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Originally published In Press as doi:10.1074/jbc.M100207200 on March 8, 2001

J. Biol. Chem., Vol. 276, Issue 20, 16720-16730, May 18, 2001
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Receptor-mediated Inhibition of G Protein-coupled Inwardly Rectifying Potassium Channels Involves Galpha q Family Subunits, Phospholipase C, and a Readily Diffusible Messenger*

Qiubo Lei, Edmund M. Talley, and Douglas A. BaylissDagger

From the Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908-0735

Received for publication, January 10, 2001, and in revised form, March 2, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

G protein-coupled inwardly rectifying K+ (GIRK) channels can be activated or inhibited by distinct classes of receptor (Galpha i/o- and Galpha q-coupled), providing dynamic regulation of cellular excitability. Receptor-mediated activation involves direct effects of Gbeta gamma subunits on GIRK channels, but mechanisms involved in GIRK channel inhibition have not been fully elucidated. An HEK293 cell line that stably expresses GIRK1/4 channels was used to test G protein mechanisms that mediate GIRK channel inhibition. In cells transiently or stably cotransfected with 5-HT1A (Galpha i/o-coupled) and TRH-R1 (Galpha q-coupled) receptors, 5-HT (5-hydroxytryptamine; serotonin) enhanced GIRK channel currents, whereas thyrotropin-releasing hormone (TRH) inhibited both basal and 5-HT-activated GIRK channel currents. Inhibition of GIRK channel currents by TRH primarily involved signaling by Galpha q family subunits, rather than Gbeta gamma dimers: GIRK channel current inhibition was diminished by Pasteurella multocida toxin, mimicked by constitutively active members of the Galpha q family, and reduced by minigene constructs that disrupt Galpha q signaling, but was completely preserved in cells expressing constructs that interfere with signaling by Gbeta gamma subunits. Inhibition of GIRK channel currents by TRH and constitutively active Galpha q was reduced by U73122, an inhibitor of phospholipase C (PLC). Moreover, TRH- R1-mediated GIRK channel inhibition was diminished by minigene constructs that reduce membrane levels of the PLC substrate phosphatidylinositol bisphosphate, further implicating PLC. However, we found no evidence for involvement of protein kinase C, inositol trisphosphate, or intracellular calcium. Although these downstream signaling intermediaries did not contribute to receptor-mediated GIRK channel inhibition, bath application of TRH decreased GIRK channel activity in cell-attached patches. Together, these data indicate that receptor-mediated inhibition of GIRK channels involves PLC activation by Galpha subunits of the Galpha q family and suggest that inhibition may be communicated at a distance to GIRK channels via unbinding and diffusion of phosphatidylinositol bisphosphate away from the channel.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

G protein-coupled inwardly rectifying K+ (GIRK1; Kir3.x) channels are targets for up- and down-regulation by receptors that couple to different classes of heterotrimeric G proteins, providing a mechanism for dynamic regulation of cellular excitability (1, 2). Activation of GIRK channels involves receptors that couple to pertussis toxin (PTx)-sensitive Galpha subunits of the Galpha i or Galpha o family, whereas inhibition of GIRK channels is mediated by receptors that couple via PTx-insensitive Galpha subunits. This dual regulation of GIRK channels has been noted in a number of cellular contexts, including atrial cells of the myocardium (3, 4), aminergic neurons of the brainstem (5, 6), and enteric neurons of the peripheral nervous system (7). Because demonstration of this dual regulation requires simultaneous activation of different receptor classes, the phenomenon may be even more widespread than is currently realized.

Of the dual components of receptor-mediated GIRK channel regulation (activation and inhibition), most emphasis has been on understanding cellular mechanisms underlying GIRK channel activation. It is now well established that, following agonist stimulation, Gbeta gamma subunits released from receptor-bound heterotrimers bind directly to GIRK channels to increase channel activity (reviewed in Refs. 1, 2, and 8). It was recently found that Gbeta gamma activation of GIRK channels requires permissive levels of membrane phosphatidylinositol bisphosphate (PIP2) and that interactive effects of these mediators on channel activity result from Gbeta gamma -mediated stabilization of PIP2 binding to GIRK channels (9-11).

