Regulation of the Inward Rectifying Properties of G-protein-activated Inwardly Rectifying K+ (GIRK) Channels by Gbeta gamma Subunits*

Leif G. Hommers, Martin J. Lohse, and Moritz BünemannDagger

From the University of Würzburg, Department of Pharmacology & Toxicology, Versbacherstrasse 9, 97078 Würzburg, Germany

Received for publication, May 30, 2002, and in revised form, October 15, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Gbeta gamma subunits are known to bind to and activate G-protein-activated inwardly rectifying K+ channels (GIRK) by regulating their open probability and bursting behavior. Studying G-protein regulation of either native GIRK (IKACh) channels in feline atrial myocytes or heterologously expressed GIRK1/4 channels in Chinese hamster ovary cells and HEK 293 cells uncovered a novel Gbeta gamma subunit mediated regulation of the inwardly rectifying properties of these channels. IKACh activated by submaximal concentrations of acetylcholine exhibited a ~2.5-fold stronger inward rectification than IKACh activated by saturating concentrations of acetylcholine. Similarly, the inward rectification of currents through GIRK1/4 channels expressed in HEK cells was substantially weakened upon maximal stimulation with co-expressed Gbeta gamma subunits. Analysis of the outward current block underlying inward rectification demonstrated that the fraction of instantaneously blocked channels was reduced when Gbeta gamma was over-expressed. The Gbeta gamma induced weakening of inward rectification was associated with reduced potencies for Ba2+ and Cs+ to block channels from the extracellular side. Based on these results we propose that saturation of the channel with Gbeta gamma leads to a conformational change within the pore of the channel that reduced the potency of extracellular cations to block the pore and increased the fraction of channels inert to a pore block in outward direction.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

G-protein-activated inwardly rectifying K+ channels (GIRKs)1 are expressed in many areas of the brain and in supraventricular myocytes of the heart (1, 2). Activation of G-protein-coupled receptors that couple to Gi-proteins such as the M2 muscarinic acetylcholine receptors (M2-mAChRs) lead to a dissociation of heterotrimeric G-proteins into activated alpha  subunits and beta gamma dimers. Gbeta gamma subunits are known to bind to GIRK channels and increase the open probability of these channels (3, 4). Cardiac IKACh channels are formed by heteromultimers of GIRK1 and GIRK4 subunits (4). The binding site of Gbeta gamma subunits to GIRK channels was mapped primarily to the C terminus of GIRK1 and GIRK4 (4-8). Cross-linking studies have demonstrated that the heterotetrameric channel can bind up to 4 Gbeta gamma subunits (9). However, despite much experimental effort the mechanism by which Gbeta gamma activates these channels is not well understood.

GIRK channels belong to the family of strong inwardly rectifying K+ channels, which are characterized by their strong inwardly rectifying current-voltage relationships. The inward rectification has been linked to the presence of intracellular Mg2+ and polyamines (10-12). These positively charged cytoplasmic ions are thought to block outward K+ currents by blocking the pore of channels from the inside (10-13); however, for a related inwardly rectifying channel Kir2.1 this hypothesis has recently been questioned (14). Inward rectification of K+ channels is not only voltage-dependent but also dependent on the extracellular K+ concentration (11). The inward rectification of these K+ channels is closely related to their function in the heart as well as in many neuronal tissues. In cardiac myocytes activation of inwardly rectifying K+ channels such as IKACh causes the cell membrane to hyperpolarize between action potentials because the conductivity for K+ generated by these channels is high at membrane potentials close to EK. This hyperpolarization induced by IKACh appears to be at least partially responsible for the negative chronotropic effect induced by vagal activity (1, 2, 15). During action potentials, however, the conductivity of IKACh for K+ declines several-fold with the rise of voltage enabling the myocyte to generate prolonged action potentials, which are critically important for cardiac function (16).

The initial observation that led to the study presented here was the discovery that the agonist-induced IKACh in cardiac myocytes were quite variable in their degree of inward rectification,2 indicating that the modulation of the open probability of these channels (15) by ACh may not be the only property of these channels that is regulated by ACh. It seemed possible that, in addition, inward rectification of these channels may be modulated as well by ACh. The present experiments have tested this possibility.

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Preparation of Feline Atrial Myocytes-- Isolation of feline atrial myocytes was performed as described (17). Animal procedures used were in accordance with guidelines of the Animal Care and Use Committee of Northwestern University. Briefly, adult cats were first anesthetized with pentobarbital sodium (70 mg/kg body weight, intraperitoneally). The heart was quickly removed and retrograde perfused with Krebs-Henseleit buffer. It was digested by perfusion with collagenase-containing solution. After 10-15 min of digestion the atria were collected and cut into small pieces, followed by a 5-min incubation with fresh enzyme solution. Isolated atrial myocytes were collected, placed in M199 (Invitrogen), and plated in cell culture dishes. The cells were kept at 37 °C under 7% CO2 until further use.

