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

Background: Basal activity of the inward rectifying potassium channel Kir2.4 is important for a variety of neuronal functions.

Results: Pertussis toxin-sensitive Gα subunits reduce basal current and surface expression of Kir2.4, whereas Gβγ increases them.

Conclusion: Heterotrimeric G-proteins regulate the surface expression of potassium channels.

Significance: This study extends the role of G-protein subunits in modulating neuronal physiology by regulating the expression of channel 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 Gα_o1. Here, we wished to determine whether and how Gα_o affects this channel. Using transfected HEK 293 cells and retinal tissue, we showed that Kir2.4 interacts with Gα_o, and this interaction is stronger with the GDP-bound form of Gα_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 Gα_o reduce the Kir2.4 basal current, whereas the active mutant has little effect. Other pertussis-sensitive Gα subunits also reduce this current, whereas Gα_s 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 Gα_o slows this down by binding Gβγ.

Introduction

Heterotrimeric G-proteins play diverse roles in biological systems. Their major role lies in coupling metabotropic receptors to a variety of effectors. In this role, G-protein subunits stimulate or inhibit a diverse number of effectors, including ion channels (reviewed by Ref. 1). In an effort to find retinal interactors for the constitutively active subunit Gα_o, the subunit that is required for the retinal ON bipolar light response, we previously fished many known G-protein modulators and the inwardly rectifying potassium channel, Kir2.4 (2–5). The Kir channel family is divided into four subfamilies (Kir1–4) with the Kir3 subfamily being directly gated by Gβγ (reviewed by Refs. 6–12). Gα also binds this subfamily and plays an important role in reducing the basal activity of this channel and priming it so that it can be gated more efficiently by Gβγ (13–16). The other Kir subfamilies are not known to be directly modulated by G-proteins, although they are modulated by phosphorylation mediated by G-protein-coupled receptors (17–19).

The Kir2 channels inwardly rectify more strongly than the other Kir subfamilies (6, 7, 20). Their general function is adjusting neuronal excitability, contributing to resting potential and metabolic processes in neural and non-neural tissues. Kir2.4 was initially cloned from the brain; it was found in several regions, most strongly in motorneurons of cranial nuclei, and the human form was found to be particularly abundant in retina, where it is expressed in most cell types (21–23). Kir2.4 subunits can form homotetrameric channels as well as heterotetramers with other members of Kir2 family such as Kir2.1, and these different stoichiometries have been suggested to provide physiological heterogeneities (24). Having fished the Kir2.4 by Gα_o, we wished to know whether these proteins functionally interact. Specifically, we wanted to determine whether the activity of Kir2.4, similar to Kir3 activity, is modulated by G-proteins. We thus recorded the activity of this channel in Xenopus oocytes with and without the co-expression of different G-protein subunits. We found that Gα_i/o severely reduces Kir2.4 surface expression and its basal current, whereas Gβγ increases them. Our data expand the repertoire of G-protein functions beyond their essential and dominant effect of coupling receptors to downstream signaling processes.

MATERIALS AND METHODS

cDNA Constructs and mRNA

Kir2.4 was amplified by RT-PCR from mouse retinal RNA prepared using a Nucleospin RNA II kit (Clontech). Reverse transcription was performed on 1 μg of total RNA with oligo-dT primers using Moloney murine leukemia virus reverse transcriptase (BD Biosciences). The primers used for PCR were as follows: 5′-agg aca gat cta gag ggg gtc t-3′ (forward) and 5′-cat cag agg ctg gaa gga ag-3′ (reverse). PCRs used 35 cycles (94 °C for 1 min, 58 °C for 30 s, and 72 °C for 2 min) and were performed on a programmable thermocycler (PerkinElmer Life Sciences). The PCR product was then subcloned into pcDNA3.1 using pcDNA3.1/V5-His TOPO TA expression vector (Invitrogen) to yield Kir2.4-pcDNA3.1.

Kir2.4-cerulean (Kir-cer, fused at the Kir2.4 N terminus) construct was prepared by amplifying the cerulean from m-Cerulean-C1 (a kind gift from Dr. Matthew Dalva, Jefferson University, Philadelphia, PA) using the following primers: 5′-agc agc aag ctt atg gtg agc aag ggc gag gag ctg-3′ (forward) and 5′-agc agc cca cga tgg gga ctt gta cag ctc gtc cat gcc-3′ (reverse). PCR product and Kir.2.4-pcDNA3.1 vector were digested with BSrG1. The following clones (in pGEMHE or its derivative pGEMHJ; high-expression oocyte vectors containing 5′- and 3′-untranslated sequences of Xenopus β-globin) were gifts from Dr. Nathan Dascal (Tel-Aviv University, Israel): Gα_o1, Gα_i1, Gα_i3, Gα_s, Gβ1, Gγ2, and m-phosducin. The Gα_o2 clone (in pAGA-2) was a gift from Dr. Lutz Birnbaumer (NIH, Research Triangle Park, NC), and it was subcloned into pGEMHJ. Gα_o1 (in pDP) was provided by Dr. David Manning (University of Pennsylvania, PA). Gα13 and Gα_q (in pCDNA) were obtained from UMR cDNA Resource Center (Rolla, MO). Gβ1 and Gγ2 cDNA (in pCDNA3.1) were provided by Dr. Kirill Martemyanov (The Scripps Research Institute). For oocyte injections, DNA plasmids containing the various clones were linearized with the appropriate restriction enzymes using a standard protocol, and mRNAs were synthesized in vitro using mMessage mMachine Kit (Ambion, USA). The injected volume of RNA mixture was 41.4 nanoliters.