In contrast to GIRK channel activation, mechanisms that contribute to receptor-mediated inhibition of GIRK channels are not well understood. It is clear that GIRK channel inhibition involves receptors that typically couple via PTx-insensitive G proteins of the Galpha q family, and it therefore seems reasonable to suspect involvement of that class of Galpha subunit. However, GIRK channel inhibition by these and/or other classes of PTx-insensitive Galpha subunits has not been directly examined. Moreover, it remains to be determined if the signal for GIRK channel inhibition derives from the alpha  subunit or beta gamma dimer of the heterotrimeric G protein. In this respect, we recently found that Gbeta 5-containing dimers can inhibit Gbeta gamma -activated GIRK channels (12), a finding that is especially intriguing in the current context of receptor-mediated GIRK channel inhibition since Gbeta 5gamma dimers associate preferentially with Galpha q-coupled receptors (13, 14). Signaling mechanisms that contribute to GIRK channel inhibition downstream of G protein subunits are also not known with any certainty, although recently it was suggested that phospholipase C (PLC) activation could contribute to GIRK channel inhibition by Galpha q-coupled receptors in atrial myocytes by decreasing membrane levels of PIP2 rather than by production of downstream mediators (15-17). It remains to be determined if a similar mechanism contributes to GIRK channel inhibition by similar receptors in other settings.

Here, we prepared mammalian cell lines expressing GIRK channel subunits together with two different classes of G protein-coupled receptors in order to recapitulate dual regulation of GIRK channels in a heterologous expression system. Using this system, we found that Galpha q-coupled thyrotropin-releasing hormone (TRH) type 1 (TRH-R1) receptors can inhibit GIRK channels pre-activated by Galpha i/o-coupled 5-hydroxytryptamine (5-HT) type 1A (5-HT1A) receptors. GIRK channel inhibition involved Galpha q family subunits, PIP2, and activation of PLC, but it was independent of downstream signaling mediators typically associated with PLC activation such as protein kinase C (PKC), inositol trisphosphate (IP3), and intracellular calcium. These data are consistent with the possibility that PLC-mediated decreases in membrane PIP2 levels provide the signal for GIRK channel inhibition by Galpha q-coupled receptors (15-17); if this is the case, PIP2 may be readily diffusible since inhibition was robust even in cell-attached patches that were not exposed to agonist. These results further indicate that Galpha and Gbeta gamma subunits, each derived from a different class of receptor, can converge with opposite effects on the same channel to dynamically regulate cellular excitability.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Stable Cell Line Expressing GIRK Channels and TRH-R1 and 5-HT1A Receptors-- The derivation and maintenance of stable human embryonic kidney HEK293 cells expressing GIRK1 (Kir3.1) and GIRK4 (Kir3.4) channels (referred to as G1/4 cells) were described previously (12). This cell line was further stably transfected with the TRH-R1 and 5-HT1A receptors (referred to as G1/4R cells). Briefly, TRH-R1 (pBS; M. C. Gershengorn, Cornell University) and 5-HT1A receptor (pGEM3z; D. K. Grandy, Vollum Institute) cDNAs were subcloned into pcDNA3 and transfected together with pBabe-Puro (K. R. Lynch, University of Virginia) into G1/4 cells and maintained under G418 (400 µg/ml; Life Technologies, Inc.) and puromycin (200 ng/ml; Sigma) selection. A G418- and puromycin-resistant cell line was selected based on strong expression of inwardly rectifying K+ currents and robust regulation of those currents by TRH and 5-HT (G1/4R cells). Both the G1/4 and G1/4R cell lines were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F-12 with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in the continued presence of G418 (G1/4 cells) or G418 and puromycin (G1/4R cells).