Cell Culture and Transfection-- Chinese hamster ovary (CHO-K1) cells were grown in Ham's F-12 medium (Invitrogen). The media were supplemented with 10% fetal bovine serum and streptomycin/penicillin (100 units each). Cells were grown under 7% CO2 at 37 °C. In all transfections for electrophysiological studies the CD8 reporter gene system was used to visualize transfected cells (18). Dynabeads coated with anti-CD8-antibodies were purchased from Dynal. CHO-K1 cells were transfected using adenovirus-mediated gene transfer (19) using the following amounts of endotoxin-free cDNAs (Qiagen)/6 cm dish: human CD8 (in pi 3; 0.15 µg; gift from Dr. G. Yellen); mouse GIRK1 (in pC1, 0.3 µg) and mouse GIRK4 (in pCDNA1, 0.3 µg; gifts from Drs. F. Lesage and M. Laszdunski); human A1-adenosine receptors (in CLDN 10B, 0.2 µg; gift from Dr. J. Linden); human M2-mAChR (0.8 µg, in pcDNA3; gift from Dr. E. Peralta); human Gbeta 1 (in pCMV5, 0.3 µg) and human Ggamma 2 (in pcDNA1, 0.3 µg; gifts from Dr. H. A. Bourne). Empty pcDNA3 was used to balance the total amount of cDNA used for transfection to 2-2.35 mg/6 cm. All assays were performed 48-72 h post transfection if not otherwise mentioned.

HEK 293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, streptomycin/penicillin (100 units each), and 1% glutamine. Cells were grown under 7% CO2 at 37 °C. In cells stably expressing GIRK1/4, the media was supplemented with 200 mg/l G418. To visualize transfected cells the CD8 reporter gene system was used as described before. Transfection was performed using the Effectene transfection kit (Qiagen) according to the manufacturer's protocol using the following amounts of endotoxin-free cDNAs (Qiagen)/6 cm dish. Experiments described in Fig. 4: 0-1.25 µg empty pcDNA3, 0.05 µg CD8, 0.5 µg human alpha 2a-adrenergic receptor (AR)-Galpha i1 fusion protein (in pcDNA3, kindly provided by Dr. G. Milligan), and GIRK1, GIRK4, Gbeta 1, and Ggamma 2 as indicated in the figure. For experiments shown in Figs. 5-7: HEK cells stably expressing GIRK1 and GIRK4 channels were transfected with 0.7 µg alpha 2a-AR-Galpha i1, 1.4 µg each of human Gbeta 1 and Ggamma 2 (in pCDNA3), and CD8 (in pi 3, 0.2 µg). Experiments were performed 40-50 h post transfection.

Solutions-- For the measurement of K+ currents an extracellular solution of the following composition was used (mM): NaCl, 120; KCl, 20; CaCl2, 2; MgCl2, 1; Hepes-NaOH, 10, pH 7.3. The internal (pipette) solution contained (mM): potassium aspartate, 100; KCl, 40; MgATP, 5; Hepes-KOH, 10; NaCl, 5; EGTA, 2; MgCl2, 1; GTP, 0.01; pH 7.3. All standard salts as well as ACh and adenosine (Ado) were purchased either from Sigma or from Merck.

Measurement of Membrane Currents-- Membrane currents were recorded under voltage-clamp conditions, using conventional whole cell patch clamp techniques (20). Patch-pipettes were fabricated from borosilicate glass capillaries, (GF-150-10, Warner Instrument Corp.) using a horizontal puller (P-95, Fleming & Poulsen). The DC resistance of the filled pipettes ranged from 3-6 MOmega . Membrane currents were recorded using either a patch-clamp amplifier (Axopatch 200, Axon Instruments) or an EPC 9 (HEKA Instruments) as described previously (21, 22). Signals were analog-filtered using a lowpass Bessel filter (1-3 kHz corner frequency). Data were digitally stored using either a Mac (Centrion 640 with pulse software) or an IBM compatible PC equipped with a hardware/software package (ISO2 by MFK, Frankfurt/Main, Germany) for voltage control, data acquisition, and data evaluation. IKACh was measured as an inward current using a holding potential of -90 mV as described (23). Voltage ramps (from -120 mV to +60 mV in 500 ms, every 10 s) were used to determine current-voltage (I-V) relationships.

All measurements were performed at room temperature. Summarized results are presented as mean values ± S.E. Student's t tests (two population) were performed to test for significance of differences between groups of data.

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INTRODUCTION
EXPERIMENTAL PROCEDURES
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The atrial muscarinic K+ current (IKACh) is regulated by muscarinic receptors, and the underlying pathway has been studied in detail by many groups (15). The inwardly rectifying properties of this channel have been the topic of many detailed studies that provided interesting insights into the mechanisms of inward rectification (10, 11). However, so far no physiological modulation of the inward rectification of this current has been reported. The following study was based on the surprising observation that the inward rectification of ACh-evoked K+ currents in feline atrial myocytes varied as a function of the agonist concentration.

