Na+ Promotes the Dissociation between Galpha GDP and Gbeta gamma , Activating G Protein-gated K+ Channels*

Ida Rishal, Tal Keren-RaifmanDagger , Daniel Yakubovich, Tatiana Ivanina, Carmen W. Dessauer§, Vladlen Z. SlepakDagger , and Nathan Dascal

From the Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel, the Dagger  Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33156, and the § Department of Integrative Biology and Pharmacology, University of Texas-Houston Medical School, Houston, Texas 77030

Received for publication, October 31, 2002, and in revised form, December 3, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

G protein-gated K+ channels (GIRK, or Kir3) are activated by the direct binding of Gbeta gamma or of cytosolic Na+. Na+ activation is fast, Gbeta gamma -independent, and probably via a direct, low affinity (EC50, 30-40 mM) binding of Na+ to the channel. Here we demonstrate that an increase in intracellular Na+ concentration, [Na+]in, within the physiological range (5-20 mM), activates GIRK within minutes via an additional, slow mechanism. The slow activation is observed in GIRK mutants lacking the direct Na+ effect. It is inhibited by a Gbeta gamma scavenger, hence it is Gbeta gamma -dependent; but it does not require GTP. We hypothesized that Na+ elevates the cellular concentration of free Gbeta gamma by promoting the dissociation of the Galpha beta gamma heterotrimer into free Galpha GDP and Gbeta gamma . Direct biochemical measurements showed that Na+ causes a moderate decrease (~2-fold) in the affinity of interaction between Galpha GDP and Gbeta gamma . Furthermore, in accord with the predictions of our model, slow Na+ activation was enhanced by mild coexpression of Galpha i3. Our findings reveal a previously unknown mechanism of regulation of G proteins and demonstrate a novel Gbeta gamma -dependent regulation of GIRK by Na+. We propose that Na+ may act as a regulatory factor, or even a second messenger, that regulates effectors via Gbeta gamma .

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

GIRK1 (Kir3) channels are crucial for the regulation of heartbeat and for inhibitory actions of many neurotransmitters in the brain. They are activated by direct binding of Gbeta gamma released from heterotrimeric G proteins following activation of G protein-coupled receptors (GPCR) (1-3). GIRK activity also crucially depends on the presence of membrane phosphatidylinositol 4,5-bisphosphate (4). Cytosolic Na+ has been shown to activate GIRK by a direct, G protein-independent mechanism. The direct activation by Na+ is fast and exhibits low affinity for Na+ with an EC50 of 30-40 mM Na+ (4-7).

GIRKs are usually heterotetramers composed of two pairs of homologous subunits. GIRK1/GIRK4 is predominant in the heart; GIRK1/GIRK2 is abundant in the brain. An aspartate, which is absent in GIRK1 but present in the proximal C terminus of GIRK2 (Asp-226) and GIRK4 (Asp-223), is crucial for fast direct gating by Na+ (6, 8).

We have noticed an additional, slow activating effect of Na+ on GIRK channels in excised patches of Xenopus oocytes. The slow activation occurred both in wild-type (WT) GIRK channels and, surprisingly, in GIRK mutants that lack the fast direct Na+ regulation. It did not require GTP but was blocked by a Gbeta gamma scavenger, suggesting mediation by Gbeta gamma . We hypothesized that Na+ promotes dissociation of the heterotrimeric Galpha GDPbeta gamma complex into free Galpha GDP and Gbeta gamma ; the latter activates GIRK. This hypothesis was supported by direct biochemical measurements. Our findings shed new light on mechanisms of regulation of G proteins and GIRK channels by Na+ and suggest that Na+ may act as a second messenger that regulates effectors via Gbeta gamma .

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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cDNA Constructs and RNA-- RNA was synthesized in vitro from the following DNAs: GIRK1F137S (9), GIRK1, GIRK2, myristoylated cbeta ARK, and Galpha i3 (10). Amounts of injected RNA were as follows: GIRK1, GIRK2, and GIRK2D226N, 0.1-0.2 ng/oocyte; GIRK1F137S, 1-5 ng/oocyte; Galpha i3, 0.5-2 ng/oocyte; cbeta ARK, 5 ng/oocyte. In GIRK1F137S experiments, an antisense oligonucleotide (50 ng) against the endogenous GIRK5 subunit was injected to prevent the formation of GIRK1/5 channels (11).