Transfection of Stable Cell Lines Expressing GIRK Channels-- Stable cell lines (G1/4 and G1/4R) were transiently transfected with plasmids that direct expression of Galpha , Gbeta , or Ggamma subunits or of minigene constructs that interfere with G protein signaling under the control of a cytomegalovirus promoter. Galpha subunits that bear a point mutation (Gln right-arrow Leu) rendering them GTPase-deficient (i.e. constitutively active, indicated as Galpha *) were used as follows: Galpha q* (in pcDNA3; W. F. Simonds, National Institutes of Health) and FLAG epitope-tagged Galpha q* (in pSKAC; R. Iyengar, Mount Sinai School of Medicine, Mount Sinai, NY); Galpha 11* (in pCI; M. I. Simon, California Institute of Technology, Pasadena, CA); wild-type Galpha 14 (in pCIS; M. I. Simon), mutated by overlap extension polymerase chain reaction to yield Q205L, subcloned into pcDNA3, and sequenced; Galpha 15* (in pBS; M. I. Simon), subcloned into pcDNA3; Galpha i1*, Galpha i3*, and Galpha z* (in pCMV5; H. Itoh, National Children's Medical Research Center, Tokyo, Japan); Galpha i2* (in pCMV7; G. L. Johnson, National Jewish Medical and Research Center); Galpha o* (in pBS; B. M. Denker, Harvard University), subcloned into pcDNA3; Galpha s* and Galpha 12* (both in pCMV5; G. L. Johnson); Galpha 13* (in pcDNA3; M. Negishi, Kyoto University, Kyoto, Japan); and Gbeta 5 and Ggamma 2 (in pcDNA3; W. F. Simonds). The green fluorescent protein (GFP)-tagged PLCbeta 1-ct (where ct is the C-terminal domain; pEGFP-C1) and RGS2 (pCI) Galpha q effector antagonist constructs were obtained from S. R. Ikeda (Guthrie Research Institute) (18). Two RGS constructs (RGS6 and RGS11) that bind specifically to Gbeta 5 (19, 20) and interfere with signaling mediated by Gbeta 5-containing Gbeta gamma dimers (21) were obtained in pcDNA3.1 (A. G. Gilman, University of Texas-Southwestern). Two additional constructs that interfere generally with Gbeta gamma signaling were also used: a beta -adrenergic receptor kinase (beta ARK) C-terminal domain construct, beta ARK-ct (in pRK5; W. J. Koch, Duke University), was subcloned in frame with a myristic acid attachment signal into pGTM, a pcDNA3 derivative (E. A. Golemis, Fox Chase Cancer Center) (22); and Galpha t (transducin) was obtained in pcDNA1 (F. L. Kolakowski, University of Texas at San Antonio). GFP-tagged constructs designed to decrease membrane PIP2 levels (PLCdelta -PH (where PH is the pleckstrin homology domain), AKT-PH, and 5'-phosphatidylinositol phosphatase (5'-PI-PTase)), provided by T. Meyer (Stanford University, Stanford, CA), were as previously described (23). Expression plasmids were transfected in 35-mm culture dishes by CaPO4 precipitation (24) together with a plasmid (pGreenLantern, Life Technologies, Inc.) that directs expression of an enhanced GFP at a test plasmid/GFP plasmid ratio of 6:1 µg.

Western Blots-- Crude cell lysates were prepared from transfected cells in the presence of a mixture of protease inhibitors (2 µg/ml each leupeptin, pepstatin, and aprotinin and 100 µM phenylmethylsulfonyl fluoride). Proteins were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted. Expression of FLAG epitope-tagged constitutively active Galpha q subunits (FLAG-Galpha q*) was detected using M2 anti-FLAG monoclonal antisera (10 µg/ml; Sigma), and GIRK channel subunit expression was determined with a rabbit anti-GIRK1 antibody (1:500 dilution; Alomone Labs). Primary antibodies were detected with horseradish peroxidase-conjugated mouse or rabbit IgG using enhanced chemiluminescence.

Electrophysiological Recordings from Transfected Cells-- Cells were plated onto glass coverslips at a confluency appropriate to obtain single cells for electrical recording 48-72 h after transfection (except where noted). Coverslips were submerged in a recording chamber at room temperature on microscopes equipped with epifluorescent optics (Zeiss Axioskop FS or Nikon TE300). Individual cells that expressed GFP were identified using standard fluorescein isothiocyanate filter sets and targeted for recording.

Patch electrodes were made from borosilicate glass capillaries (Clark Electromedical) and connected to the head stage of an Axopatch 200A patch-clamp amplifier (Axon Instruments, Inc.). For whole cell recordings, pipettes (2-4 megaohms) were filled with an internal solution containing 120 mM KCH3O3S, 4 mM NaCl, 1 mM MgCl2, 0.5 mM CaCl2, 10 mM HEPES, 10 mM EGTA, 3 mM MgATP, and 0.3 mM Tris-GTP (pH 7.2). The external solution contained 137 mM NaCl, 6 mM KCl, 10 mM HEPES, 2 mM CaCl2, 2 mM MgCl2, and 10 mM glucose (pH 7.3). For cell-attached patch recordings, the same external solution was used, but pipettes were pulled to a higher resistance (7-10 megaohms) and contained 130 mM KCl, 5 mM EGTA, 2 mM MgCl2, and 10 mM HEPES (pH 7.3).