The Inward Rectification of Feline Atrial IKACh Was Modulated by Stimulus Strength-- IKACh in isolated feline atrial myocytes was measured in response to two different concentrations of ACh either in the inward or outward direction using the whole cell patch technique. The membrane potential was clamped to either -90 mV or +60 mV in the presence of 20 mM extracellular K+. When the holding potential was negative (-90 mV) to the potassium equilibrium potential (EK) (Fig. 1A), superfusion of the cell with 0.1 µM ACh gave rise to inward currents that were about 70% in amplitude compared with currents activated by 10 µM ACh. In contrast, at +60 mV outward currents induced by 0.1 µM ACh were barely detectable and were only about 10% in amplitude compared with currents activated by 10 µM ACh. I-V curves of ACh-induced currents (Fig. 1B) were determined by subtracting currents measured in the absence of agonist from currents measured in the presence of agonist in response to linear voltage ramps from -120 mV to +60 mV. I-V curves of the currents elicited by 0.1 µM ACh or by 10 µM ACh exhibited inward rectification and identical reversal potentials close to the EK (Fig. 1B), suggesting that the currents generated by either concentration of ACh were attributable to activation of IKACh. However, inward rectification of the current activated by 0.1 µM ACh was found to be considerably stronger than that of the current activated by 10 µM (Fig. 1B). Plotting GIRK conductance (normalized to the amplitude measured at -90 mV) against voltage shows a 15-20 mV shift to more positive potentials when currents were activated by 10 µM compared with 0.1 µM ACh (Fig. 1C).


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Fig. 1.   The inward rectification of feline atrial IKACh is dependent on stimulus strength. ACh-induced changes in whole cell currents of freshly isolated feline atrial myocytes were measured in the presence of 20 mM extracellular K+ (EK ~ -50 mV). Currents were activated in response to either submaximal (0.1 µM) or saturating (10 µM) concentrations of ACh at -90 mV and +60 mV as indicated (A). Current-voltage curves of ACh induced currents as shown in B were calculated after subtraction of background currents. The voltage dependencies of IKACh conductance in the presence of low or high agonist concentrations are plotted in C. These results were representative for similar experiments performed in four different atrial myocytes obtained from two different myocyte preparations.

Heterologously Expressed GIRK Currents Exhibited Strong Inward Rectification When Activated via Endogenous G-proteins-- Whole cell currents were measured in CHO-K1 or HEK 293 cells transfected with GIRK 1 and 4 as described before to prove that GIRK channels are responsible for the observed currents in feline atrial myocytes (21, 24). Gi-coupled receptors such as the M2-mAChR or A1 adenosine receptors activated GIRK currents in response to agonist (Fig. 2, A and B). Although the size of the currents increased as a function of agonist concentration (Fig. 2A), no stimulus-dependent change in inward rectification of GIRK currents was observed (Fig. 2B). Indeed, agonist-induced currents all exhibited strong inward rectification very similar to atrial IKACh activated with low doses of agonist (compare Figs. 2B and 1B). Similar results were observed in transiently transfected HEK 293 cells using M2-mAChR, A1 adenosine receptors, or alpha 2A adrenergic receptors (data not shown and Refs. 21 and 22).


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Fig. 2.   Gbeta gamma modulates inward rectification of heterologously expressed GIRK currents. HEK 293 cells were transiently transfected with cDNAs for GIRK1, GIRK4, and A1 adenosine receptors and with Gbeta 1gamma 2 (C, D, E, F, indicated as +Gbeta gamma ) or without additional exogenous G-protein subunits (A, B, E, F, indicated as agonist). GIRK currents activated via A1 adenosine receptors or by co-expression with Gbeta gamma subunits were measured using whole cell voltage clamp recording similar as described in the legend to Fig. 1. GIRK current-voltage curves were calculated by subtracting either background currents in the absence of agonist (B) or currents insensitive to 1 mM Ba2+ (D). Normalized GIRK current conductance in cells co-transfected with or without Gbeta gamma subunits were plotted against voltage (E). To quantify the degree of inward rectification, an inward rectification factor was defined (Fir = I(Erev - 50 mV)/I(Erev + 50 mV)) (F). Summarized data were compared for adenosine-evoked currents in the absence of exogenous Gbeta gamma (agonist) and Ba2+-sensitive currents evoked by heterologous expression of Gbeta gamma (F) (n = 9 each, the two groups were significantly different at p < 0.05).

