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J Biol Chem. 2013 Mar 8;288(10):7420-9. doi: 10.1074/jbc.M112.412791. Epub 2013 Jan 21.

Kir2.4 surface expression and basal current are affected by heterotrimeric G-proteins.

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1
Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

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.

PMID:
23339194
PMCID:
PMC3591649
DOI:
10.1074/jbc.M112.412791
[Indexed for MEDLINE]
Free PMC Article
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