Electrophysiological data were acquired and analyzed using the pCLAMP suite of programs (Axon Instruments, Inc.). Series resistance was typically <10 megaohms and compensated by ~70-80%. Membrane voltages were corrected for a 10-mV liquid junction potential. To record inwardly rectifying whole cell currents, cells were held at -50 mV, and a slow voltage ramp command (Delta -90 mV, 0.1 V/s) was applied at 0.1 Hz. Membrane current was filtered at 0.5-1 kHz and sampled at 1-2 kHz. Measured variables included holding current at -50 mV and slope conductance, obtained from a linear fit to current-voltage data over the range of -100 to -120 mV. The effect of TRH on GIRK channel conductance was determined following subtraction of endogenous currents, defined by their resistance to 0.2 mM Ba2+. Single channel data were filtered at 2 kHz (four-pole Bessel) and collected on line using gap-free recording with a 10-kHz sampling frequency. The slope of current amplitudes obtained from gaussian fits to all-points histograms at three different patch potentials was used to determine single channel conductance. Analysis of the effects of agonist stimulation on GIRK channel activity in cell-attached patches was performed using Fetchan (Axon Instruments, Inc.) and the nPo freeware program (provided by J. L. Sui). All data are presented as means ± S.E., and each data point includes results obtained from at least two transfections.

Drugs and Reagents-- Compounds were prepared as concentrated stock solutions, stored at -20 °C, and diluted to the indicated concentrations in recording or pipette solutions, as needed. 5-HT (serotonin, Sigma) was made up as a 10 mM stock solution with 100 mM ascorbate and applied to cells at 50 µM. TRH (100 µM stock solution; Peninsula Laboratories, Inc.) was applied at 100 nM. Toxins from Bordetella pertussis (132 µg/ml) and Pasteurella multocida (50 µg/ml) were generously provided, respectively, by E. L. Hewlett (University of Virginia) and L. J. Eaton (List Biological Laboratories, Inc.); cells were incubated in toxins overnight at 2 and 1 µg/ml, respectively. The PLC inhibitor U73122 and its inactive analog, U73343, were obtained from Research Biochemicals Inc. and prepared as 2 mM stock solutions in dimethyl sulfoxide; preincubation with U73122 or U73443 (both at 1 µM) in the culture medium was begun at least 30 min prior to recording to test effects on receptor-mediated GIRK channel inhibition and ~36-48 h before recording to test effects in cells transfected with Galpha q*. The bisindolylmaleimide PKC inhibitor (GF 109203X, Calbiochem) was stored as a 5 mM stock solution, and continuous exposure to 5 µM bisindolylmaleimide was started >= 30 min before recording. The phorbol ester phorbol 12,13-dibutyrate (PDBu; Research Biochemicals Inc.) and its inactive analog, 4alpha -PDBu (Alexis), were prepared as 1 mM stock solutions in dimethyl sulfoxide and applied in the bath at 1 µM. IP3 (10 mM; Alexis) was diluted to 100 µM in pipette solution.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Dual Modulation of GIRK1/4 Channel Currents in a Stable HEK293 Cell Line-- To study mechanisms contributing to receptor-mediated inhibition of recombinant GIRK channels in the context of a mammalian cell system, we prepared a stable HEK293 cell line that expresses Kir3.1/3.4 (GIRK1/4) channels. As we described previously for G1/4 cells, hyperpolarizing ramp voltage commands evoked substantial inwardly rectifying currents under non-stimulated conditions that were due, in large part, to channel activation by free Gbeta gamma subunits endogenous to those cells (12). In cells transfected with the Galpha i/o-coupled 5-HT1A receptor and Galpha q-coupled TRH-R1, whether expressed transiently in G1/4 cells or stably in G1/4R cells, the dual regulation of GIRK channels that is seen in various native cell systems (5-7) was fully recapitulated, i.e. GIRK channel currents pre-activated by Galpha i/o-coupled receptors were inhibited by Galpha q-coupled receptors. Thus, as illustrated in the representative cell of Fig. 1A, the basal inwardly rectifying conductance was increased by 5-HT (50 µM), and this enhanced conductance was inhibited by TRH (100 nM). Although it was often more convenient to use the G1/4R cell line stably expressing the 5-HT1A and TRH-R1 receptors, we obtained essentially identical results with transient receptor expression; therefore, where available, data from experiments using transient and stable receptor expression were combined. Averaged data from cells treated with both 5-HT and TRH (n = 9) revealed that 5-HT caused an increase in GIRK channel conductance (42.2 ± 8.7% of the initial conductance), whereas TRH inhibited GIRK channel conductance (80.4 ± 3.8% of the conductance in 5-HT); when referenced to the initial GIRK channel conductance before 5-HT, the inhibition by TRH was often >100% (average of 118.3 ± 11.5%, n = 9), indicating that signaling from 5-HT1A and TRH-R1 receptors converges on the same GIRK channels, but with opposite effects.