Heterologously Expressed GIRK Currents Exhibited Weakened Inward Rectification When Activated via Co-expressed Gbeta gamma -- Agonist-induced GIRK currents obtained from cells heterologously transfected with GIRK1/4 were activated via endogenous G-proteins (Fig. 2, A and B). It seemed likely that the pool of endogenous G-proteins might have been limiting for the extent of maximal GIRK current activation. Therefore, Gbeta 1gamma 2 subunits were co-expressed with GIRK channels. GIRK currents were constitutively active due to Gbeta gamma subunits. The amplitude of GIRK currents was determined via inhibition by Ba2+ (Fig. 2C). In most cases, activation of co-expressed A1 adenosine receptors by 10 µM Ado induced no further stimulation of Ba2+-sensitive GIRK currents, indicating a maximal stimulation of GIRK channels by Gbeta gamma . Under these conditions, total Ba2+-sensitive GIRK currents compared with control conditions (activation via receptor and endogenous G-proteins) were about 2-fold larger in amplitude (147 ± 12.7 pA/pF with co-expressed Gbeta 1gamma 2 versus 71.2 ± 20.7 pA/pF activated via A1 adenosine receptors and endogenous G-proteins) and exhibited a weaker inward rectification (Fig. 2D versus Fig. 2B). Comparing the conductance-voltage relationship revealed a shift to more positive voltages for GIRK currents activated by heterologously expressed Gbeta gamma subunits compared with GIRK currents activated by agonist only (Fig. 2E). To quantify the relative inward rectification of GIRK currents the ratio of GIRK current conductance in outward versus inward direction (Ba2+-sensitive GIRK currents at reversal potential (Erev) ± 50 mV) was calculated. The ratio of outward/inward currents of Ba2+-sensitive GIRK currents activated by heterologously expressed Gbeta gamma was significantly increased compared with Ba2+-sensitive GIRK currents activated via A1 adenosine receptors and endogenous G-proteins (0.39 ± 0.11, n = 9 versus 0.14 ± 0.05, n = 8) (Fig. 2F). The voltage-dependence of GIRK currents maximally activated by heterologous expression of Gbeta gamma was comparable with atrial IKACh activated by saturating concentrations of ACh (10 µM), whereas agonist-induced GIRK currents activated via endogenous G-proteins exhibited similar strong inward rectification as submaximally activated atrial IKACh. This result indicated that the inwardly rectifying properties of GIRK channels were modulated depending on the internal Gbeta gamma concentration.

In a minority of cells transfected with Gbeta gamma subunits addition of adenosine to stimulate A1 adenosine receptors resulted in a further increase in GIRK currents (Fig. 3A), indicating submaximal stimulation of GIRK channels by heterologously expressed Gbeta gamma . Under these circumstances, basal Gbeta gamma -induced GIRK currents exhibited strong inward rectification, whereas addition of adenosine resulted in a pronounced weakening of inward rectification (Fig. 3B), demonstrating that inward rectification of heterologously expressed GIRK currents can be modulated via stimulation of G-protein-coupled receptors similar to atrial myocytes. Taken together, these results suggested that Gbeta gamma at submaximal concentrations induces strong inwardly rectifying GIRK currents, whereas at maximal concentrations, Gbeta gamma -evoked GIRK currents exhibited weakened inward rectification.


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Fig. 3.   Agonist-mediated modulation of inward rectification in cells submaximally stimulated with transfected Gbeta gamma . In a minority of cells transfected with Gbeta gamma , stimulation of co-expressed A1 adenosine receptors caused, in addition to the constitutively active GIRK currents, a further increase in GIRK currents (A). I-V curves for the Ba2+-sensitive currents in the absence (a-c) and presence of 1 µM adenosine (Ado, b-c) as well as the currents that were stimulated by Ado in addition to the constitutively active currents (b-a) were determined (B). Note a substantial weakening of GIRK current inward rectification in response to adenosine.

The Ratio of Gbeta gamma to GIRK Channel Expression Is Critical for Regulation of the Inward Rectification of GIRK Currents-- If the ratio of GIRK channels versus available Gbeta gamma in the cells is important for the degree of inward rectification as suggested by these results, it should be possible to achieve a high ratio of endogenous Gbeta gamma to GIRK channels by lowering the GIRK channel expression. Contrarily, a strengthening of the inward rectification should occur when GIRK channel expression is increased relative to the Gbeta gamma expression. We attempted to counteract the Gbeta gamma mediated weakening of inward rectification by transfecting HEK 293 cells with steady amounts of Gbeta gamma subunits but increasing amounts of GIRK1 and GIRK4 subunits as illustrated in Fig. 4. Gbeta gamma expression in the presence of co-expressed GIRK channels decreased cell survival. Therefore, we choose to co-express an alpha 2A-adrenergic receptor fused to a Gialpha 1-protein (26) to reduce constituitive Gbeta gamma signals. GIRK channels were stimulated using saturating concentrations of norepinephrine (10 µM), and subsequently GIRK currents were blocked by superfusion of the cells with 1 mM BaCl2 to determine background currents. The ratio of outward to inward GIRK currents significantly declined with increasing amounts of GIRK channels transfected (Fig. 4, upper panel). The GIRK current density measured at -90 mV increased with increasing amounts of GIRK1/4 channel expression (Fig. 4, lower panel), suggesting that Gbeta gamma expression was not limiting for maximal inward GIRK currents in cells transfected with 0.1 µg cDNA/5 cm dish of GIRK1/4. These results supported the hypothesis that inward rectification of GIRK channels is modulated dependent on the ratio of Gbeta gamma subunits relative to GIRK channels. In addition, we tried to lower GIRK expression relative to endogenous G-proteins by prolonging the time after transfection and found a significant increase in the ratio of outward to inward currents from day 3 to 4 post-transfection in transiently transfected CHO cells (Iout/Iin: 0.22 ± 0.06 d.4 versus 0.095 ± 0.025 d.3) accompanied by a small reduction in GIRK current density determined at -90 mV (43 ± 13 pA/pF, d.4 n = 12 compared with 64 ± 10 pA/pF, d.3 n = 6). This weakening of inward rectification of GIRK currents reflected most likely a decrease in GIRK channel expression in the individual cells, resulting in an increase of the ratio of G-proteins versus GIRK channels.


