To test the effect of Gβγ, phosducin (9 µM), a scavenger of this dimer, was dialyzed under dark adaptation. The basal current increased significantly (n=5) and the light ON response increased slightly as well.When rod bipolar cells were dialyzed with a mutated phosducin (thus lose the ability to scavenge Gβγ; n=3), no clear change was observed. This is consistent with Gβγ closing the channel: by scavenging Gβγ with phosducin, the channel opens to allow more cations flow into the cell.
Our data suggest that Gβγ, instead of Gαo1, closes the channel. Activation of Gαo1, however, is required to release Gβγ to close the channel.
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