Purpose:
ON bipolar neurons in retina hyperpolarize in response to glutamate released by photoreceptors through a G-protein cascade. This cascade is initiated by activation of mGluR6, that activates the G protein Go, and eventually closes the TRPM1 channel. Two G-protein beta subunits, Gβ3 and Gβ5, are present in all ON bipolar cells. In this study we investigated the role of Gβ3 in ON bipolar cells at the molecular and functional level.
Methods:
Expression of ON bipolar cell cascade elements was examined in the Gnb3-/- retina by immunocytochemistry and Western blots. Electron microscopy on WT and Gnb3-/- retina was performed by standard methods. ON bipolar cell function was assessed by electroretinographic (ERG) recordings under scotopic and photopic conditions and by whole cell recordings. Co-immunoprecipitation using the Gαo antibody was performed followed by Western blotting for detection of Gβ3 in the complex.
Results:
Absence of Gβ3 caused dramatic reduction of:1. The levels of the G-protein subunits, Gαo and Gγ13 .2. Immunostainng of several cascade proteins that in the WT retina concentrate in the dendritic tips. These include mGluR6, RGS11, TRPM1, and Gβ5.3. The number of intact synaptic triads with two horizontal cell processes and at least one bipolar dendrite (reduced to ~1/3rd).4. Scotopic ERG b-wave (totally absent).5. Current response to light (totally absent by whole cells recordings from rod bipolar cells).6. Photopic ERG b-wave (reduced, but not totally eliminated).Some of the effects listed above (reduction in punctate staining of cascade modulators, effect on triads, and absence of scotopic b-wave) also result due to absence of Gβ5; however Gαo, Gβ3, and Gγ13 are largely unaffected in this mutant. Further probing the role of Gβ3, we showed that co-immunoprecipitation using Gαo antibody on a wild type retinal homogenate also precipitates Gβ3.
Conclusions:
Gβ3 is the partner of Gαo in the G-protein heterotrimer, it is required for the normal light ON response in rod bipolar and cone ON bipolar cells, and it is required for the correct assembly of the postsynaptic proteins associated with the mGluR6 cascade.
Keywords: signal transduction • immunohistochemistry • gene/expression