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Fig. 4.   The ratio of Gbeta gamma subunits to GIRK channel is important for regulation of inward rectification. HEK 293 cells were transiently transfected with indicated amounts of cDNAs encoding for GIRK1, GIRK4, Gbeta 1, and Ggamma 2 as well as constant amounts of cDNA of a alpha 2A-AR-Galpha i1-fusion protein and the CD8-reporter gene. 40-48 h post transfection whole cell currents were recorded in response to voltage ramps either in the presence of 10 µM norepinephrine (maximal GIRK activation) or 1 mM Ba2+ (to specifically block GIRK currents). Summarized data for the degree of GIRK current inward rectification (Fir = I(Erev + 50 mV)/I(Erev - 50 mV)) obtained under the indicated conditions is illustrated in the upper panel (n = 5-9, of 2-3 transfections). Corresponding maximal GIRK current densities measured in inward direction (-90 mV holding potential) are shown in the lower panel. Differences from the results shown in the first column (0.1 µg cDNA of GIRK1/4; 0.5 µg cDNA for Gbeta 1gamma 2) that reached significance are indicated (*, p < 0.05; **, p < 0.01).

The Weakened Inward Rectification Was Not Accompanied by Changes in Slow Blocking Kinetics of Outward GIRK Currents Attributed to Polyamine-induced Inward Rectification-- For further analysis experimental conditions were chosen to consistently induce either strong inward rectifying currents (control) or weak inward rectifying currents (Gbeta gamma -induced) in HEK cells stably expressing GIRK1 and 4. Strong inward rectifying currents were induced via agonist stimulation of alpha 2A adrenergic receptors in the absence of exogenous Gbeta gamma , whereas weak inward rectifying currents were evoked by additional co-transfection of Gbeta 1gamma 2. As described above the current model of the inward rectifying mechanism is a voltage-dependent open channel block by internal Mg2+ and polyamines such as spermine and spermidine. To test whether an alteration of the polyamine- and Mg2+-induced open channel block was the cause for the observed weakening of inward rectification, blocking and unblocking kinetics were determined using whole cell recording. According to Refs. 13, 27, and 28, the polyamine block is responsible for the time-dependent (slow) activation and inactivation of K+ currents through GIRK channels (or other inward rectifier channels) in response to voltage steps, whereas current block induced by internal Mg2+ occurs almost instantaneously. Therefore, whole cell currents resulting from voltage steps (-120 mV to 60 mV; 60 mV to -120 mV) were measured to determine the time constants of polyamine block onset and offset. In case inward rectification was weakened due to lowered polyamine block affinity, a faster polyamine unbinding from the channel and/or a slower-polyamine binding to the channel should be observed. In contrast, if Mg2+ block was altered, the fraction of channels blocked instantaneously in outward direction should be decreased, whereas changes in blocking and unblocking kinetics should not be observed. Background currents were determined by inhibiting GIRK channels via Ba2+ and subtracted from each measured whole cell current. A second-order exponential function was used to fit the current curves and determine time constants. Comparison of currents measured under control (strong inward rectification) and Gbeta gamma over-expressed (weak inward rectification) conditions showed no striking alteration of the slow blocking kinetics (Fig. 5A). As expected, the unblocking appeared to be faster (Fig. 5B), however, this effect did not reach statistical significance (1.28 ms ± 0.1 versus 1.05 ms ± 0.46; 10 ms ±0.95 versus 9.1 ms ± 2.8). In contrast to the proposition, blocking of the channel in the outward direction (reflecting binding of polyamines) was faster, too (4.9 ± 0.97 ms versus 2.75 ± 0.62 ms; 58.8 ± 16 ms versus 36 ± 9.3 ms). Normalizing to the maximum inward current revealed that the probability of channel opening at voltages positive to EK was increased under weak inward rectifying conditions compared with control conditions. Normalizing to the outward maximum current demonstrated that the same percentage of channels underwent a slow blockade under control as well as under weak inward rectifying conditions. Because the fraction of channels instantaneously blocked in the outward direction was lower when Gbeta gamma was over-expressed the potency of internal Mg2+ to block the channels might have been reduced. Therefore, we increased internal Mg2+ up to 20 mM to compensate for a reduced potency of Mg2+ to block GIRK channels, however, no change in inward rectification was observed (data not shown).


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Fig. 5.   The slow component of outward current block is not altered by co-expression of Gbeta gamma subunits. Illustrated are representative current recordings measured in response to voltage steps (-120 mV, 60 mV, and -120 mV, as indicated) from cells, which did (red) or did not express exogenous Gbeta gamma (black). Currents were normalized either to the maximal inward (upper and lower right panel) or outward (lower left panel) currents, and the time course of the onset of outward current block (lower left panel) as well as the recovery from outward current block (lower right panel) was fitted best by a bi-exponential decay. Summarized data for the resulting time constants are illustrated in the figure (n = 5-7).

Affinity for Ba2+ Block Was Reduced under Weak Inward Rectifying Conditions-- A hallmark for strong inward rectifier potassium channels is a high affinity block by external Ba2+. Studies using crystal structures of the bacterial KcsA channel complexed with Ba2+ have located a single Ba2+-binding site on the cytosolic side of the selectivity filter (29, 30). In close proximity to this site are some of the residues that have been implicated to be critical for strong inward rectification (11, 28). To test if Gbeta gamma mediates a conformational change of the GIRK channel that causes weakening of inward rectification by altering structures close to the selectivity filter, we questioned whether or not GIRK channel block by Ba2+ was affected by Gbeta gamma . Whole cell currents at a holding potential of -90 mV were measured in the presence of 1 µM, 10 µM, 40 µM, 140 µM, 1 mM, and 2 mM extracellular Ba2+ under strong and weak inward rectifying conditions (Fig. 6). Ba2+ effectively inhibited GIRK currents under both conditions, however, the potency of Ba2+ to block GIRK currents was substantially decreased when channels were maximally activated by Gbeta gamma (IC50: 73 µM versus 20 µM; Hill coefficient: n = 1.14 versus n = 2). These results strongly suggested that interaction with Gbeta gamma subunits induced conformational changes of GIRK channel structures close to the Ba2+-binding site.


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Fig. 6.   Concentration-response curve for Ba2+-induced GIRK current inhibition. The inhibition of steady GIRK currents (holding potential: -90 mV) in response to extracellular Ba2+ was determined in CHO-K1 cells transfected with the same set of cDNAs as described in the legend to Fig. 2. Curve fitting using conventional dose-response equations (Origin 6.1 software) determined the concentration for half-maximal inhibition of GIRK currents to be 30 µM Ba2+ for agonist-activated (strong inward rectifying) currents and 70 µM for Gbeta 1gamma 2-activated currents. Hill slopes were n = 1.1 (agonist) and 2.0 (+Gbeta 1gamma 2), respectively (n = 3-5).

Cs+-induced Block of GIRK Channels Was Attenuated under Weak Inward Rectifying Conditions-- Inwardly rectifying K+ channels can be blocked efficiently by external Cs+. This block is highly voltage-dependent and most prominent at negative potentials (11, 31). Binding sites for Cs+ in the channel have been mapped to pore-lining residues of transmembrane domain 2 (M2) (32) and to a site close to selectivity filter (32). Therefore, possible Gbeta gamma -dependent modulation of GIRK current block by external Cs+ (3 mM) was studied (Fig. 7). At a membrane potential of -90 mV, whole cell GIRK currents were inhibited under control (strong inward rectifying) conditions by 85 ± 2.6%, whereas whole cell currents in the presence of heterologously expressed Gbeta gamma (weak inward rectifying conditions) were inhibited only by 28 ± 4% (Fig. 7, A-C). To verify whether or not the attenuation of the Cs+ block by co-expression of Gbeta gamma was correlated to the Gbeta gamma -mediated weakening of inward rectification, the degree of inward rectification (defined as Fir = I(Erev - 50 mV)/I(Erev + 50 mV)) was plotted against the potency of Cs+ to block GIRK channels. We obtained a close inverse correlation of the degree of inward rectification and the ability of Cs+ to block GIRK currents (Fig. 7D). This result suggested that a Gbeta gamma -mediated conformational change of GIRK channels caused the reduced inward rectification and was mechanistically coupled to a reduction of the Cs+ block. We further analyzed the voltage-dependencies of the Cs+ block by comparing GIRK currents activated via endogenous G-proteins and selected GIRK currents activated via co-expressed Gbeta gamma subunits, but exhibiting a different degree of inward rectification (most likely due to different expression levels of Gbeta gamma subunits). Background-subtracted, current-voltage relationships of strong inward rectifying (Fir = 0.10; no Gbeta gamma co-transfected) and medium and weakly inward rectifying currents (Fir = 0.14, Fir = 0.20; both with co-expression of Gbeta gamma ) were determined in the presence or absence of 3 mM external Cs+ and fitted according to the Woodhull model (33, 34) (Fig. 7E).


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Fig. 7.   Gbeta gamma over-expression attenuates Cs+-induced inward current block of GIRK channels. Current-voltage relationships of GIRK currents were recorded in the presence or absence of 3 mM Cs+ in the bath solution. GIRK currents were evoked either via alpha 2A adrenergic receptors and endogenous G-proteins (A and C, blue-colored bar) or by co-expression of Gbeta gamma (B and C, red-colored bar). The potency of 3 mM Cs+ to block GIRK currents at -90 mV were plotted against the degree of inward rectification in cells expressing or not expressing exogenous Gbeta gamma (D). The voltage dependence of the current block induced by 3 mM extracellular Cs+ was determined in dependence of the degree of inward rectification (E). Representative experiments have been selected for strong (Fir = 0.1, control; no Gbeta gamma transfected), medium, and weak (Fir = 0.14, Fir = 0.20; both with co-transfection of Gbeta gamma subunits) inwardly rectifying currents and were fitted according to the Woodhull model (Equation 1 under "Results").