Xenopus Oocyte Preparation and Electrophysiology-- Xenopus oocytes were prepared, injected with RNA, and incubated in NDE-96 solution (96 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 2.5 mM sodium pyruvate, 50 µg/ml gentamycin, 5 mM Hepes/NaOH, pH = 7.6). Patch-clamp measurements of GIRK activity were done at -80 mV as described previously (10). The electrode solution contained 146 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 2 mM NaCl, 10 mM Hepes/KOH. The bath (500 µl) contained a Na+-free solution (130 mM KCl, 2 mM MgCl2, 10 mM Hepes/KOH, 1 mM EGTA, 2 mM Mg-ATP). The pH of all solutions was 7.4-7.6. NaCl was added in 50 µl of bath solution and mixed manually. Patches that showed >50% rundown of activity within 6 min after addition of Na+ (about 10% of all patches) were excluded from the study. Currents were filtered at 2 kHz and sampled at 5 kHz using the Axotape software (Axon Instruments). The results were analyzed as described previously (10). In each patch, the Na+-induced changes in activity (-fold activation) were calculated as -fold change in total open probability, NPo, relative to basal NPo measured during the last minute before the addition of Na+.

Biochemistry and Immunocytochemistry-- The DNA of GST-Galpha i3 was constructed by inserting the coding sequence of human Galpha i3 into the EcoRI-NotI sites of pGEX-4T-1 vector (Amersham Biosciences). The protein was amplified and purified from Escherichia coli using glutathione-Sepharose affinity beads. For pull-down assays, GST-Galpha i3 was incubated for 30 min at 4 °C in binding buffer (50 mM Tris, 5 mM MgCl2, 1 mM EDTA, 0.05% Tween 20, pH 7.0 with 150 mM KCl or 150 mM NaCl) with either 100 µM GDP or GTPgamma S. [35S]Methionine-labeled Gbeta gamma was synthesized in reticulocyte lysate, diluted 1:4, and incubated with GST-Galpha i3 in 300 µl of the above buffer (with 150 mM NaCl or KCl) for 30 min at room temperature in the presence of GDP or GTPgamma S. The mixture was incubated with glutathione-Sepharose beads and washed, and bound proteins were eluted with 15 mM glutathione and analyzed on a 12% SDS-polyacrylamide gel followed by Coomassie Blue staining and autoradiography as described previously (12).

Surface plasmon resonance (SPR) analysis of GST-Galpha i3-Gbeta gamma interaction was carried out on Biacore 2000 (Biacore AB, Uppsala, Sweden) using anti-GST antibodies (90 ng) covalently attached to the surface of a CM5 sensor chip as described previously (13). Following the capture of GST-Galpha i3, Gbeta gamma was injected across the sensor surface at a flow rate of 15 ml/min in series of concentrations in a buffer containing 50 mM Tris-Cl, 5 mM MgCl2, 1% CHAPS buffer, and either 100 mM KCl or 100 mM KCl. Intermediate concentrations of Na+ were obtained by mixing the two buffers. The kinetic data were analyzed using BIAEvaluation 3.1 software (Biacore AB) and Sigmaplot (SPSS Inc.).

The level of GIRK1F137S protein in plasma membrane (Fig. 4C) was measured as described previously (10). Briefly, large oocyte membrane patches were attached to coverslips, fixated, stained by a specific GIRK1 antibody (Alomone Labs, Jerusalem, Israel), and visualized using a Cy3-conjugated rabbit IgG (Jackson Immunoresearch Laboratories) using a Zeiss LSM 410 confocal microscope.

Statistics-- Data are presented as mean ± S.E.; pairwise comparisons were done using a two-tailed t test.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

WT GIRK1/GIRK2 channels were expressed in Xenopus oocytes, and Na+-induced activation was studied in excised patches (Fig. 1A). GIRK activity was first recorded in cell-attached configuration. Then the patch was excised ("inside-out") with its intracellular membrane surface facing the bath solution, which was Na+- and GTP-free but contained Mg-ATP to preserve membrane phosphatidylinositol 4,5-bisphosphate (4). After excision, the basal activity declined to a new steady level within 0.5-2 min (10). 3-4 min after excision, 40 mM NaCl was added to the bath solution, causing a 6.7 ± 2.5-fold increase (n = 8) in channel activity. In agreement with previous studies (7), this fast Na+ activation was not affected by coexpression of the Gbeta gamma scavenger cbeta ARK (myristoylated C terminus of beta -adrenergic receptor kinase; data not shown). In addition, in many WT GIRK1/GIRK2 patches, a slow, late activation was observed. It started after about 1 min and developed over the next 3-6 min (Fig. 1A).