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  		Fig. 1.   Dual regulation of GIRK channels in G1/4R cells. A, dual regulation of GIRK channel currents in HEK293 cells expressing GIRK1/4 channel subunits, the Galpha i/o-coupled 5-HT1A receptor, and Galpha q-coupled TRH-R1. Left panel, conductance was determined as the slope of current-voltage curves between -100 and -120 mV under control conditions, during application of 5-HT (50 µM) and during application of TRH (0.1 µM) in the continued presence of 5-HT. Right panel, sample traces reveal the inwardly rectifying profile of current responses to hyperpolarizing ramp voltage commands. Note that 5-HT activated an inwardly rectifying current that was nearly completely blocked by TRH. B, inhibition of GIRK channel currents by TRH is PMT-sensitive, but is unaffected by PTx. Left panel, shown is the time course of the effect of TRH on basal conductance in a control cell (closed circles) and in a cell treated overnight with PMT (1 µg/ml; open circles). Right panel, shown are summary data on the effect of TRH in control cells (n = 41) and in cells treated with PMT (n = 15) and PTx (2 µg/ml, overnight; n = 6) on TRH-induced inhibition of conductance (percent of control conductance; means ± S.E.). The asterisk denotes a statistically significant difference from control cells (p < 0.05 by ANOVA with post hoc Bonferroni's test).

It is well known that activation of GIRK channels by 5-HT1A and other receptors in native systems is mediated by PTx-sensitive G proteins of the Galpha i/o class. Likewise, we found that GIRK channel activation by 5-HT1A receptors was completely blocked by PTx (2 µg/ml, overnight; 56.2 ± 7.3% activation in control cells (n = 15) versus 11.9 ± 3.8% inhibition after PTx (n = 16)). On the other hand, receptor-mediated GIRK channel inhibition in native systems is PTx-insensitive; and accordingly, we found no effect of PTx on TRH-induced GIRK channel inhibition (Fig. 1B, right panel). Because TRH-R1 is typically associated with G proteins of the Galpha q family, we tested the effects of P. multocida toxin (PMT) (Fig. 1B, left panel), which appears to directly activate and permanently uncouple Galpha q subunits from receptors (25, 26); compared with the typical TRH-induced inhibition of GIRK channel conductance in a control cell (closed circles), the inhibition by TRH was completely blocked in a representative cell treated with PMT (1 µg/ml, overnight; open circles). The degree of inhibition of GIRK channel conductance by TRH was >55% in nearly all control cells (38 of 41 cells tested; 93%), whereas it reached that level in only three PMT-treated cells (of 15 cells tested; 20%); as shown in Fig. 1B (right panel), the averaged percent inhibition of GIRK channel conductance by TRH was significantly decreased by PMT (34.5 ± 5.7%, n = 15) compared with control cells (72.6 ± 2.0%, n = 41; p < 0.05). Thus, these data suggest that TRH-R1 couples to Galpha q family subunits to mediate inhibition of GIRK channels.

Galpha q Family Subunits and Gbeta 5-containing Gbeta gamma Dimers Are Capable of GIRK Channel Inhibition-- Receptor-mediated modulation of native GIRK channel currents is invariably associated with Galpha q-coupled receptors (reviewed in Ref. 1). To determine if members of the Galpha q family can cause inhibition of GIRK channels independent of receptor activation, we transfected constitutively active (i.e. GTPase-deficient, Gln right-arrow Leu mutant) Galpha q* subunits into G1/4 cells. We chose to use constitutively active Galpha subunits because effects of wild-type Galpha subunits could result from promiscuous coupling to receptors; from activity due to intrinsic GDP/GTP cycling; or indirectly, from effects due to sequestering free Gbeta gamma subunits.