<UP>I</UP>(<UP>E</UP>)=<UP>I<SUB>0</SUB></UP>(<UP>E</UP>)<FENCE><FR><NU><UP>1</UP></NU><DE><UP>1+exp</UP>[<UP>−</UP>(<UP>zF/RT</UP>)<UP>&dgr;</UP>(<UP>E−E<SUB>Block1/2</SUB></UP>)]</DE></FR></FENCE> (Eq. 1)
The half-blocking voltage EBlock1/2 was shifted in the negative direction by up to -30 mV by Gbeta gamma (EBlock1/2 = -64 mV for Fir = 0.10; EBlock1/2 = -79 mV for Fir = 0.14; EBlock1/2 -93 mV for Fir = 0.20). Interestingly, the apparent voltage dependence of the Cs+-induced current block as indicated by the electrical distance delta  was up to 3-fold steeper under weak inwardly rectifying conditions (delta  = 2.2 for Fir = 0.10; delta  = 3.0 for Fir = 0.14; delta  = 6.9 for Fir = 0.20), suggesting a deeper penetration of Cs+ into the pore or a change in voltage-dependent binding parameters for Cs+ within the pore. This result strongly suggests that Gbeta gamma induced a significant conformational change within the GIRK channel pore.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Weakening of Inward Rectification of IKAch in Feline Atrial Myocytes Is Due to Binding of Gbeta gamma to GIRK Channels-- This study discovered that G-proteins do not only activate GIRK channels, but in addition also regulate the degree of inward rectification of these channels. In isolated atrial myocytes from adult cats, submaximally activated IKACh exhibited strong inward rectification, whereas maximal stimulation resulted in a 2-3-fold weakening of the inward rectification of IKACh. The fact that GIRK channels heterologously expressed in cell lines devoid of any other measurable inward rectifying currents were modulated in their inward rectifying properties by co-expression of Gbeta gamma subunits, strongly suggested that the inward rectification of GIRK channels themselves can be modulated by Gbeta gamma and that the observed stimulus-dependent weakening of IKACh inward rectification was the result of a Gbeta gamma -mediated modulation of GIRK channels.

The maximal activation of GIRK channels expressed in HEK 293 or CHO cells was clearly limited by the availability of endogenous Gbeta gamma subunits as the co-expression of Gbeta gamma subunits boosted GIRK currents 2.5-4-fold (Figs. 2 and 4 and Refs. 21 and 35). This may explain why in the absence of Gbeta gamma co-expression no agonist-mediated modulation of GIRK inward rectification was observed (unless GIRK channel expression was very low). In cells exhibiting strong inwardly rectifying GIRK currents despite expression of exogenous Gbeta gamma , additional stimulation via Galpha i-coupled receptors led to a dramatic weakening of the inward rectification of these currents (Fig. 3). This observation suggested that modulation of inward rectification was not an artifact of Gbeta gamma over-expression. At a constant expression level of Gbeta gamma subunits an increase of GIRK expression strengthened inward rectification and increased inward current density (Fig. 4), suggesting that the ratio of available Gbeta gamma subunits to expressed GIRK channels is critical for regulating inward rectification. Taken together these results point to a bimodal regulation of GIRK channels by Gbeta gamma subunits: at submaximal concentrations Gbeta gamma increased the open probability of GIRK channels (as demonstrated before (15, 36)), whereas at saturating concentrations Gbeta gamma weakened inward rectification of GIRK channels giving rise to a substantial increase in outward K+ current conductance.

The Physiological Role of Weakened Inward Rectification-- The inward rectification of IKACh channels is important for their physiological function to stabilize the membrane potential at negative voltages but not for blocking the generation of the plateau phase of action potentials (16). Because under physiological conditions net-potassium flux through this channel will always be in outward direction, one would predict that 2-3-fold increases in potassium outward currents, due to weakening of the inward rectification as observed in this study, will have a great impact on the shape and duration of supraventricular action potentials. It seems likely that the local in vivo concentration of ACh in the synaptic cleft can reach levels high enough, at least for very short periods, to cause weakening of inward rectification of atrial IKACh, because high frequency stimulation of the vagal nerves can induce a hyperpolarization in atrial tissue similar in amplitude as if directly evoked by ACh in the low µM range (37).

The Weakening of Inward Rectification Is Not Due to a Reduced Polyamine Affinity-- It has been shown that open channel block by polyamines and Mg2+ ions contributes to inward rectification in GIRK channels. Therefore, changing the inward rectification in the observed way may be related to polyamine and/or Mg2+-binding properties to the channel. Mg2+ is known to block instantaneously, whereas polyamine block exhibits slow voltage-dependent blocking and unblocking kinetics (13, 27, 28). In whole cell patch clamp experiments the polyamine block is found to be responsible for the slow inactivation/activation of GIRK currents measured resulting from voltage steps (28). Our investigation of polyamine block revealed no striking alteration of the blocking/unblocking time constants in the presence of Gbeta gamma over-expression. If a decrease of the polyamine affinity had been the cause for weakened inward rectification, a major increase in the blocking time constants and/or a major decrease in the unblocking time constant should have been observed. However, we found the contrary. Under weak inwardly rectifying conditions blocking time constants were slightly decreased and no major differences in unblocking time constants was observed. The observed weakening of inward rectification could be attributed to a decrease of the fraction of channels that were blocked instantaneously in outward direction (Fig. 3), pointing to attenuation of either the Mg2+-induced channel blockade or some yet unknown intrinsic outward current block (14). However, no change in inward rectification was observed when increasing internal Mg2+ up to 20 mM to compensate for a possibly reduced potency. So far, there is no direct experimental evidence to attribute the weakening of inward rectification to altered binding properties of polyamines or Mg2+ to the channels. However, we cannot exclude that Mg2+-induced outward current block was completely impaired in weak inwardly rectifying GIRK channels.

The Weakening of Inward Rectification Is Associated with a Reduction of Ba2+ and Cs+ Affinity-- In Kir2.1 channels there exists an overlap between sites important for inward rectification and blocking by external cations such as Cs+ and Ba2+ (11, 38). Therefore, Gbeta gamma -induced reduction of the affinity of Ba2+ to block GIRK currents supports the assumption that weakening of inward rectification is induced by conformational changes in the pore region of GIRK channels. Extracellular Cs+ is known to block strongly inwardly rectifying K+ channels in a highly voltage-dependent manner. The Cs+-binding site is also located within the channel pore, probably deeper in the channel than the blocking site for Ba2+. Similarly to Ba2+-induced GIRK channel block, Cs+-induced block was attenuated under weak inward rectifying conditions and the weakening of inward rectification correlated to weakening of Cs+-induced current block. Gbeta gamma -induced weakening of inward rectification was correlated as well with a stronger voltage dependence of Cs+ block and a shift to more negative potentials. These Gbeta gamma -mediated changes in the pore blocking properties of GIRK channels compare well to the differences of the pore blocking properties between members of the week and strong inwardly rectifying K+ channel family. Weak inwardly rectifying K+ channels exhibit usually a weaker affinity for Cs+ and Ba2+ compared with strong inwardly rectifying K+ channels (11, 28, 39). Based on these results we propose that maximal activation of GIRK channels by Gbeta gamma subunits induces a conformational change within the channel pore that tunes GIRK channels from a strong to a weak inwardly rectifying channel.

Gbeta gamma Induces a Conformational Change in the Pore of GIRK Channels-- How does binding of Gbeta gamma to the channel induce a conformational change of the channel that leads to reduced affinity for Cs+ and Ba2+? The fact that Gbeta gamma binds near the intracellular C terminus makes it unlikely that the cations and Gbeta gamma subunits share common binding sites within the pore. Considering that a tetrameric channel can bind up to four Gbeta gamma subunits (9) and the open probability is gradually regulated by at least three Gbeta gamma -binding sites (40), we propose that binding of the third or more likely the fourth Gbeta gamma subunit to the channel may force the channel into a weak inward rectifying conformation. How could this work? Recently L.Y. Jan and co-workers (41) presented convincing data, which suggested that opening of GIRK channels requires a rotation of the M2 transmembrane helix. Because the residues important for cation pore block are located either on the M2 helix or are in close proximity to the M2 helix, a rotation of these helices may likely alter the position of these residues. Assuming that the model of Jan and co-workers is correct and binding of a Gbeta gamma subunit to a GIRK channel subunit causes a rotation of the M2 helix of this particular GIRK channel subunit, it is obvious that the structures close to the cation-binding site(s) within the channel pore of a tetrameric GIRK channel will be different depending on how many Gbeta gamma subunits are bound. According to Refs. 36 and 42, single channel characteristics in respect to open and closed times GIRK channels are different depending on the concentration of available Gbeta gamma subunits. If strong inward rectification and high affinity Ba2+ and Cs+ block require one or two of the four M2 helices not to be rotated, rotation of the last two helices (induced by binding of the 3rd or 4th Gbeta gamma subunit to the tetrameric channel) could potentially weaken inward rectification. This working hypothesis needs to be verified in future studies.

The G-protein-mediated regulation of inward rectification of atrial and heterologously expressed GIRK channels described in this study represents to our knowledge the first description of a regulatory mechanism that alters the inward rectifying properties of an ion channel. Furthermore, we demonstrate that binding of Gbeta gamma subunits to the channel alter the conformation at known cation-binding sites within the channel pore, supporting the hypothesis that Gbeta gamma might gate the channel at the selectivity filter rather than at a cytoplasmic gate.

    ACKNOWLEDGEMENTS

We thank Dr. M. Hosey and Dr. B. TenEick for generous support of this work, which included providing lab space, equipment, and materials as well as scientific advice. Feline atrial myocytes were kindly provided by C. Hansen. The skillful assistance of M. Frank for work related to cDNA cloning and cell culture accelerated this project significantly.

    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft Research Fellowship BU 1133/1 (to M. B.) and a Leibniz award (to M. J. L.).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.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, University of Würzburg, Versbacherstrasse 9, 97078 Würzburg, Germany. Tel.: 49-931-201-48854; Fax: 49-931-201-48539; E-mail: m-buenemann@toxi.uni-wuerzburg.de.

Published, JBC Papers in Press, October 25, 2002, DOI 10.1074/jbc.M205325200

2 M. Bünemann, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: GIRK, G-protein-activated inwardly rectifying K+ channels; CHO, Chinese hamster ovary; Ado, adenosine; ACh, acetylcholine; M2-mAChRs, M2 muscarinic acetylcholine receptors; AR, adrenergic receptor; M2, transmembrane domain 2.

    REFERENCES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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