Cell Culture, Transfection, and Co-immunoprecipitation

Human embryonic kidney (HEK) 293 cells were cultured in minimal essential medium supplemented with Penstrep (Invitrogen) and 10% heat-inactivated fetal bovine serum at 37 °C in a 5% CO₂ incubator. Cells were transiently transfected with Kir2.4 (in pcDNA3.1) and Gα_o1 (in pDP) using FuGENE 6 transfection reagent (Invitrogen). Cells were harvested 24 h later. HEK 293T-transfected cells or mouse retinal homogenates were collected in lysis buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 5 mm MgCl₂, 100 μm GDP, 30 mm PMSF) with or without AlF₄⁻ (30 μm AlCl₃ and 10 mm NaF). The cells were homogenized at low speed and centrifuged at 8,000 × g in an Eppendorf centrifuge for 5 min. The supernatant was precleared by adding 20 μl of protein G agarose beads (Invitrogen), centrifuging, and collecting the supernatant. The precleared supernatant was incubated with mouse anti-Gα_o and protein G agarose beads on a rotator at 4 °C for ∼12 h. The beads with protein complexes were then pulled down by centrifuging (10,000 × g), washed thoroughly in lysis buffer, resuspended in Laemmli buffer, boiled, and spin filtered. The proteins were run on 10% SDS-PAGE gel and transferred to a nitrocellulose membrane using semi-wet transfer apparatus (Bio-Rad). Blots were then incubated sequentially in the following: 10% nonfat dry milk in PBS containing 0.1% Tween 20 (PBST) at 4 °C for 1 h, primary antibodies (against Gα_o1 or Kir2.4, both raised in rabbit) in PBST at 4 °C overnight, PBST, and secondary antibodies linked to HRP. Protein bands were detected by SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology).

Antibodies

We used mouse anti-Gα_o (mAb 3073) from Millipore (Billerica, MA); rabbit anti Gα_o, a gift from Dr. Manning (University of Pennsylvania); rabbit anti-Kir2.4, a gift from Dr. Rudiger Veh (Institut fur Integrative Neuroanatomie, Berlin, Germany; (see Ref. 5); guinea pig anti-mGluR6 (Neuromics, Inc., Northfield, MN); rabbit anti-GFP from Millipore (Temecula, CA); rabbit anti-Gβ1(SC-379, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-rabbit Fab fragments linked to HRP from Protos (Burlingame, CA), and anti-mouse F(ab′)2 linked to Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA).

Electrophysiology

Xenopus oocytes were isolated and prepared as described previously (25) or were obtained already prepared from the laboratory of Dr. Zhe Lu (University of Pennsylvania). All experiments were carried out in accordance with the University of Pennsylvania Institutional Animal Care and Use Committee. Oocytes were injected with mRNA and incubated for 2–3 days in physiological ND96 solution (96 mm NaCl; 2 mm KCl; 1 mm CaCl₂; 1 mm MgCl₂; and 5 mm Hepes/NaOH; pH 7.5, and adjusted using NaOH) supplemented with streptomycin (100 μg ml⁻¹) and penicillin (62.75 μg ml⁻¹). Experiments were performed at room temperature (20–22 °C). Kir2.4 currents were measured with a two-electrode voltage clamp using Oocyte clamp OC-725 (Warner Instruments). Electrodes were filled with 3 m KCl, and their resistances were ∼2 megohms. High K⁺ 24 solution was as follows: 24 mm KCl; 74 mm NaCl; 1 mm CaCl₂; 1 mm MgCl₂; 5 mm Hepes; pH 7.5, adjusted with KOH. High K⁺ 96 solution consisted of the following: 96 mm KCl; 2 mm NaCl; 1 mm CaCl₂; 1 mm MgCl₂; 5 mm Hepes; pH 7.5, adjusted with KOH. Cells were clamped to −80 mV, and the holding current was measured under different conditions. For conductance measurements, a voltage ramp was applied from −150 to +30 mV for 2 s. Current signals were filtered at 1 kHz; data acquisition and analysis were done using pCLAMP software (Molecular Devices). In each experiment (i.e. batch of oocytes), the average current of uninjected oocytes was subtracted from the current recorded for each injected oocyte, and this was normalized to the average current of the group injected only with Kir2.4 mRNAs. These normalized currents were then averaged across experiments and subjected to a Student's t test.

Quantifying Kir2.4-cer Surface Expression

Oocytes injected with Kir2.4-cerulean were imaged with a confocal microscope (Olympus FV-1000) under water immersion 10× objective (numerical aperture of 1). The focal plane was adjusted to obtain the brightest image (usually at the largest perimeter). To capture a representative average of the expression throughout the oocytes, we took care to place the oocytes so that the imaged equator contained both the animal and the vegetal hemispheres of the oocytes. An annulus was drawn around the fluorescent region at the oocyte perimeter, and the average intensity was calculated (with Fluoview 1000; dynamic range, 12 bit). A circular region at the middle of the oocyte was used to calculate the mean background fluorescence, and this value was subtracted from the average perimeter value. In each experiment, uninjected oocytes were also measured, and their average fluorescent value was also subtracted from the fluorescent value of each injected oocyte. All imaging parameters (e.g. laser power, offset, gain, etc.) remained the same for all oocytes.

We initially assessed several methods for quantification taking care to include the full oocyte in the field of view. In one method, we took the average value of the whole field without drawing regions of interest. In another method, we imaged the top surface of the oocyte in case this revealed a larger area and a better representation of the expression. And yet, in two other methods, we focused until we obtained the largest circle or the brightest circle. We then plotted fluorescent values versus basal current and computed the correlation between these parameters; the best correlation was obtained when the brightest large circle was in focus, hence the choice of this method as described in the previous paragraph.

Immunostaining

Wild type (WT) C57BL/6J mice were purchased from Charles River Laboratories; the Gnb3-null mouse was described in Ref. 26, and the Gnao1-null mouse was described in Ref. 3. Mice were treated in compliance with federal regulations and University of Pennsylvania policy. Mice were deeply anesthetized by intraperitoneal injection of a mixture of 100 μg/gm ketamine and 10 μg/gm xylazine; the eyes were enucleated, and the mouse was killed by anesthetic overdose. Both male and female mouse tissues were used.

The eyeball was fixed in 4% paraformaldehyde for 10 min and cryoprotected and embedded in a mixture of two parts 20% sucrose in phosphate buffer and one part tissue freezing medium. The eye was cryosectioned radially at 10–15-μm thickness. Retinal cryosections or fixed HEK cells were soaked in diluent containing 1.5% normal goat serum, 5% sucrose, and 0.5% Triton X-100 in phosphate buffer. Samples were incubated in primary antibodies at 4 °C for 3 days (retina) or overnight (HEK cells); washed; incubated for 3 h in secondary antibodies conjugated to a fluorescent marker; rinsed; and mounted in Vectashield (Vector Laboratories, Burlingame, CA). Retinal slices or transfected HEK cells were photographed under a confocal microscope (Olympus FL1000) with a ×60 lens with zoom 2. For retinal immunostaining comparison, a set of age-matched wild type and null retinas were simultaneously processed and imaged under the same settings.

Evaluation of Kir2.4 Distribution in Transfected HEK cells

To determine the number of cells with membrane localization of Kir2.4, we visually inspected transfected cells throughout their depth and simply counted those that showed an outline and those that did not (i.e. showed only an accumulation of stain in the cytoplasm). To get a representative intensity profiles, we drew a line along the diameter of each cell expressing Kir2.4 and plotted the intensities along this line. The starting point of each line was slightly outside the membrane (as detected by staining for Gα_o1 or Gβ) at a region where no large intracellular Kir2.4 expression was present (so that Kir2.4-cer expression at this point was either sharp or absent; see examples in Fig. 7, J and K). The line then progressed through the cell to the other end passing through an intracellular region with high level of expressed Kir2.4. The intensity profile of the line provides a semiquantitative visual display of the relative distribution of Kir2.4 near or at the membrane (on the left of the graph) to that in the cytoplasm (broad staining on the right). Using a Matlab program, multiple lines from multiple cells were aligned according to the membrane location, as determined by the peak of the staining for Gα_o or Gβγ.

FIGURE 7.

G-proteins affect intracellular distribution of Kir2.4. A–F, confocal images of transfected HEK cells. A, cells were transfected with Kir2.4-cer alone and visualized by their fluorescent signal; B–D and F, cells were transfected with Kir2.4-cer + Gβ1 +Gγ2 and visualized as indicated; E, cells were transfected with Kir2.4-cer + Gα_o1 and visualized as indicated. Note in B and C the exquisite colocalization of Kir2.4 (cyan) and Gβ1 (red). F, an example of cell transfected with Kir2.4-cer + Gα_o1 with a line drawn through it; the line begins at the lower point. G, intensity profiles for Kir2.4 and Gα_o along the line in F. In this cell, no Kir2.4 expression at or near the membrane could be detected. H, examples of intensity profiles for Kir-cer (top) and Gβ1(bottom) for three cells (drawn in three gray shades) co-transfected with Kir-cer + Gβ1 + Gγ2. I, average (Ave.) intensity profiles for 32 cells transfected and viewed as described above. J, three examples of intensity (int.) profiles for Kir-cer (top) and Gα_o (bottom) for cells co-transfected with plasmids encoding these proteins. K, average intensity profiles for 36 cells transfected and viewed as indicated. Dotted lines show membrane locations as determined by staining for Gα_o or Gβ1. The scale bar in E4 applies to all D and E panels.

RESULTS

Kir2.4 Interacts with Gα_o

Kir2.4 was identified as an interactor for Gα_o in the yeast two-hybrid system, so it was important to determine whether these two proteins also interact in the mammalian system. We addressed this question by performing co-immunoprecipitation on HEK cells transfected with mouse Kir2.4 and Gα_o. We chose to use a mouse sequence because most of our physiologically relevant work is performed on mouse retina. Amplifying and sequencing the coding region of this message showed the sequence to be identical to that for mouse embryonic Kir2.4 transcript, a provisional RefSeq record in the GenBank (accession no. NM_145963). The coding sequence was 96 and 88% identical to the rat and human Kir2.4 sequence, respectively.

Immunoprecipitating Gα_o with a monoclonal antibody pulled down Kir2.4, whereas omitting anti-Gα_o did not (Fig. 1A, five experiments). When retinal samples were used for co-immunoprecipitation, only three of five experiments produced Kir2.4 bands. Nevertheless, in these three experiments, more Kir2.4 was pulled down when we omitted ALF4⁻, an activator of Gα that promotes a conformation mimicking the transition stage for GTP hydrolysis (Fig. 1B). The interaction between these two proteins raises the possibility that Gα_o, most likely in its GDP-bound form, modulates Kir2.4 channel activity.

FIGURE 1.

Kir2.4 co-immunoprecipitates with Gα_o in HEK cells and in retina. Gα_o was precipitated with a monoclonal antibody in HEK cells (A) and retinal tissue (B), and Kir2.4 was probed with rabbit anti-Kir2.4. Top panels show the Gα_o bands; lower panels show the pulled Kir2.4 bands. Anti-Kir2.4 typically reveals a monomer and a dimer. In HEK cells, both were often pulled; in retina, only the monomer was precipitated. RH, retinal homogenate (20 μg protein was loaded). Some retinal samples were pretreated with AlF₄⁻ to stabilize Gα_o in the active transition state; Kir2.4 co-immunoprecipitation is better in the absence of AlF₄⁻.

Properties of Mouse Kir2.4 Current

Because our Kir2.4 sequence was cloned from mouse retina, we first tested its characteristics. Messenger RNA of Kir2.4 (1 ng) was injected into Xenopus oocytes and the basal current of the channel was measured using two-electrode voltage clamp recordings. In all experiments, oocytes were clamped to −80 mV in ND96 solution, and the holding current was measured before and after switching to 24 mm external K⁺ concentration (henceforth called high K) or occasionally to 96 mm K⁺ concentration (high K96). Upon switching the medium from ND96 to high K or to high K96, the oocyte showed an increase in inward current that was accompanied by an increase in slope conductance (Fig. 2, A and B). This increase in current occurred in two phases, a fast phase that probably depended on the perfusion rate, and a very slow phase that lasted more than 15 min (Fig. 2A). This two-phase response has been reported previously for human retinal Kir2.4 (22). Notably, although the current was dramatically reduced when the solution switched from high K back to ND96, a residual “memory” of the high K⁺ effect remained as the current did not return to its initial low level even after 20 min. Consistent with earlier reports (21, 22), this K⁺ current was blocked by barium: 2 mm reduced the current by 60% and 5 mm totally eliminated it (Fig. 2C). Although K⁺-selective channels are usually not blocked by extracellular Na⁺, the delayed rectifying potassium channel in bullfrog sympathetic neurons is an exception (27). Thus, we tested whether the slow increase in inward current after switching to high K⁺ was due to release from Na⁺ block by replacing Na⁺ with the impermeant cation NMDG⁺. We found that neither total substitution of Na⁺ with NMDG⁺ (Fig. 2D) nor switching from 24 mm NMDG⁺ + 72 mm Na⁺ solution to Hi K⁺ solution (i.e. without changing Na⁺ concentration; data not shown) affected this slow conductance increase, suggesting that the behavior is not due to Na⁺ block.

FIGURE 2.

Kir2.4 cloned from mouse retina shows its characteristic behavior. A, sample current recorded from an oocyte that was injected with Kir2.4 (1 ng) and clamped to −80 mV. In this experiment, after ∼10 s in ND96 (ND), the solution was changed to high K⁺ 24 (Hi K24) for 1 min, then to ND96 briefly, then to high K⁺ 96 (Hi K96) for 1 min, and finally to ND96. Ramps from −130 to +30 mV were applied at the ends of ND96 and at the beginnings and ends of high K⁺ 24 and high K⁺ 96 applications; they are seen as the fast downward trace followed by an upward deflection. Immediately after the solution is changed to high K, the inward current shows a fast increase (F) and then a slower increase (slow). When the solution is switched back to ND96, the base line does not return to its initial value quickly. B, ramp responses are shown in an expanded time scale for ND96, high K⁺ 24, and high K⁺ 96. C, when 5 mm barium replaces 5 mm Na⁺ (K24+Ba), the current that is evoked by high K⁺ 24 is completely blocked. D, replacing Na⁺ with NMDG⁺ (NM) does not affect the K⁺-induced current. Time scale in D also applies to C.

Kir2.4 Basal Current Is Reduced by Gα_o1

We wondered whether Kir2.4 might be modulated by heterotrimeric G-proteins in a manner similar to Kir3. We therefore investigated the effect of Gα_o1 on the Kir2.4 basal current and inward conductance. Because the inward current under high K⁺ did not plateau in a reasonable time, measurements of this basal current were taken one and/or 2 min after switching solutions. For conductance measurements, we ramped the oocytes from −150 mV to +30 mV and calculated the slope of the I-V curve in the linear range (Fig. 2B). When small amounts of Gα_o1 were co-expressed with Kir2.4, the basal current decreased dramatically in a dose-dependent manner (Fig. 3A, total of three experiments). Injection of 0.2 ng of Gα_o1 reduced the current to ∼40% of the control level, and injection of 0.5 ng decreased it to ∼10%. Higher amounts did not reduce the current further; current was insignificantly higher than that with 0.5 ng of Gα_o1 but still significantly lower than Kir2.4 alone. For the rest of our experiments, unless otherwise stated, we used 1 ng of Gα_o1. Co-injecting Gα_o1 also affected the zero-crossing potential, i.e. the potential at which no net current passes through the membrane and thus reflects the resting potential of the oocyte. Although uninjected oocytes showed an average potential of ∼−15 mV, oocytes expressing Kir2.4 under ND96 showed −27 mV (due to a maintained outward K⁺ current), and those expressing Kir2.4 + Gα_o1 showed a potential similar to uninjected oocytes of ∼−15 mV, suggesting a reduced basal outward K⁺ current. The reduction in current and conductance induced by the wild type form was also observed with a mutant form of Gα_o1 that renders this subunit constitutively inactive (G204A) but was not seen with the constitutively active form (Q205L) (Fig. 3B). As for this experiment and for all experiments henceforth, the observed effects remained the same regardless of whether we measured current at 1 or 2 min after the switch to high K, and regardless of whether we measured inward current or slope conductance. Therefore, the following figures show only the relative basal currents.

FIGURE 3.

Inactive but not constitutively active Gα_o decreases basal Kir2.4 current. A, average normalized currents for oocytes injected with Kir2.4 mRNA (1 ng) and the indicated amounts of Gα_o mRNA. For current recordings, oocytes were clamped at −80 mV. For all bar graphs, error bars indicate S.E., and numbers above bars indicate number of oocytes recorded for each group. *, significant (p < 0.05) difference from the group injected with only Kir2.4; **, highly significant (p < 0.01) difference. Wild type Gα_o at relatively low amounts decreases the basal current in a dose dependent manner because current with 0.5 ng was significantly different from 0.2 ng of Gα_o. Current with 2 or 4 ng was not significantly different from 0.5 or 1 ng. B, average normalized currents (gray bars) and slope conductances (white bars) for oocytes injected with Kir2.4 mRNA (1 ng) alone, or with Kir2.4 and 1 ng of the following: wild type, constitutively inactive (G204A), or constitutively active (Q205L) Gα_o. The reduction in basal current of oocytes co-injected with wild type or inactive Gα_o are highly significant when compared with the current in oocytes co-injected with active Gα_o, indicated by brackets.

We next determined the specificity of Gα. We injected several species of Gα subunits and found that all the Gα_i/o tested, Gα_o2, Gα_i1, and Gα_i3, were as effective as Gα_o1 in reducing the Kir2.4 basal current, with the difference between these groups and the group injected with only Kir2.4 being highly significant (Fig. 4). In contrast, Gα_s dramatically increased the Kir current (by 2.4-fold; p ≪ 0.01). Gα_q also increased the current, but to a lesser extent than Gα_s (1.5-fold) and without significance, whereas Gα₁₃ did not change it. In the remaining experiments, we used only Gα_o1 (and refer to it as Gα_o)

FIGURE 4.

Gα_o/i reduces basal current, whereas Gα_s increases it. Normalized basal currents (V = −80 mV) for oocytes injected with Kir2.4 (1 ng) alone or with Kir2.4 and one of the indicated Gα subunits (1 ng). Error bars are S.E., and significance is as described in the legend to Fig. 3.

Kir2.4 Basal Current Is Increased by Gβγ

Following the model of Kir3, we hypothesized that the reduction of Kir2.4 current by co-expressing Gα_o/i is due to scavenging Gβγ that might be required to gate the channel. Thus, we tested the effect of co-expressing Gβ1γ2 and found that it increased the basal current in a dose-dependent manner (Fig. 5A). Injecting 1 ng of Gβ1 + 0.2 ng of Gγ2 increased the current by ∼2-fold (five experiments). Doubling the amount of each subunit increased the current 5-fold (three experiments), and tripling it increased the current almost 9-fold (one experiment). Moreover, Gβγ counteracted the effect of Gα_o when these subunits were co-injected, and this effect was dose-dependent as well. Injecting 5 ng of Gβ +1 ng of Gγ in addition to 1 ng of Gα_o increased the current more than 2-fold relative to that of Kir2.4 alone, and more than 8-fold relative to that of Kir + 1 ng Gα_o (Fig. 5B, compare second bar to rightmost bar). When we compare the ratio of oocytes injected with Kir2.4+Gβγ to Kir2.4 alone, and that of Kir2.4+Gα_o+Gβγ to that of Kir2.4+Gα_o, it appears that higher expression of Gβγ are needed to lift the base line when Gα_o is co-expressed because the potency of Gα_o is greater than Gβγ. Although 1 ng of Gα_o decreases the current by ∼5-fold relative to Kir alone, 1 ng of Gβ + 0.2 ng of Gγ increase the current by only ∼2-fold. To determine whether the effect of Gβγ is specific and whether endogenous Gβγ would have a similar effect to exogenously added subunits, we injected phosducin, a molecule that is known to scavenge Gβγ without affecting GTP binding to Gα. This reduced the current to ∼50% (with 1 ng of phosducin) or ∼20% (with 2 and 4 ng). The effect of phsoducin could be reversed by adding Gβγ (Fig. 5C, altogether four experiments). Our findings suggest that Gβγ modulates Kir2.4 activity and pertussis toxin-sensitive Gα acts to scavenge Gβγ. This interpretation is consistent with inactive Gα being more effective than active Gα because the inactive form has a much higher affinity for Gβγ. However, it may be inconsistent with the finding that Gα_s increases basal expression.

FIGURE 5.

Gβγ increases the basal current of Kir2.4 when injected either alone or with proteins that sequester it. A, normalized basal currents for oocytes injected with mRNA for Kir2.4 (1 ng) alone or Kir2.4 + indicated amount (in ng) for Gβ1 + one-fifth of this amount for Gγ2; (V = −80 mV). A t test was performed against Kir2.4 alone. Error bars are S.E., and significance is as described in the legend to Fig. 3. B, normalized basal currents for oocytes injected with Kir2.4 (1 ng) alone, Kir2.4 + Gα_o (1 ng), or Kir2.4 + Gα_o + indicated amount for Gβ1 + 1/5 of this amount for Gγ2. A t test compares currents between oocytes with Kir2.4 + Gα_o to those with additional Gβγ (indicated by brackets). C, normalized basal currents for oocytes injected with Kir2.4 (1 ng) alone, Kir2.4 + additional phosducin (phos) as indicated (in ng), or Kir2.4 + phosducin + Gβγ as indicated. When no bracket is shown, t test compares that particular group to the Kir2.4 group.

Kir2.4 Surface Expression Is Reduced by Gα_o

If Gβγ gates the Kir2.4 channel as it gates Kir3, it should be possible to activate the channels by co-expressing a G-protein-coupled receptor. Thus, we co-expressed the muscarinic receptor M2, Gα_o, and the Kir2.4 or Kir3.1/2 channels. When the receptor and Gα_o were expressed with Kir3.1/2, application of 20 micromolar acetylcholine activated the channel. However, when they were co-expressed with Kir2.4, no such activation could be observed (data not shown). Similar experiments using the metabotropic glutamate receptor mGluR6 instead of the muscarinic receptor showed that glutamate evoked Kir3.1/2 current (see Ref. 2) but not Kir2.4 current. Thus, we suspected that the observed G-protein modulation of the Kir2.4 channel results indirectly by affecting trafficking or expression. We tested this hypothesis by expressing Kir2.4 fused to cerulean (Kir-cer).

We first performed some control experiments. We confirmed that Kir-cer has the same channel properties as the wild type Kir2.4 (data not shown). We also determined that our measurements of surface fluorescence were meaningful. To this end, we expressed different amounts of Kir-cer mRNA and recorded the surface fluorescence to verify that the fluorescence is linearly related to the injected amount (Fig. 6, A and B). Next, we injected oocytes with 5 or 10 ng of Kir-cer and used nine oocytes from each group to measure both the basal current and the surface fluorescence. We found that the current correlated well with the fluorescence (Fig. 6C), confirming the validity of the method for measuring surface expression.

FIGURE 6.

Heterotrimeric G-protein subunits affect Kir2.4 surface expression. A, confocal image of an oocyte injected with 5 ng of Kir-cer and imaged 3 days later under a 10× water immersion objective. B, the measured surface fluorescence is linearly related to the amount of injected Kir2.4 mRNA. R² is the correlation coefficient for these data. C, the measured surface fluorescence correlates well with the inward current. Data points are for 5 and 10 ng of injected Kir2.4 from a different experiment than in B. D, normalized (Norm.) basal currents for oocytes injected with various mRNAs for kir-cer (4 ng) alone (condition A, 4 kir-cer), and 4 ng kir-cer + various amounts of G-protein subunits (condition B, 0.5 ng of Gα_o; condition C, 0.5 ng of Gα_o+10 ng of Gβ1 + 2 ng of Gγ2; condition D, 1 ng of Gα_o; condition E, 1 ng of Gα_o+ 5 ng of Gβ1 + 1 ng of Gγ2; or condition F, 2 ng of Gα_o1). E, normalized surface fluorescence (Norm. fluor.) for the same oocyte groups as in D. Oocytes with high amounts of Gα display negative values because the autofluorescence of uninjected oocytes is relatively high and variable, and here it happened to be higher than the fluorescence of these groups. Unless otherwise indicated by brackets, asterisks indicate the significant difference compared with results for Kir-cer injection alone; a single asterisk indicates p < 0.05 and double asterisks indicate p < 0.01. F, normalized fluorescence measurements for each group in each experiment is plotted against the normalized basal current showing that these are correlated (R² = 0.92); data are taken from the experiments in D and E; n = three experiments (conditions B and F were repeated twice).

To determine the effect of G-protein subunits on surface expression of the Kir2.4 channel, we measured the inward current and surface fluorescence for groups of oocytes that were co-injected with mRNA for Gα_o1 and Gβγ. Injection of 1 ng of Kir2.4-cer yielded very low fluorescent measurements that were similar to the measured autofluorescence. We therefore had to inject greater amounts (but not too great to keep the current within a reasonable range of less than 2 μA for Kir2.4 alone); we therefore settled on 4 ng of Kir2.4-cer mRNA. We found that 0.5 ng of Gα_o mRNA reduced the basal current down to 23% of Kir alone, and the surface fluorescence to 13% (Fig. 6, D and E). One ng of Gα_o mRNA further reduced the current to 4% and the fluorescent to noise level. Adding Gβγ mRNA to 0.5 ng of Gα_o mRNA increased both fluorescent intensity (by 4-fold) and basal current (by 2-fold). The relative increase in current and fluorescence was not identical due to limitations in determining surface expression when expression is low and gets close to autofluorescence level shown by uninjected oocytes. Nevertheless, the basal current correlated pretty well with the measured surface expression (R² = 0.92; Fig. 6F). We therefore suggest that all the effects that we have observed with the basal current could be explained by the G-protein subunits having affected the expression or the trafficking of Kir2.4.

Differential Distribution of Kir2.4 in Transfected HEK Cells

To test whether G-protein subunits affect Kir2.4 localization in mammalian cells, we transfected HEK293 cells with Kir-cer alone, Kir-cer + Gα_o1, or Kir-cer + Gβ1γ2 (Fig. 7). To identify cells that were cotransfected, slides were immunostained for Gα_o or Gβ1. Expression of Kir-cer varied in different cells; in few cells, a clear outline of the cells could be seen, suggestive of membrane localization; in some, the expression was restricted to the intracellular milieu, where it concentrated near the nucleus (presumably on the Golgi apparatus; as in Fig. 7E); and in others, Kir-cer appeared in both of these locations (as in Fig. 7, A–D). Interestingly, when Kir2.4 was co-expressed with Gβγ, these proteins perfectly colocalized within intracellular microdomains and on the plasma membrane (Fig. 7, B–D). We counted the number of cells in which a clear outline appeared and those in which Kir-cer was restricted to the cytosol and computed the percentage of those with membrane localization to the total number. In cells expressing only Kir-cer, the percentage was 67% (63 cells); in cells expressing Kir-cer+Gα_o1 (Fig. 7E), it was 39% (44 cells); and in those expressing Kir2-cer+Gβ1γ2 (Fig. 7D), it was 78% (n = 37). To further assess these results by a different method, we drew lines across cells and plotted the intensities along these lines (Fig. 7, F and G). This representation provides a semiquantitative visual display of the relative distribution of Kir2.4 near or at the membrane (on the left of the graph) to that in the cytosol (broad staining on the right). In all cases, cytosolic expression was much stronger than membranal staining. However, when Kir2.4 co-expressed with Gβγ, in most cells staining intensity was sharp near the membrane (Fig. 7, H and I). When Kir2.4 was co-expressed with Gα_o1, membranal staining was weaker (Fig. 7E, J and K). Thus, we conclude that as in oocytes, Gα_o overexpressed in HEK cells appeared to reduce surface expression, whereas Gβγ increases it. Unlike in oocytes where it was possible to image Kir-cer surface expression without contribution from cytosolic expression, imaging HEK cells does not provide the required resolution to discriminate these because cytosolic expression near the membrane will be lumped with that of the membrane. Thus, it is possible that the effect in HEK cells is larger than seen. It should also be noted that HEK cells express Gβ1 endogenously, perhaps contributing to Kir2.4 surface expression when transfected alone.

Kir2.4 in the Dendritic Tips of Retinal ON Bipolar Cells Is Reduced in the Absence of Gβ3

To see whether our findings in oocytes are also relevant to neural tissue, we immunostained wild type retina and Gnb3 (gene encoding Gβ3)-null retina for Kir2.4. We have previously shown that Kir2.4 is expressed in the dendritic tips of ON bipolar cells (5). The antibody against Kir2.4 revealed punctate staining that often showed two populations of puncta, a brightly stained population that we judge to be noise and a faintly stained population that colocalized well with the staining for mGluR6, the G-protein-coupled receptor that mediates the light response in these cells (Fig, 8, left column). In Gnb3-null retinas (four sets of experiments), the puncta that stained for Kir2.4 and colocalized with mGluR6 were greatly reduced in their intensity. Staining of bipolar cell somas in the inner nuclear layer was also slightly reduced in the null retina but to a lesser extent than the dendritic tips. As shown previously, staining for mGluR6 was also slightly reduced (Fig. 8, right column) (26). Unlike Gnb3-null retina, staining of Gnao1 (gene encoding Gα_o1)-null retina was not much different from the wild type (data not shown).

FIGURE 8.

Gβ3 deletion affects Kir2.4 localization in retinal ON bipolar cells. Double immunostaining of radial retinal sections for Kir2.4 (green) and mGluR6 (red) in wild type (left column) and Gnb3-null (right column) retinas. OPL, outer plexiform layer; INL, inner nuclear layer. With either Kir2.4 or mGluR6 staining, puncta intensity (arrows) is reduced in the null retina; the dotted circle on the KO image represents an unstained dendritic tip. Somas in the inner nuclear layer mainly represent ON bipolar cells, and their staining was also slightly reduced.

DISCUSSION

We show here that different types of G-protein subunits affect the basal current and surface expression of Kir2.4. Although Gα_o/i reduces this current, Gα_s and Gβγ increase it. We also show that the effects on basal current are correlated with the effects on surface expression, and we suggest that the former results from the latter. We further show that, when co-expressed, Gα_o and Kir2.4 interact in retina and in HEK cells. Given that Gα and Gβγ affect expression in an opposing manner, it would be informative to know which of these subunits mediates the surface expression. On the one hand, it may be that free Gβγ helps traffic the channel to the plasma membrane. On the other hand, it is possible that Gα_o-GDP binds Kir2.4 and prevents it from moving to the plasma membrane or sends it for degradation, as may be suggested by preliminary results showing that not only surface expression but also total expression of Kir2.4 is reduced by co-injecting Gα_o.

Gβγ as a Player in Trafficking

Our measurements of currents and fluorescence can most easily be explained assuming that mediation by the Gβγ dimer; i.e. free Gβγ helps transport the channel to the plasma membrane. Thus, co-expression of Gβγ with Kir2.4 increases channel expression because Gβγ is overexpressed and a good fraction of these subunits remain as free Gβγ. Adding Gα_o reduces channel expression because Gα_o sequesters endogenous free Gβγ and leaves a smaller fraction of these dimers available for trafficking. Co-expressing Gβγ and Gα yields intermediate currents whose magnitude depends on the ratio of the expressed subunits. Most significantly, phosducin, which binds Gβγ without affecting the activity state of Gα, reduces the current, and this effect is reversed by co-expressing Gβγ. Our interpretation is also consistent with the finding that Gα_oGA (inactive), and not Gα_oQL (active), reduces the current because only Gα_oGA significantly binds Gβγ and sequesters it. If we assume the alternative explanation that only Gα_o-GDP mediates the observed effects, it would be difficult to explain how overexpression of Gβγ increases Kir2.4 expression (unless we assume that a fraction of endogenous Gα-GDP exists as a monomer and is being sequestered by Gβγ), how phosducin reduces the current, and how Gβγ reverses the effect of phosducin. One puzzling question is why Gα_s does not decrease basal current because it should be able to bind Gβγ just as well as Gα_o/i. This may be explained by additional factors that affect surface expression such as cAMP, whose concentration can be increased by overexpression of Gα_s (e.g. Ref. 28).

The effect of G-proteins on trafficking has been studied in several systems. Co-expressing Kir3.2 with Gα_i3 or Gβγ in oocytes affects Kir3.2 surface expression in a similar way as it affects Kir2.4 (16). The function of Gβγ in controlling trafficking was also studied (reviewed by Refs. 29 and 30). The mechanism of its action is attributed mostly to the direct stimulation of protein kinase D located in the Golgi apparatus (31–33). In a more recent study where the effect of Gβγ on transport was investigated, Gβγ was shown to localize to the Golgi in HeLa cells and to facilitate transport of cargo to the plasma membrane. This transport was inhibited by gallein, a small molecule inhibitor of Gβγ, and by a Gβγ scavenger (GRK2ct) that was directed to the Golgi (34). A role for Gβγ in trafficking is also supported by studies in neural tissue. We have recently shown that in retinal ON bipolar cells lacking Gβ3 (a subunit that mediates their light responses by coupling the metabotropic mGluR6 receptor to the TRPM1 channel), numerous elements that play an important role in the mGluR6 cascade are down-regulated or redistributed (26). This includes not only the G-protein subunit partners, but also the receptor mGluR6 and the channel TRPM1. The most dramatic effect was seen on the expression of the GTPase activating complex (comprised of Gβ5, RGS7/11, and R9AP). In wild type, this complex was highly localized to the dendritic tips of the ON bipolar cells, but in Gβ3-null cells, all of the elements of this complex were hardly detectable. In this study, we added Kir2.4 to the list of proteins whose localization is affected by deletion of Gβ3. Some of these effects on localization may be indirect, resulting from a lack of activity. However, some are probably direct because we see a greater effect in cells lacking Gβ3 than those lacking Gα_o1, another subunit required for the light response of this cell (3 and this work) (40).

Possible Role of Gα_o

Although all of our data can be qualitatively explained by assuming that Gβγ alone traffics Kir2.4, it is still possible that Gα_o/i contributes directly to the stability of Kir2.4, to its trafficking to the plasma membrane, or to regulation of its basal current. The role of Gα in stability has been shown in Neuro-2A cells where Gα_o/i2-GDP directed Rap1GAPII for proteasomal degradation (thereby activating Rap1 and inducing neurite outgrowth) (35). The function of Gα in trafficking has been shown in LLC-PK1 cells, where overexpression of Gα_i3 tonically represses trafficking of heparin sulfate proteoglycan through the Golgi apparatus (36). The direct effect of Gα_i3 on Kir current (which is independent of its role as a Gβγ donor) has been shown in Xenopus oocytes where it binds GIRK1, reduces the basal current of Kir3.1/2 channels and simultaneously improves the activation of the channel by Gβγ (15, 16). Interestingly, Gα_i3 does not bind Kir3.2, so it does not affect the basal current of homomeric Kir3.2.

In light of these diverse possible roles for Gα subunits, it is possible that Gα_o/i in oocytes also affects the basal current of Kir2.4 independently of its effect through Gβγ. Some support for this notion comes from the interaction we observe between Gα_o1 and Kir2.4 in retina and in HEK cells transfected with these genes. Similar to Kir3.1, whose current is affected primarily by the GDP-bound form of Gα, interaction of Gα_o with Kir2.4 is stronger in the Gα-GDP conformation (as indicated by stronger interaction in the absence of AlF₄⁻)_, and this is consistent with the current being reduced by Gα_o1GA but not Gα_o1QL. In addition, although the interaction we observe using co-immunoprecipitation could have occurred through Gβγ, the interaction we initially saw in yeast should have been direct.

Possible Implication of Kir2.4 Regulation in Neurons

The properties of the Kir2.4 channels have mostly been studied in expression systems where the channel is probably a homomer, but Kir2.4 has been shown to form a functional channel with Kir2.1 (24). Although the Kir2.4 homomer (or monomer) largely accumulates at the Golgi apparatus with only a small fraction being targeted to the plasma membrane, a large fraction of Kir2.1 is targeted to the plasma membrane. The targeting signal in the Kir2.1 sequence has been identified and has been shown to target also the Kir2.4-Kir2.1 heteromer to the plasma membrane (37). In retina, many cell types including horizontal cells and certain types of bipolar cells are found to exhibit Kir current (38, 39). Correspondingly, immunostaining for Kir2.4 is abundant in retina, and ON bipolar cells display a punctate pattern with high concentrations at their somas and their dendritic tips where they receive input from photoreceptors (Ref. 5 and this work). If Kir2.4 does oligomerize with another Kir2 member in ON bipolar cells, its trafficking is likely controlled by the nature of the oligomer with additional regulation by the G-proteins in these cells. Given that Kir2.4 is strongly inwardly rectifying, it will contribute little to the membrane potential at rest or during depolarization because its outward conductance is probably too small. However, when neurons are depolarized for a long time, the extracellular K⁺ concentration increases. Then the Kir channel can prevent excessive K⁺ accumulation with its inward current and can help recover the resting membrane potential. Thus, targeting Kir to the active regions within a cell via G-proteins may represent an efficient mechanism because the activity of many neurons is already mediated by G-proteins.