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 G alpha(o1). Here, we wished to determine whether and how G alpha(o) affects this channel. Using transfected HEK 293 cells and retinal tissue, we showed that Kir2.4 interacts with G alpha(o), and this interaction is stronger with the GDP-bound form of G alpha(o). 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 alpha(o) reduce the Kir2.4 basal current, whereas the active mutant has little effect. Other pertussis-sensitive G alpha subunits also reduce this current, whereas G alpha(s) increases it. G beta gamma increases the current, whereas m-phosducin, which binds G beta gamma without affecting the state of G alpha, 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 G beta 3, localization of Kir2.4 in the dendritic tips is reduced. We conclude that G beta gamma targets Kir2.4 to the plasma membrane, and G alpha(o) slows this down by binding G beta gamma.