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Fig. 1.   Fast and slow activation of GIRK channels by Na+. A, activation of the WT GIRK1/2 channels by 40 mM NaCl: full time course of a representative experiment. Periods of patch excision and addition of NaCl, accompanied by noise, are blanked. Channel openings are downward. B, slow activation of GIRK1F137S channels by 40 mM NaCl. Upper trace, full time course of the experiment; lower traces zoom on shorter periods of activity. C, activation of GIRK1F137S channels by 40 mM NaCl is blocked by coexpression of cbeta ARK: a representative record. D, summary of effects of 40 mM NaCl on GIRK1F137S. Numbers of experiments are shown above bars. *, p < 0.05. c.a., cell-attached.

To explore the slow effects of Na+, we utilized the GIRK1 pore mutant GIRK1F137S, which forms functional homotetrameric channels (9) and lacks the C-terminal aspartate crucial for the direct Na+ effect. Fast activation of homotetrameric GIRK1F137S channels by 40 mM Na+ was very weak, 1.82 ± 0.36-fold (n = 7) (Fig. 1B). It might reflect a weak direct effect of Na+ on the GIRK1F137S channel. In seven of eight patches, the activity continued to increase over the next several minutes, reaching a maximum after 4-7 min. This slow activation, measured during a 3-min period between 4 and 6 min after addition of Na+, was 3.9 ± 1-fold (n = 7) above basal (Fig. 1D). It was fully blocked by coexpression of cbeta ARK (Fig. 1, C and D), implying a Gbeta gamma -dependent mechanism. We assumed that the additional 2-fold slow increase in GIRK activity (compared with the 1st min after addition of Na+) reflects an increase in cellular concentration of free Gbeta gamma , [Gbeta gamma ]. Comparable slow activation (2.62 ± 0.45-fold, n = 6) was observed already at 10 mM Na+, which causes little fast activation in WT GIRK channels (5, 14) and no fast activation in GIRK1F137S (1.31 ± 0.16-fold, see Fig. 4).

How could Na+ activate GIRK in a Gbeta gamma -dependent manner? GPCRs activate G proteins by promoting the exchange of GDP for GTP at the Galpha subunit followed by dissociation of Galpha GTP from Gbeta gamma (15). Cl- also promotes GTP binding to Galpha in vitro (16), but Cl- concentration in our bath solution was already saturating for this effect before the addition of NaCl. Most importantly the activation of GIRK1F137S by NaCl was achieved in a GTP-free solution, ruling out a mechanism involving GDP-GTP exchange. Another source of free Gbeta gamma , which does not require GTP, is the basal equilibrium between the Galpha beta gamma heterotrimer and free Galpha GDP and Gbeta gamma (17):
<UP>G</UP>&agr;<SUB><UP>GDP</UP></SUB>&bgr;&ggr; ⇆ <UP>G</UP>&agr;<SUB><UP>GDP</UP></SUB>+<UP>G</UP>&bgr;&ggr;

<UP><SC>Reaction </SC></UP>1
We hypothesized that Na+ promotes the dissociation of the Galpha beta gamma heterotrimer into free Galpha GDP and Gbeta gamma by increasing the KD of Reaction 1. This will increase the concentration of free Gbeta gamma , [Gbeta gamma ], activating GIRK in the absence of GTP. The observed time course of Na+ activation (minutes) is consistent with the slow dissociation rate constant of the Galpha beta gamma heterotrimer (~0.001 s-1) (17).

