title: Kir2.4 surface expression and basal current are affected by heterotrimeric G-proteins.
abstract: Kir2.4, a strongly rectifying potassium channel that is localized to neurons and is especially abundant in retina, was fished with yeast two-hybrid screen using a constitutively active Gαo1. Here, we wished to determine whether and how Gαo affects this channel. Using transfected HEK 293 cells and retinal tissue, we showed that Kir2.4 interacts with Gαo, and this interaction is stronger with the GDP-bound form of Gαo. Using two-electrode voltage clamp, we recorded from oocytes that were injected with Kir2.4 mRNA and a combination of G-protein subunit mRNAs. We found that the wild type and the inactive mutant of Gαo reduce the Kir2.4 basal current, whereas the active mutant has little effect. Other pertussis-sensitive Gα subunits also reduce this current, whereas Gαs increases it. Gβγ increases the current, whereas m-phosducin, which binds Gβγ without affecting the state of Gα, reduces it. We then tested the effect of G-protein subunits on the surface expression of the channel fused to cerulean by imaging the plasma membranes of the oocytes. We found that the surface expression is affected, with effects paralleling those seen with the basal current. This suggests that the observed effects on the current are mainly indirect and are due to surface expression. Similar results were obtained in transfected HEK cells. Moreover, we show that in retinal ON bipolar cells lacking Gβ3, localization of Kir2.4 in the dendritic tips is reduced. We conclude that Gβγ targets Kir2.4 to the plasma membrane, and Gαo slows this down by binding Gβγ.
authors: Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors: Pyroja
authors: P
authors: Sulaiman
authors: Ying
authors: Y
authors: Xu
authors: Marie E
authors: ME
authors: Fina
authors: Shanti R
authors: SR
authors: Tummala
authors: Hariharasubramanian
authors: H
authors: Ramakrishnan
authors: Anuradha
authors: A
authors: Dhingra
authors: Noga
authors: N
authors: Vardi
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D005260:Female; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009154:Mutation; D009865:Oocytes / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012160:Retina / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D014981:Xenopus
chemical_list: D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; C489465:Kir2.4 channel; D024661:Potassium Channels, Inwardly Rectifying; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins