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J Biol Chem, Vol. 275, Issue 2, 921-929, January 14, 2000

The G Protein  Subunit Has a Key Role in Determining the Specificity of Coupling to, but Not the Activation of, G Protein-gated Inwardly Rectifying K⁺ Channels^*

Joanne Louise Leaney, Graeme Milligan^§¶, and Andrew Tinker

From the  Centre for Clinical Pharmacology, Department of Medicine, University College London, Rayne Institute, 5 University Street, London WC1E 6JJ and the ^§ Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow G12 8QQ, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In neuronal and atrial tissue, G protein-gated inwardly rectifying K⁺ channels (Kir3.x family) are responsible for mediating inhibitory postsynaptic potentials and slowing the heart rate. They are activated by G dimers released in response to the stimulation of receptors coupled to inhibitory G proteins of the G_i/o family but not receptors coupled to the stimulatory G protein G_s. We have used biochemical, electrophysiological, and molecular biology techniques to examine this specificity of channel activation. In this study we have succeeded in reconstituting such specificity in an heterologous expression system stably expressing a cloned counterpart of the neuronal channel (Kir3.1 and Kir3.2A heteromultimers). The use of pertussis toxin-resistant G protein  subunits and chimeras between G_i1 and G_s indicate a central role for the G protein  subunits in determining receptor specificity of coupling to, but not activation of, G protein-gated inwardly rectifying K⁺ channels.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

G protein-gated, inwardly rectifying K⁺ channels were first identified in atrial myocytes where they were shown to be activated by acetylcholine at muscarinic m₂ receptors (1), and stimulation of this current is responsible for slowing of the heart rate in response to vagal nerve stimulation. It was subsequently shown that this activation was sensitive to pertussis toxin (PTx),¹ implicating the family of G_i/o proteins (2-4). It is now apparent that G protein-gated inwardly rectifying K⁺ currents are present in many neuronal cell types, and they are involved in the postsynaptic inhibitory effects of stimulating G_i/o-coupled receptors such as GABA_B and adenosine A₁ (5).

Cloning efforts from a number of laboratories have revealed the molecular counterparts of these currents (6-10). The channel is a heteromultimer of members of the inwardly rectifying Kir3.x family of potassium channels. Co-expression of Kir3.1 with Kir3.2, Kir3.3, or Kir3.4 results in currents that show many of the basic characteristics of the native channels in neurons and atria (11-13). Homomultimers of splice variants of Kir3.2 may also form in certain neuronal regions (14).

Channel activation of these currents in native tissues and of the cloned counterparts in heterologous expression systems was shown to be membrane-delimited (4, 15), involving a direct interaction with the G protein dimer (16). This finding was initially controversial, but there are now numerous lines of evidence that point to the involvement of the subunit as the direct channel activator (17). Recent studies have focused on domains and residues involved in G protein dimer binding (18-24) and the cell biology of channel biogenesis (25-27).

The question remains as to how receptor specificity is achieved and what are the key determinants involved in this. In native tissues only G_i/o-coupled receptors, and not G_s-coupled receptors, activate these channels, and yet both should liberate free G protein upon receptor stimulation. Receptor selectivity does not lie at the level of the G protein dimer since a variety of different subunit combinations have been shown to be similarly effective at potentiating these currents (28). In this study we have used a mammalian expression system (HEK293 cells) to investigate the neuronal-type Kir3.1+3.2A channels and their modulation by a variety of G protein-coupled receptors. We have demonstrated the activation of Kir3.1+3.2A channels by G and by agonist stimulation of G_i/o-coupled receptors but not by G_s-coupled receptors. We present evidence to suggest a key role for the G subunit in determining the specificity of coupling between receptor and channel.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture and Transfection

HEK293 cells (human embryonic kidney cell line) were maintained in minimum essential medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin (stocks: 10,000 units/ml penicillin, 1 mg/ml streptomycin), at 37 °C in humidified 95% O₂, 5% CO₂. A lipid-based transfection procedure was used (LipofectAMINE; Life Technologies, Inc.) and monoclonal cell lines established by picking single colonies of cells following transfection and growth under selective pressure. For the channel-expressing HKIR3.1/3.2 line, 727 µg of G418 (Life Technologies, Inc.) was used and for channel+receptor-expressing lines, we used a dual selection strategy with 727 µg of G418 and 364 µg/ml Zeocin (Invitrogen). For transient expression of receptors, G proteins, and other components, cells were co-transfected with cDNA (100-150 ng) encoding the humanized, red-shifted variant of jellyfish green fluorescent protein (pEGFP-N1; CLONTECH) and cells visualized using a Nikon Diaphot epifluorescent microscope.

Molecular Biology

Standard molecular cloning and mutagenesis techniques were employed throughout. All G protein-coupled receptors and G protein subunits, mutants, and chimeras were cloned into pcDNA1, pcDNA3, or pcDNA3.1/Zeo(+) (Invitrogen). All cDNAs were sequenced to confirm their identity. For the expression of Kir3.1+3.2A, a bicistronic vector was engineered that enabled both subunits to be expressed in the same vector. This vector contained two restriction enzyme polylinkers around a picornavirus internal ribosome entry site (IRES). An IRES-containing plasmid (pIRES1hyg) was obtained from CLONTECH, and the IRES element was amplified using PCR with oligonucleotides containing additional restriction enzyme sites inserted at the 5' and 3' ends using PCR. This fragment was subcloned into pcDNA3. The channel subunits were then subcloned into the newly engineered pcDNA3 and a stable HEK293 cell line established (HKIR3.1/3.2). For some experiments, we made stable cell lines that expressed both channel and receptor. In these instances, receptors (in pcDNA3.1/Zeo(+)) were transfected into the stable Kir3.1+3.2A line and selected for using a dual selection strategy. Point mutants of G_i1 (C351G and C351I) were made as in Ref. 29. COOH-terminal chimeras between G_i1 and G_s were made using a PCR-based approach with a high fidelity DNA polymerase (Vent; New England Biolabs) and 18-25 cycles. To replace the COOH-terminal 6 amino acids, a single round of PCR was used and appropriate 5' and 3' restriction enzyme sites added. For the chimeras where the COOH-terminal 13, 16, and 20 amino acids of G_i1 were replaced with G_s, two rounds of PCR were employed. The first round replaced the COOH-terminal 10, 13, and 17 amino acids, respectively, of G_i1 with those of G_s, and appropriate 5' restriction enzyme sites while the second round added the remaining 3 amino acids, stop codon, and 3' restriction enzyme sites.

Biochemistry

Western Blotting-- SDS-polyacrylamide gel electrophoresis and transfer of proteins to nitrocellulose was performed using a Bio-Rad minigel system according to the manufacturer's instructions. Cells were harvested into phosphate-buffered saline and total cell homogenate lysed with an equal volume of 2× gel loading buffer and probed with a primary polyclonal rabbit antibody to G_s (Santa Cruz) and the appropriate secondary antibody before visualization of bands using the ECL Western blot analysis kit (Amersham Pharmacia Biotech).

Adenylate Cyclase Assay-- For the measurement of A_2A and ₁ receptor-mediated accumulation of cAMP, the cell lines HKIR3.1/3.2/A2A and HKIR3.1/3.2/B1 were grown to 20% confluence in six-well dishes. Cells were pre-labeled with 5 µCi of [³H]adenine/well (in minimum essential medium) overnight at 37 °C and then incubated with adenosine deaminase (1 unit; for A_2A receptors only) and the phosphodiesterase inhibitor Ro20-1724 (100 µM) in serum-free medium, for 30 min at 37 °C. Agonist (1 µM NECA for A_2A; 10 µM isoprenaline for ₁) was then added and incubated for 15 min at 37 °C. Medium was then aspirated and cells washed with serum-free medium. Reactions were terminated by the addition of 2.5% perchloric acid and 0.1 mM cAMP at 4 °C. [³H]cAMP was isolated by sequential chromatography using Dowex 50-alumina columns. Each fraction was collected in a scintillation vial containing 5 ml of Ultima Gold MV scintillant (Packard). A Beckman LS6000TA liquid scintillation counter was used to count radioactivity. Reactions were done in triplicate, and data are expressed as percentage conversion of [³H]ATP to [³H]cAMP.

Radioligand Binding-- Cells were washed and harvested into binding buffer (50 mM Tris-HCl, 1 mM MgCl₂, pH 7.4) in the presence of protease inhibitors. They were then hypotonically shocked (10 mM Tris-HCl) and put on ice for 15 min, after which time an equal volume of 500 mM sucrose plus 10 mM Tris-HCl was added to restore tonicity. Cells were homogenized using a glass-on-glass Dounce homogenizer. The homogenate was spun at 4 °C and the cell pellet resuspended in binding buffer. Specific binding was assessed using the radioligands [³H]DPCPX (A₁) and [³H]CGS21680 (A_2A) and nonspecific binding determined in the presence of non-labeled R-PIA. Binding reactions were incubated for at least 2 h at room temperature before the reaction was terminated by the addition of 1 ml of ice-cold binding buffer. Bound ligand was separated from non-bound ligand by vacuum filtration onto Whatman GF-B filters, which were then washed four times with 2 ml of ice-cold binding buffer. Bound radioactivity was determined by liquid scintillation. Points were determined in triplicate, and data were pooled. Binding data were fitted using non-linear regression to a saturation binding isotherm.

Electrophysiology

Membrane currents were recorded using the whole-cell configuration of the patch clamp technique (30) with an Axopatch 200B amplifier (Axon Instruments). Patch pipettes were pulled from filamented borosilicate glass (Clark Electromedical) and had a resistance of 1.5-2.5 megohms when filled with pipette solution (see below). Prior to filling, the tips of patch pipettes were coated with a Parafilm/mineral oil suspension. Records were were filtered at 1 kHz, digitized at 5 kHz, and data acquired and analyzed using a Digidata 1200B interface (Axon Instruments) and the pClamp suite of software (version 6.0; Axon Instruments). Cell capacitance was approximately 15 pF, and series resistance (<10 megohms) was at least 75% compensated. Recordings of membrane current were commenced after an equilibration period of approximately 5 min. Currents were measured at the end of each voltage step. All data are presented as mean ± S.E., and current densities are measured at 100 mV (unless otherwise stated). Student's t tests were performed to examine statistical significance, and * indicates p  0.05.

Materials and Drugs

Solutions were as follows (concentrations in mM): pipette solution, 107 KCl, 1.2 MgCl₂, 1 CaCl₂, 10 EGTA, 5 HEPES, 2 MgATP, 0.3 Na₂GTP (pH 7.2); bath solution, 140 KCl, 2.6 CaCl₂, 1.2 MgCl₂, 5 HEPES (pH 7.4). All materials for cell culture were obtained from Life Technologies, Inc. and Invitrogen. Radioligands were purchased from NEN Life Science Products and antibodies from Santa Cruz. Molecular biology reagents were obtained from New England Biolabs or Roche Molecular Biochemicals. All chemicals were from Sigma, Calbiochem, or RBI. Oligonucleotides were from Genosys Biotechnologies. Drugs were made up as concentrated stock solutions and kept at 20 °C or 80 °C.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Characterization of Kir3.1+3.2A Cell Line-- Channel-forming subunits, receptors, and G proteins were expressed in a mammalian cell line (HEK293). These cells are electrically silent (in non-transfected cells, current density measured in symmetrical K⁺ solutions at 100 mV was 9.6 ± 1.4 pA/pF, n = 24) and do not appear to possess significant endogenous inwardly rectifying K⁺ currents. The native neuronal channel is a heterotetrameric structure thought to comprise both Kir3.1 and Kir3.2 subunits. In initial experiments, the cDNAs encoding Kir3.1 and Kir3.2A were transiently transfected into cells in separate vectors (pcDNA3). No inwardly rectifying K⁺ currents were observed when individual subunits were transfected alone (Fig. 1A), but transfection of both subunits together resulted in the expression of strong inwardly rectifying K⁺ currents. We subcloned both Kir3.1 and Kir3.2A subunits into a single bicistronic vector around an IRES (Fig. 1B), which allowed the expression of both subunits on a single plasmid. A monoclonal stable line expressing Kir3.1+3.2A (hereafter designated as HKIR3.1/3.2) was then established using this vector, and currents were recorded using the whole cell configuration of the patch clamp technique. The inwardly rectifying K⁺ currents were blocked by external Ba²⁺, and an example of these currents and the corresponding current-voltage relationship is shown in Fig. 1 (C and D).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Kir3.1+3.2A channels stably expressed in HEK293 cells exhibit Ba2+ sensitivity and are enhanced by G but not G subunits. A, the top portion of this panel shows the voltage protocol used to elicit membrane currents: cells were held at 0 mV and stepped to potentials between 100 and +50 mV, in 10-mV increments for 100 ms (for solutions, see "Experimental Procedures"). Transfection of either Kir3.1 or Kir3.2A alone was insufficient to elicit strong inwardly rectifying K+ currents. B, the IRES-containing vector used for establishing stable cell lines expressing Kir3.1+3.2A, showing the restriction enzyme sites between which the cDNA for Kir3.1 and Kir3.2A were cloned. C, a typical example of Kir3.1+3.2A currents recorded from the HKIR3.1/3.2 cell line is shown (upper traces). Mean basal current density measured at 100 mV was 112 ± 17 pA/pF (n = 41). Currents were reversibly inhibited by external Ba2+ (middle traces); 1 mM inhibited currents by 81 ± 2% (n = 17). The lower traces show recovery after removal of Ba2+. D, corresponding current-voltage relationship for the data shown in C. E, the inclusion of GTPS in the pipette solution enhanced basal current density from 103 ± 27 pA/pF to 229 ± 52 pA/pF (n = 9, p < 0.01). Transfection of exogenous G also increased current density from 112 ± 17 pA/pF (n = 41) to 701 ± 226 pA/pF (n = 14, p < 0.01), in contrast to the reduction in current density observed with the transfection of  subunits from Gi1 (41 ± 6 pA/pF, n = 10, p = 0.05), Gi2 (20 ± 3 pA/pF, n = 10, p = 0.01) or the PTx-insensitive mutant Gi1C351G (34 ± 8 pA/pF, n = 8, p = 0.05; see "Results").

G Protein Regulation of Kir3.1+3.2A Channels-- The family of Kir3.x channels are regulated by heterotrimeric G proteins. To mimic the activation of G proteins, we used the non-hydrolyzable analogue of GTP, GTPS. Basal currents were potentiated by the inclusion of 500 µM GTPS in the pipette solution (Fig. 1E). It has been established that free G subunits but not G subunits mediate channel activation through a direct protein-protein interaction between determinants on the G dimer and cytoplasmic domains on the Kir3.x subunit (18-24, 31). The currents expressed in our monoclonal cell line demonstrate behavior that is consistent with this body of work. It was possible to transiently transfect the HKIR3.1/3.2 line and identify transfected cells after co-transfection with green fluorescent protein using epifluorescence. Transient transfection of exogenous ₁₂ subunits significantly increased basal current density, whereas overexpression of G subunits from G_i1 and G_i2 significantly reduced basal current densities (Fig. 1E). These data confirm that G activates the channel, while the reduction in current density by overexpression of  subunits is consistent with sequestration of free endogenous dimers. Interestingly, G_s also acted to suppress basal current (in the absence of receptors) to an extent similar to that observed with G_i subunits (data not shown). However, when receptors (either G_i/o- or G_s-coupled) were co-expressed, no suppression of basal current was seen.

Potentiation of Kir3.1+3.2A Currents by G_i/o-coupled Receptors-- We next investigated the issue of receptor specificity of channel activation by transiently transfecting a number of G_i/o- and G_s-coupled receptors (A₁ and A_2A adenosine receptors, _2A and ₁ adrenergic receptors, and D_2S and D₁ dopaminergic receptors, respectively) into the HKIR3.1/3.2 line. The concentrations of agonists used in the present study (A₁, A_2A: 1 µM NECA, _2A: 3 µM noradrenaline, ₁: 10 µM isoprenaline, D_2S: 10 µM quinpirole, D₁: 1 µM SKF38393) were manyfold greater than K_D values from radioligand binding studies published in the literature, such that receptors would exhibit full occupancy by agonist. Fig. 2A shows current potentiation by the adenosine receptor agonist, NECA, in the HKIR3.1/3.2 line after the expression of A₁ receptors. Stimulation of all three G_i/o-coupled receptors, A₁, _2A, and D_2S, potentiated currents and these increases were abolished by pre-treatment of the cells with PTx (Fig. 2B, upper panel) confirming that channel stimulation was mediated by the G_i/o family of G proteins. In contrast, agonist stimulation of the G_s-coupled receptors, A_2A, ₁, and D₁, was unable to significantly activate currents except for a small increase in current density observed with stimulation of the ₁ adrenergic receptor (Fig. 2B, lower panel). Interestingly this effect was PTx-sensitive, suggesting that ₁ can also couple to G_i/o proteins to activate the channel. PTx treatment to inhibit any G_i/o proteins that might be competing for G_s combined with the overexpression of G_s still did not allow the G_s-coupled receptors to activate the Kir3.1+3.2A currents (Fig. 2C).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Agonist stimulation of Gi/o-coupled receptors leads to the enhancement of Kir3.1+3.2A currents. A, activation of A1 receptors by 1 µM NECA increased Kir3.1+3.2A currents. Currents were elicited as illustrated in Fig. 1A. This shows the effects of 1 µM NECA on Kir3.1+3.2A currents. The left panel shows control, the middle panel is in the presence of 1 µM NECA, and the right panel is after washing NECA off. B, summary of the effects of stimulating three pairs of G protein-coupled receptors. The upper panel shows data for Gi/o-coupled receptors and the lower panel shows data for Gs-coupled receptors. Maximal concentrations of agonists (1 µM NECA (A1 and A2A), 3 µM noradrenaline (2A), 10 µM isoprenaline (1), 10 µM quinpirole (D2S), and 1 µM SKF38393 (D1)) were applied for 20-40 s. For PTx experiments, cells were exposed to PTx (100 ng/ml), post-transfection, for at least 16 h. C, Kir3.1+3.2A currents were not activated by Gs-coupled receptors, even in the presence of excess Gs. Cells were transfected with receptor and Gs and then treated with PTx.

To ascertain that this observation of specificity of receptor coupling was not an anomaly of the expression system, for example, due to stronger or more efficient transient expression of G_i/o-coupled receptors than G_s-coupled receptors, stable monoclonal cell lines were established that expressed both channel subunits and a receptor (A₁ denoted as HKIR3.1/3.2/A1, A_2A denoted as HKIR3.1/3.2/A2A, _2A or ₁ denoted as HKIR3.1/3.2/B1). Mean basal current density in these cell lines was no different to that measured in HKIR3.1/3.2 after transient transfection of receptors. Similarly to the responses seen in the transiently transfected cells, only the stable lines expressing G_i/o-coupled receptors (i.e. A₁ and _2A) were able to potentiate Kir3.1+3.2A currents (data not shown).

Confirmation of Expression of Functional Receptors Coupled to an Intact Second Messenger System-- The lack of potentiation of currents by stimulation of G_s-coupled receptors was not due to low expression levels of G_s since overexpression, together with relevant receptor in PTx-treated cells, did not significantly enhance currents (Fig. 2C). In order to confirm that these cells express endogenous G_s, we performed Western blots on total cell homogenate from the HKIR3.1/3.2/A1 and HKIR3.1/3.2/A2A lines. Clearly, the HKIR3.1/3.2/A1 line expressed G_s. In the HKIR3.1/3.2/A2A line, expression of G_s was lower but was revealed upon longer exposure of the blot. Transfection of the G_s-containing plasmid led to increased protein expression (Fig. 3A).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Biochemical approaches used to investigate functional expression of Gs-coupled receptors. A, Western blotting was used to investigate the expression of Gs in the A1 (HKIR3.1/3.2/A1 line) and A2A (HKIR3.1/3.2/A2A line) stable lines compared with a control protein (Gs: 1, 10 ng). We used equivalent loading of sample in each lane. Gs was detected in the HKIR3.1/3.2/A1 line, although expression was lower in the HKIR3.1/3.2/A2A stable line. Expression was enhanced when Gs was transiently transfected. B, [3H]DPCPX and [3H]CGS21680 saturation binding to A1 (open circles) and A2A receptors (solid circles) in the HKIR3.1/3.2/A1 and HKIR3.1/3.2/A2A cell lines. The following binding parameters were obtained: A1, KD = 0.37 ± 0.06 nM and Bmax = 8.67 ± 0.33 pmol/mg protein; A2A, KD = 13.5 ± 6.3 nM and Bmax = 4.23 ± 0.54 pmol/mg protein. Non-transfected cells did not exhibit any specific binding (not shown). Data were pooled from at least six experiments, with each counted in triplicate. C, adenylate cyclase activation via A2A and 1 receptors in lines HKIR3.1/3.2/A2A and HKIR3.1/3.2/B1, respectively. Total [3H]cAMP conversion was measured as described under "Experimental Procedures." A2A receptors were stimulated for 15 min by 1 µM NECA and 1 receptors by 10 µM isoprenaline. Data are from four to six experiments, each being performed in triplicate.

It is clear that the G_i/o-coupled receptors were functionally expressed since K⁺ currents were potentiated by stimulation of these receptors. However, since no potentiation was observed with the G_s-coupled receptors, it was important to determine that these receptors were actually being expressed at the cell membrane and were functionally coupled to downstream second messenger pathways.

We investigated the biochemical characteristics of the monoclonal lines HKIR3.1/3.2/A1, HKIR3.1/3.2/A2A, and HKIR3.1/3.2/B1. Equilibrium radioligand binding experiments using [³H]DPCPX (A₁) and [³H]CGS21680 (A_2A) were performed on homogenates from the HKIR3.1/3.2/A1 and HKIR3.1/3.2/A2A stable lines. Non-transfected cells did not exhibit binding of either radioligand (data not shown). Both HKIR3.1/3.2/A1 and HKIR3.1/3.2/A2A lines exhibited saturation of binding of appropriate ligand (Fig. 3B). It was noted that the A_2A receptor was stably expressed at lower levels than the A₁ receptor; this is an observation that has also been made in CHO cells (32).

To confirm signaling to a downstream effector, namely adenylate cyclase, agonist-induced cAMP accumulation was measured in the HKIR/3.1/3.2/A2A and HKIR3.1/3.2/B1 lines. A large increase in cAMP levels was observed in both lines in response to receptor stimulation (Fig. 3C).

Coupling of Channels to PTx-insensitive G Protein Mutants-- Thus far, we report that, as in native tissue, Kir3.1+3.2A currents were strongly activated by G but not G subunits and by stimulation of G_i/o- but not G_s-coupled receptors. To further investigate this specificity of channel activation, we took advantage of the PTx sensitivity of the G_i/o family of G proteins. These G proteins have a conserved cysteine residue in the  subunit 4 amino acids from the COOH terminus, which is ADP-ribosylated by PTx (33). Mutation of this residue prevents modification by PTx, thus resulting in a PTx-insensitive G protein (34, 35). G_i1 was mutated at this position to glycine or isoleucine (29). After transiently transfecting receptors and mutant G_i1 subunits into the HKIR3.1/3.2 line, cells were treated with PTx to preclude coupling between receptor and endogenous G_i/o, but still allow the study of coupling between receptor and mutant G_i1. Expression of these mutants alone did not enhance membrane currents and in fact significantly reduced basal current density similarly to wild type G_i1 (Fig. 1E) and in PTx-treated cells, stimulation of any of the G_i/o-coupled receptors tested were unable to enhance Kir3.1+3.2A currents (see Fig. 2B). When the mutant G_i1 subunits, G_i1C351G and G_i1C351I, were co-expressed, agonist stimulation of receptor led to a large enhancement of currents (Fig. 4A). This was observed with both A₁ (Fig. 4B) and _2A (Fig. 4C) receptors. In analogous experiments, expression of G_s in PTx-treated cells did not support coupling between Kir3.1+3.2A and any receptors (Figs. 2C and 4B). Thus, the PTx-insensitive G_i1 subunits were able to rescue signaling between G_i/o-coupled receptors and Kir3.1+3.2A in PTx-treated cells.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Kir3.1+3.2A channels can be activated through the stimulation of Gi/o-coupled receptors coupled to PTx-insensitive Gi1 mutants. A, this portion of the figure shows the effects of stimulating A1 receptors (1 µM NECA) in PTx-treated cells (upper panel) and with the transfection of Gi1C351G (middle panel) or Gi1C351I (lower panel). Traces indicate current before (basal), during (1 µM NECA) and after (Wash) receptor stimulation. B, this bar chart summarizes the data obtained with the A1 receptor and Gi1C351G, Gi1C351I, and Gs. Mutation of Cys to either Gly (Gi1C351G) or Ile (Gi1C351I) was able to support coupling between both receptors and Kir3.1+3.2A. Expression of Gs did not support coupling, as expected, between A1 receptor and Kir3.1+3.2A. C, a similar pattern of responses was observed with stimulation of the 2A receptor (3 µM noradrenaline).

A COOH-terminal Chimera between G_i1 and G_s Completely Swaps Coupling between Receptor and Kir3.1+3.2A Channels-- Our data suggest that G is important in the determination of receptor selectivity of channel activation. This hypothesis was further investigated by the use of chimeric G proteins. Since the COOH-terminal 20 amino acids of G protein  subunits are implicated in the selectivity of interactions with G protein-coupled receptors (36), we made a series of COOH-terminal chimeras between the G proteins G_i1 and G_s, where between 6 and 20 amino acids of G_i1 were replaced with the corresponding residues of G_s. These experiments were done in PTx-treated cells to prevent interaction of receptor with endogenous G_i/o, and the data are shown in Fig. 5. Strikingly, a 13-amino acid chimera (GiGs13) allowed a complete swap of receptor coupling; A_2A receptor stimulation by NECA strongly enhanced currents to a similar level observed with A₁ receptor stimulation under control (non-PTx-treated) conditions (Fig. 5A). Further replacements, GiGs16 and GiGs20, had similar, although not as profound, effects (Fig. 5B). The effects of the GiGs13 chimera were not unique to the A₁/A_2A receptors; this chimera was also able to swap receptor/channel coupling from the _2A to the ₁ adrenergic receptor (Fig. 5C). Thus, we have managed to potentiate the Kir3.1+3.2A currents via G_s-coupled receptors by using chimeric G proteins between G_i1 and G_s.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   COOH-terminal chimeras between Gi1 and Gs can swap the specificity of channel activation from Gi/o- to Gs-coupled receptors. A shows current traces obtained in response to stimulation of either the A1 (upper panel) or the A2A (lower panel) receptor, when the chimera GiGs13 is co-expressed. Experiments were done in PTx-treated cells to preclude coupling between receptor and endogenous Gi/o. B summarizes data for the A1 and A2A receptors and a series of COOH-terminal chimeras. The upper two bar charts indicate current density measured in response to stimulation of A1 (left) and A2A (right) receptors by 1 µM NECA under control conditions (non-PTx-treated). Below are bar charts summarizing data for a series of Gi1-Gs chimeras, the schematics of which are shown adjacent to the graphs. C, the GiGs13 chimera was also investigated with the adrenergic receptors, 2A (3 µM noradrenaline) and 1 (10 µM isoprenaline).

Disruption of Coupling between the A₁ Receptor and Kir3.1+3.2A by a COOH-terminal Chimera between G_s and G_i1-- Further evidence that supports a key role for the G protein  subunit is a dominant negative effect that we observed with a COOH-terminal chimera between G_s and G_i1; this chimera is mainly G_s but has the COOH-terminal 13 amino acids of G_i1 (GsGi13). This acted to significantly disrupt coupling between the A₁ receptor and Kir3.1+3.2A (a reduction of approximately 70%; Fig. 6). NECA-induced currents in control cells were 217.0 ± 38.9 pA/pF (n = 15), whereas in cells where GsGi13 was expressed, induced currents were significantly reduced: 68.6 ± 21.3 pA/pF (n = 20, p = 0.045). This dominant negative effect is likely to be specific, as overexpression of G_s with the A₁ receptor did not suppress basal current (Fig. 4B).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   A COOH-terminal chimera between Gs and Gi1 (GsGi13) disrupts coupling between the A1 receptor and the Kir3.1+3.2A channel. This shows a schematic of the GsGi13 chimera, together with a bar chart indicating current density measured in response to stimulation of the A1 receptor by 1 µM NECA under control conditions, and with the co-expression of GsGi13.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reconstitution of Specificity of Activation of Kir3.1+3.2A Channels-- One of the aims of this study was to examine whether the specificity of Kir3.1+3.2A channel activation by G protein-coupled receptors, widely observed in native tissue such as neuronal and atrial cells, could be reconstituted in an heterologous expression system. By making a stable channel-expressing mammalian cell line (HKIR3.1/3.2), we were able to reconstitute the specificity of channel activation. We have shown that only G_i/o-coupled receptors, but not G_s-coupled receptors, activate Kir3.1+3.2A channels in our HEK293 cell line. Even overexpression of G_s did not lead to current enhancement through G_s-coupled receptors, either in PTx-treated or non-treated cells, suggesting that G_s does not participate in the activation of these channels. Thus, in our system, G dimers derived from inhibitory heterotrimeric G proteins are able to activate Kir3.1+3.2A heteromultimeric currents while the stimulatory G_s is not.

A number of studies have demonstrated that, under given conditions, it is possible to activate Kir3.x or native currents through G_s-coupled receptors (37-39). However, some issues arise in relation to these and our work. Often a single receptor pathway has been used. Ruiz-Velasco and Ikeda (38) found they could activate Kir3.1+3.2 and Kir3.1+3.4 channels heterologously expressed in superior cervical ganglion neurons through the vasoactive intestinal peptide (VIP) receptor. VIP does not exclusively stimulate G_s-coupled receptors: it has been shown to couple to G_i1/2 (40) and also to elevate intracellular Ca²⁺ levels (41). The VIP-mediated effects were enhanced by pre-treatment with PTx, leading to the authors' suggestion that the uncoupling of PTx-sensitive G proteins allowed more PTx-insensitive G proteins to activate the channels. We found that PTx had no effects on the inability of G_s-coupled receptors to activate these channels and did not observe channel activation by G_s-coupled receptors either in the presence or absence of PTx. Lim et al. (37) found potentiation of currents though the ₂ adrenergic receptor but it is now appreciated that this receptor is able to couple to G_i (42, 43). Promiscuous coupling of receptors to more than one G protein is apparent with many receptors (44-47). One explanation advanced for the specificity of coupling is that there is more G_i/o than G_s in most cells; indeed, in the above studies, it was often necessary to overexpress G_s and in some instances the methods used to elevate G_s were not direct. Sorota et al. (39) used infection with an adenovirus, and this may have nonspecific and toxic effects on the cells. In our hands, even the overexpression of G_s did not lead to coupling.

Specificity of coupling may not be an absolute phenomenon and may depend on the relative levels of expression of channel, G protein, and receptor. Even though the current densities in our heterologous system are higher than seen in native neurons, we are still able to demonstrate an essentially absolute selectivity mechanism more analogous to that seen in native tissue. In the studies detailed above, no direct comparison is made with a G_i/o-coupled pathway under equivalent conditions. Indeed, in the work of Sorota et al. (39) on the native current in atrial myocytes, the levels of current activation though the  adrenergic receptor are lower than that seen though the muscarinic receptor, even after the overexpression of G_s.

A Key Role for the G Subunit in Determining Specificity of Channel Activation-- We have three lines of evidence that the G subunit is the key determinant of selectivity of channel activation by G_i/o-linked heptahelical receptors. First, the transfection of PTx-resistant mutants of G_i1 was able to rescue coupling between G_i/o-coupled receptors and Kir3.1+3.2A, but overexpression of G_s with G_s-coupled receptors under similar conditions was not able to do so. Second and most tellingly, it was possible to swap coupling between these families of receptor by exchanging only a few amino acids on the COOH terminus of G_i with those of G_s. Finally, we were able to disrupt coupling between a G_i/o-coupled receptor (A₁) and Kir3.1+3.2A by using a G_s chimera that contained the COOH-terminal 13 amino acids of G_i1. A similar strategy was used by Gilchrist et al. (48), who constructed "minigenes" of the COOH-terminal 11 amino acids of G_i1 and showed selective disruption of M₂ muscarinic receptor coupling to Kir3.1+3.4.

Schreibmayer et al. (49) showed inhibition of G₁₂-induced currents by activated G_i1 but not G_i2 or G_i3. In contrast, we have found that G subunits inhibited basal (non-agonist-stimulated) currents (presumably due to sequestration of free subunits) but that PTx-insensitive mutants of G_i1 actually supported coupling between receptor and channel. However, there are a number of methodological differences in expression system and experimental design; for example, our studies are performed in the whole cell configuration, while those studies were largely performed in excised patches in the inside-out configuration.

Expression in PTx-treated cells of a COOH-terminal chimera, in which 13 amino acids of G_i1 were replaced with the corresponding residues of G_s, swaps the receptor coupling profile, i.e. G_s-coupled receptors can now stimulate the channel whereas G_i/o-coupled receptors cannot. With 16 and 20 amino acid replacements, a swap of coupling was still observed, although it was not quite so profound. This apparent "drop-off" could simply be due to less efficient protein folding of these chimeras, and a similar finding was observed by Conklin et al. (50). Several investigators have used a chimeric G protein approach to alter the fidelity of receptor activation, i.e. receptors not normally coupled to a particular class of G protein then become able to stimulate that G protein (50-53). It has been shown that substitution of as few as 4 amino acids can alter receptor signaling to G proteins (50). Replacing the COOH-terminal 4 amino acids of G_q with those of G_i2 allowed the stimulation of phospholipase C by D₂ dopaminergic and A₁ adenosine receptors, which normally couple exclusively to G_i. However, whether a swap of coupling occurred was not established, i.e. that G_q-coupled receptors are no longer able to stimulate phospholipase C activity with the G_q/G_i chimera. Our data suggest that it is possible with some receptor groups to achieve a clean switch by COOH-terminal replacement. In addition, the carboxyl terminus of G subunits may not be the sole determinant of mediating specificity as other regions have been implicated in receptor interactions, notably at the NH₂ terminus (36, 54). It is likely that the extent of the involvement of the COOH terminus and other regions of the G subunits varies with receptor; indeed, this was found by Conklin et al. (50) with the A₁ and D₂ receptors and their coupling to G_q/G_i chimeras.

Our data demonstrate that we can reconstitute specificity of coupling between seven helix receptors and Kir3.1+3.2A channels. Basal currents are enhanced by the overexpression of the G dimer but not the G subunit. The pivotal role of the G dimer in channel activation is not being contested. We have no evidence of channel activation via G_s-coupled receptors, even when PTx-sensitive G proteins are uncoupled and/or G_s is overexpressed. By using single point mutations of G_i we can still activate the channel via G_i/o-coupled receptors in PTx-treated cells and by making chimeras between G_i1 and G_s we can swap coupling from G_i/o- to G_s-coupled receptors and Kir3.1+3.2A. Our finding that COOH-terminal G_i1/G_s chimeras could swap receptor coupling to channel strongly implicates the  subunit as playing an important role.

Do similar considerations apply to other effectors where G subunits are the direct mediators such as the inhibition of voltage-gated calcium channels of the N and P/Q type (55-58)? It is apparent that calcium channel inhibition can be mediated through a number of receptors coupling to a number of different families of G proteins so the mechanisms may be different (59, 60).

The molecular mechanisms underlying receptor specificity of Kir3.x activation are largely unknown. It has been proposed without strong supportive evidence that there is compartmentalization of signaling components: either between receptor and channel, G protein and channel, or all three. Our data suggest a key role for the  subunit and seem to make it unlikely that there is compartmentalization between receptor and channel as receptor selectivity can be switched with G_i-G_s chimeras. So what might the possible relationship be between the G subunit and the channel complex? This could be via a direct protein-protein interaction or indirectly involving other accessory proteins. The heterotrimeric G protein has been found to bind to the NH₂ terminus of Kir3.1 (18, 61), but whether G_i-containing heterotrimers were preferentially bound in comparison to G_s-containing heteotrimers was not addressed. Our data are an important insight in understanding how receptor selectivity is achieved in a G-activated system and provides a basis for future mechanistic studies.

	ACKNOWLEDGEMENTS

We thank the following people for providing us with cDNA: H. Bourne (G_s), B. Conklin (G_i2), L. Jan (_2A receptor and Kir3.1), B. Kobilka (₁ receptor), M. Lazdunski (Kir3.2A), T. Palmer (A₁ and A_2A receptors), and W. Xu (D₁ and D_2S receptors) and the members of the HFSP collaboration for fruitful discussions (L. Jan, Y. Kurachi, E. Reuveny). We also thank Z. Hafeez and other members of the team for technical help and L. Clapp for helpful comments.

	FOOTNOTES

^* This work was supported by the Human Frontiers Science Program and the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence concerning G_i1 point mutants and G should be addressed. E-mail: gbca32@udcf.gla.ac.uk.

To whom all other correspondence should be addressed. Tel.: 44-171-209-6174; Fax: 44-171-813-2846 or 44-171-209-6212; E-mail: a.tinker@ucl.ac.uk.

	ABBREVIATIONS

The abbreviations used are: PTx, pertussis toxin; PCR, polymerase chain reaction; IRES, internal ribosome entry site; VIP, vasoactive intestinal peptide; pF, picofarads; GTPS, guanosine 5'-O-(thiotriphosphate); NECA, 5'-N-ethylcarboxyamidoadenosine; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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