To scrutinize this hypothesis, we examined the effect of Na+ on binding of Gbeta 1gamma 2 to a GST-fused Galpha i3 protein, GST-Galpha i3 (Fig. 2A), using a pull-down assay. Gbeta gamma was synthesized in vitro in reticulocyte lysate. Only GST-Galpha i3-GDP, but not GST or GST-Galpha i3-GTPgamma S, bound Gbeta gamma in this assay (Fig. 2B), confirming specificity and functional activity of GST-Galpha i3. In three individual experiments like that shown in Fig. 2C (a and b), we examined the dose dependence of binding of Gbeta gamma to GST-Galpha GDP. The binding was dose-dependent and reached saturation at about 20 µl of Gbeta gamma -containing lysate. The affinity of binding was lower in high Na+ than in high K+ solution, but the differences were too small for a statistically reliable estimation of the affinity shift. Therefore, we compared the binding at a low [Gbeta gamma ] (2 µl of Gbeta gamma -containing lysate) and at saturating [Gbeta gamma ] (40 µl of lysate) (summarized in Fig. 2C, c). With 40 µl of lysate, the extent of binding in 150 mM NaCl was the same as in 150 mM KCl (102.6 ± 8.2%, n = 10). However, at 2 µl of lysate, the binding in the high Na+ solution was reproducibly weaker than in high K+ (65.9 ± 4.2%, n = 14, p < 0.001 compared with 40 µl of lysate). At doses of Gbeta gamma lower than 2 µl of lysate (where a greater effect of Na+ was expected), the signal in the autoradiograms was too weak for a reliable measurement. The ~34% decrease in Gbeta gamma binding caused by Na+ at 2 µl of lysate (which gave ~37% of maximum binding in high K+) corresponds to a 1.9-fold change in KD. The effect of Na+ was dose-dependent (Fig. 2D, a and b) with a Ki of 13.6 mM Na+ (Fig. 2D, c).


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Fig. 2.   High Na+ reduces the binding of Gbeta gamma to GST-Galpha i3. A, Coomassie Blue staining of GST and GST-Galpha i3 used in the experiment shown in B. B, pull down of [35S]methionine-labeled Gbeta gamma by GST-Galpha i3-GDP but not GST or Galpha i3-GTPgamma S (a representative PhosphorImager autoradiogram). C, binding of variable amounts of Gbeta gamma to GST-Galpha i3-GDP in solutions with 150 mM K+, 0 Na+ (K) or 150 mM Na+, 0 K+ (Na). a, a representative experiment. b, quantitation of the results shown in a relative to maximal signal (150 mM K+, 40 µl of lysate). c, Na+ reduces binding of Gbeta gamma at low, but not saturating, [Gbeta gamma ]. **, p < 0.01. D, binding of 2 µl of Gbeta gamma lysate to GST-Galpha i3-GDP depends on [Na+]. Intermediate concentrations of Na+ were obtained by mixing the parental buffers (150 mM KCl and 150 mM NaCl). a, a representative autoradiogram. b, summary of three separate experiments. Band intensity at each [Na+] was normalized to the maximal signal, observed at 0 mM Na+ (150 mM K+). c, the Na+ dose-response curve, fitted to the Michaelis-Menten equation with a Ki of 13.6 mM (solid line). The effect of Na+ (decrease in binding) at each [Na+] was presented as percentage of maximal effect (decrease in binding produced by 150 mM Na+).

The effect of Na+ on Galpha GDP-Gbeta gamma interaction was further studied by SPR. Recombinant Gbeta 1gamma 2 was injected in buffers with different concentrations of NaCl (substituted for KCl) across the sensor surface with GST or GST-Galpha i3 immobilized via anti-GST antibody (13). Gbeta gamma reveals a relatively high degree of nonspecific binding to GST in this assay (13, 18). However, the extent of Gbeta gamma binding to GST-Galpha i3-GDP was always greater than to GST (Fig. 3, A and B). Also the wash-out of Gbeta gamma from GST was fast as expected for a low affinity process, whereas unbinding of Gbeta gamma from GST-Galpha i3-GDP displayed a slower kinetic component, characteristic for a high affinity interaction. The net specific signal of Gbeta gamma -Galpha i3 binding, obtained by subtraction of the GST signal (see Ref. 13), displayed rise and decay phases that were reasonably well fitted by single exponential functions (Fig. 3C). As expected, GST-Galpha i3-GTPgamma S bound Gbeta gamma much weaker than GST-Galpha i3-GDP (Fig. 3D). When the Na+ concentration was increased, the net binding of Gbeta gamma to GST-Galpha i3-GDP was reduced in a dose-dependent manner with a Ki of 8 mM Na+ (Fig. 3, E and F). Although these data clearly show specific binding of Gbeta gamma to Galpha i3, the contribution of nonspecific binding, particularly in the first few seconds postinjection and sample wash-out, impaired the precise determination of the kinetic constants in Na+ and K+. A limited kinetic analysis on the subtracted traces showed that the dissociation constant of Reaction 1, KD, was increased by 10 mM Na+ by ~2-fold (two separate sets of experiments). The calculated 2-fold change is in very good agreement with the pull-down results. The SPR experiments support the conclusion that Na+ reduces the affinity of interaction between Galpha -GDP and Gbeta gamma .


