Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
Our data suggest that Gβγ, instead of Gαo1, closes the channel. Activation of Gαo1, however, is required to release Gβγ to close the channel.
Keywords: retina: neurochemistry • bipolar cells • signal transduction