Kir2.4 Surface Expression and Basal Current Are Affected by Heterotrimeric G-Proteins

Pyroja Sulaiman,Ying Xu,Marie E Fina,Shanti R Tummala,Hariharasubramanian Ramakrishnan,Anuradha Dhingra,Noga Vardi

doi:10.1074/jbc.m112.412791

Pyroja Sulaiman, Ying Xu + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m112.412791

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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.

Highlights

Basal activity of the inward rectifying potassium channel Kir2.4 is important for a variety of neuronal functions
We found that the surface expression is affected, with effects paralleling those seen with the basal current
Kir2.4 Interacts with G␣o—Kir2.4 was identified as an interactor for G␣o in the yeast two-hybrid system, so it was important to determine whether these two proteins interact in the mammalian system. We addressed this question by performing co-immunoprecipitation on Human embryonic kidney (HEK) cells transfected with mouse Kir2.4 and G␣o

Summary

Introduction

Basal activity of the inward rectifying potassium channel Kir2.4 is important for a variety of neuronal functions. Results: Pertussis toxin-sensitive G␣ subunits reduce basal current and surface expression of Kir2.4, whereas G␤␥ increases them. 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 G␣o1. 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 G␣o reduce the Kir2.4 basal current, whereas the active mutant has little effect. We 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. We conclude that G␤␥ targets Kir2.4 to the plasma membrane, and G␣o slows this down by binding G␤␥

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Conclusion