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Fig. 3.   Analysis of Gbeta 1gamma 2 interaction with GST-Galpha i3-GDP or GST-Galpha i3-GTPgamma S by SPR. GST-Galpha i3-GDP (150 ng) was captured on one channel of Biacore, GST-Galpha i3-GTPgamma S was captured on another channel, and recombinant GST was immobilized on a reference channel. A and B, representative traces showing the SPR signal (in resonance units, RU) upon binding of 350 nM Gbeta 1gamma 2 to the GST and GST-Galpha i3-GDP channels in 0 mM Na+ (A) and in 10 mM Na+ (B). The different Na+ concentrations were obtained by mixing the parental buffers containing 100 mM KCl (0 Na+) or 100 mM NaCl (0 K+). C, net GST-Galpha i3-GDP SPR signal after subtraction of the nonspecific binding on the GST channel. Rise and decay phases were fitted to monoexponential functions (solid lines). D, comparison of SPR signal at the GST-Galpha i3-GDP and GST-Galpha i3-GTPgamma S channels (nonspecific binding to GST on the reference channel was subtracted). E, net binding of Gbeta gamma to GST-Galpha i3-GDP is reduced by Na+: a summary of three separate measurements. F, the Na+ dose-response curve, fitted to the Michaelis-Menten equation with a Ki of 8 mM (solid line). The effect of Na+ (decrease in specific Gbeta gamma binding), at each [Na+], was presented as percentage of maximal decrease in binding. In two measurements, maximal effect occurred at 50 mM Na+ and in one measurement at 20 mM.

The findings so far support the hypothesis that the slow activation of GIRK by Na+ is due to elevated [Gbeta gamma ] that dissociated from Galpha GDP. This hypothesis not only explains the block of the slow effect of Na+ by beta ARK but also allows testable predictions. Thus, mass law dictates that coexpression of Galpha should shift the equilibrium in Reaction 1 to the left, increasing [Galpha beta gamma ] and reducing free [Gbeta gamma ]. This should reduce the basal activity of GIRK, but now addition of Na+ should cause a larger relative increase in [Gbeta gamma ] than in control conditions. This prediction is independent of initial conditions chosen to describe channel activation by Gbeta gamma .2 However, it is important not to overexpress Galpha to very high levels at which free Galpha GDP can scavenge Gbeta gamma and blunt any Gbeta gamma -induced effects. We produced mild overexpression of Galpha i3, 3-4-fold over the resting cellular level (by injecting 5 ng of Galpha i3 RNA or less), that does not hinder the activation of GIRK by Gbeta gamma (10).

In agreement with the prediction, in Galpha i3-expressing oocytes, slow activation of GIRK1F137S by 10 mM Na+ was 10.3 ± 2.3-fold (n = 10), significantly greater than without Galpha i3 (Fig. 4A and summarized in Fig. 4B). Slow activation by Na+ was still fully blocked by coexpression of cbeta ARK (Fig. 4, A and B). Another GIRK channel mutant known to lack the direct Na+ activation, GIRK1/GIRK2D226N (14), was also slowly activated by 10 mM Na+ in Galpha i3-expressing cells (11.6 ± 2.8-fold, n = 9), and this effect was inhibited by cbeta ARK ("activation" by 1.04 ± 0.13-fold, n = 4). The dramatic effect of Galpha i3 on slow Na+-induced activation strongly supports the G protein-dependent character of this phenomenon. The extent of slow activation by 10 mM Na+, in the presence of Galpha , is comparable to the direct effect of 40 mM Na+, although it is still severalfold smaller than the 20-800-fold activation by saturating doses of Gbeta gamma (10). A simple kinetic model based on our hypothesis and on known or estimated parameters of Galpha GDP-Gbeta gamma and Gbeta gamma -GIRK interactions correctly described the slow GIRK activation by Na+. The calculations showed that a ~1.8-fold increase in KD of Reaction 1 by 10 mM Na+ fully accounted for the 2-fold slow activation of GIRK1F137S under control conditions and for an ~8-fold activation after coexpression of Galpha i3.2


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Fig. 4.   Coexpression of Galpha i3 enhances the NaCl-induced activation of GIRK1F137S. A, examples of the effect of 10 mM NaCl on GIRK1F137S activity in a cell expressing only the channel (a), channel and Galpha i3 (b), and channel, Galpha i3, and cbeta ARK (c). B, summary of the effects of 10 mM Na+ and coexpression of Galpha i3 and cbeta ARK. *, p < 0.05 by one-way analysis of variance; star , p < 0.05; star star , p < 0.01 as compared with 1st min by t test. C, upper panel, confocal images of GIRK1F137S protein in large patches of plasma membrane (field, 25 × 25 µm): representative oocytes of one batch. GIRK1F137S and Galpha i3 RNAs (5 ng/oocyte) were injected. Lower panel, summary of experiments presented in the upper panel. Intensity of labeling (OD units) was measured with TINA software (Raytest, Straubenhardt, Germany). **, p < 0.001. D, dose dependence of slow Na+ activation (measured during 1 min of maximal activity between 4-8 min after addition of NaCl). GIRK1F137S (5 ng) was coexpressed with Galpha i3 (0.5-5 ng of RNA/oocyte). c.a., cell-attached.

It appeared (although a systematic study has not been conducted) that coexpression of Galpha i3 did not cause the expected reduction in basal activity of GIRK1F137S (e.g. compare the cell-attached levels of channel activity in Fig. 4A, a and b). The unexpectedly high basal activity could reflect an increase in membrane levels of GIRK1F137S caused by Galpha i3 as described for WT GIRK1/GIRK2 (10). Indeed immunostaining of the expressed channels in large plasma membrane patches (Fig. 4C) showed a net 6.4-fold increase of GIRK1F137S protein by Galpha i3 (after subtraction of background observed in uninjected oocytes) compared with channels expressed alone.

The dose dependence of the slow Na+ effect was studied in oocytes coexpressing GIRK1F137S and Galpha i3. Maximal activation was observed at 10 mM Na+ (Fig. 4D). At higher [Na+] the effect was smaller, consistent with the previously reported inhibition, at high [Na+], of GIRK channel constructs lacking the fast Na+ activation (6). The mechanism of this inhibition is unknown. The half-maximal activation dose (EC50) appeared to be slightly above 5 mM Na+, but considering the interference of the inhibitory effect at high [Na+], the actual EC50 of slow Na+ activation is probably higher.

    DISCUSSION
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ABSTRACT
INTRODUCTION
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Na+ Regulates Basal Equilibrium between Free and Galpha GDP-bound Gbeta gamma -- Our results strongly suggest that Na+ reduces the affinity of interaction between Galpha GDP and Gbeta gamma . This hypothesis initially arose following the observation of slow, Gbeta gamma -dependent activation by Na+ of mutant GIRK channels lacking the direct Na+ modulation. We then demonstrated weakening of Galpha GDP-Gbeta gamma binding using direct in vitro binding assays. Although the detected change in affinity was small, the results obtained by two independent methods (a pull-down assay and surface plasmon resonance) were in good agreement. Both methods, as well as estimates obtained from patch-clamp data, indicate that Na+ causes a ~2-fold decrease in the affinity of interaction between Galpha i3-GDP and Gbeta gamma . The EC50 or Ki of the Na+ effect estimated by electrophysiological and the two biochemical methods are also in good agreement (~6-14 mM), falling within the physiological range of [Na+]in. The proposed hypothesis made it possible to explain the observed inhibition of the slow Na+-induced activation by the Gbeta gamma scavenger, cbeta ARK, and to predict a novel physiological effect: a dramatic enhancement of slow Na+ activation of GIRK by coexpressed Galpha . Interestingly Xenopus oocytes possess an unusually high basal level of free Gbeta gamma (19, 20), and coexpression of Galpha may actually mimic the "usual" cellular condition of low [Gbeta gamma ].

Na+ has long been known to modulate the binding of agonists to many GPCRs, uncoupling these receptors from G proteins. Initially Na+ had been suspected to regulate G proteins directly, but later studies demonstrated a pivotal role of Na+ interaction with a conserved aspartate residue located in the transmembrane region of many GPCRs (for review, see Ref. 21). Finally, although NaCl has been shown to promote GDP-GTP exchange at the G protein, the active agent was Cl- rather than Na+ (16). Thus, no direct effects of Na+ on G proteins are known, and this report is the first demonstration of such regulation.

Two Mechanisms of Regulation of GIRK by Na+-- Na+ regulation of GIRK channels is considered of high physiological importance since it is believed to determine part of their basal activity in intact cells and to underlie the negative chronotropic effect of cardiac glycosides in the heart (2, 5). Our data imply that, in GIRK channels, Na+ acts both directly and indirectly (via Gbeta gamma ) to modulate basal activity. The EC50 of the well characterized, direct, Gbeta gamma -independent activation by Na+ is 30-40 mM. The slow, Gbeta gamma -dependent activation of GIRK by Na+ has a definitely lower EC50: maximal effect is attained at 10 mM Na+. Despite the small magnitude of Galpha GDP-Gbeta gamma affinity change by Na+, 10 mM Na+ caused an impressive 4-10-fold activation of GIRK. Therefore, the slow mechanism may contribute substantially to the basal activity of the channel in intact cells where the resting [Na+]in is 5-10 mM.

The observed features of slow Na+ modulation of GIRK conform to the model in which [Gbeta gamma ], elevated because of the direct effect of Na+ on Galpha beta gamma dissociation, activates the channel. Yet at present we cannot exclude contribution of additional mechanisms, for instance activation of a nucleotide diphosphate kinase (NDPK), which catalyzes the transfer of phosphate from ATP to GTP. This might, in principle, promote Galpha beta gamma dissociation and activate GIRK (see the discussion in Ref. 2). However, Na+ has been reported to inhibit NDPK (22) and should have inhibited GIRK if NDPK were involved.

Na+ as a Second Messenger or a Servo-type Intracellular Regulator-- Sodium ions are crucial for neuronal activity as carriers of depolarization and also regulate many physiological processes (fluid balance and secretion, cardiac contraction, glutamate-induced neuronal excitation, etc.) by a variety of molecular mechanisms from a direct binding of Na+ (GIRK and Na+-dependent transporters and exchangers (23)) to activation of Src by Na+ via an unknown intermediate (N-methyl-D-aspartate receptors (24)). Basal [Na+]in is tightly regulated by pumps and exchangers, being maintained in resting cells below 10 mM. Yet [Na+]in often widely fluctuates. In neurons, even relatively short periods of synaptic activity produce very large increases in [Na+]in, reaching ~30 mM in apical dendrites and >100 mM in dendritic spines (25). These considerations raise the possibility that Na+ may, in some cases, act as a second messenger that regulates intracellular targets when a substantial change in concentration occurs.

Our findings suggest a new mechanism of Na+-dependent regulation of cellular processes: via Gbeta gamma . The effective range of Na+ concentrations that regulate the Galpha beta gamma left-right-arrows  GGalpha GDP + Gbeta gamma equilibrium is close to the resting physiological range of [Na+]in. Therefore, regulation of G proteins by Na+ may be a servo-type mechanism that sensitively responds to bidirectional changes in [Na+]in rather than to increases alone. We propose that Na+ regulation of the dynamic equilibrium between bound and free Galpha GDP and Gbeta gamma can have a substantial biological effect by regulating a host of effectors of Gbeta gamma (1), some of which are second messenger-generating enzymes that may further amplify Na+ effects. This establishes a possible novel way of communication between electrical activity and other cellular processes.

    ACKNOWLEDGEMENTS

We are grateful to A. G. Gilman for encouragement and to Bernard Attali, Sagit Peleg, and Ilana Lotan for critical reading of the manuscript. We thank the colleagues who kindly provided the original cDNA constructs: D. Logothetis (GIRK1F137S), E. Reuveny (cbeta ARK), M. I. Simon (G protein subunits), E. Liman (pGEM-HE), and R. Murrell-Lagnado (GIRK1D226N).

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM56260 (to N. D.), GM60419 (to C. D.), and GM60019 (to V. S.) and grants from the United States-Israel Binational Science Foundation (to N. D., V. S., and C. D.).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 should be addressed. Tel.: 972-3-6405743; Fax: 972-3-6409113; E-mail: dascaln@post.tau.ac.il.

Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.C200605200

2 D. Yakubovich, I. Rishal, and N. Dascal, unpublished results.

    ABBREVIATIONS

The abbreviations used are: GIRK, G protein-gated K+ channel; cbeta ARK, myristoylated C-terminal part of beta -adrenergic receptor kinase; [Gbeta gamma ], free cellular concentration of Gbeta gamma ; GPCR, G protein-coupled receptor; WT, wild-type; GST, glutathione S-transferase; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; NDPK, nucleotide diphosphate kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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