As evident in the sample records from representative cells shown in Fig. 2A, basal GIRK channel currents were substantially smaller in a cell transfected with FLAG epitope-tagged Galpha q* compared with a control G1/4 cell. Currents were essentially identical in cells transfected with either FLAG-tagged or untagged Galpha q*, and data from experiments with those constructs were combined in subsequent analyses. As shown in the averaged data of Fig. 2C, GIRK channel currents were significantly decreased in G1/4 cells transfected with Galpha q*; indeed, the conductance in cells transfected with Galpha q* approximated that seen in parental HEK293 cells not expressing GIRK channels (data not shown), suggesting that channel activity was essentially completely eliminated by Galpha q*. Likewise, GIRK channel currents were drastically reduced in cells transfected with constitutively active mutants of all other Galpha q family members tested, including Galpha 11*, Galpha 14*, and Galpha 15* (Fig. 2C). Expression of GIRK channel subunits was apparently unaffected by Galpha q* transfection inasmuch as we found no difference in GIRK1 levels by Western blot analysis (Fig. 2A, inset). In addition, this inhibitory effect was specific to Galpha q family subunits since GIRK channel currents were unaffected by representative constitutively active members of all other major classes of Galpha proteins. These included all members of the Galpha i/o family (Galpha i1*, Galpha i2*, Galpha i3*, and Galpha o*) as well as the PTx-insensitive Galpha s*, Galpha 12*, Galpha 13*, and Galpha z* subunits (Fig. 2C).


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  		Fig. 2.   GIRK1/4 channel currents are inhibited by Galpha q family subunits and Gbeta 5-containing Gbeta gamma dimers. A, sample current traces from a representative control G1/4 cell and from a cell expressing FLAG-Galpha q*. The basal GIRK channel conductance was essentially completely eliminated in the cell expressing FLAG-Galpha q*. Inset, immunoblots of G1/4 cell lysates from control cells and from cells transfected with FLAG-Galpha q*. There was no effect on GIRK1 channel expression (upper panel) in cells expressing FLAG-Galpha q* (lower panel). B, sample current traces from a control G1/4 cell and from a cell expressing Gbeta 5gamma 2 dimers. Conductance was reduced in the cell expressing Gbeta 5gamma 2. C, averaged data (means ± S.E.) from cells expressing the indicated constitutively active Galpha subunits and Gbeta 5gamma 2. Asterisks denote statistically significant differences from control cells (p < 0.05 by ANOVA with post hoc Bonferroni's test; the number of cells in each group is shown in parentheses). Note that among constitutively active Galpha subunits, only members of the Galpha q family inhibited GIRK channel conductance; beta 5gamma 2 also inhibited GIRK channel conductance. NS, not significant.

Gbeta gamma subunits also convey signaling information to effectors following receptor activation (reviewed in Refs. 1, 2, and 8). Many Gbeta and Ggamma subunit genes have been identified by molecular cloning, and it is now well established that multiple different combinations of Gbeta gamma are capable of activating GIRK channels, including dimers composed of Gbeta 1, Gbeta 2, Gbeta 3, and Gbeta 4, together with various Ggamma subunits (12, 27, 28). By contrast, we recently found that Gbeta 5 forms Gbeta gamma dimers that cause inhibition, rather than the customary Gbeta gamma -mediated activation of GIRK channel currents (12). As shown in the representative records of Fig. 2B and in averaged data of Fig. 2C, GIRK channel currents were consistently smaller in cells transfected with Gbeta 5gamma 2 than in control cells not expressing exogenous G protein subunits. The observation that Gbeta 5-containing dimers are unique among Gbeta gamma dimers in causing GIRK channel inhibition is particularly cogent in the current context of GIRK channel inhibition by Galpha q-coupled receptors since Gbeta 5-containing dimers associate preferentially with Galpha subunits of the Galpha q family (13, 14). Together, these data indicate that Galpha q family subunits and Gbeta gamma dimers that contain Gbeta 5 are capable of inhibiting GIRK channel currents.

Receptor-mediated GIRK Channel Inhibition Involves Galpha q, but Does Not Require Gbeta gamma Dimers-- Our experiments using chronic exogenous expression of G protein subunits appear to constrain the possible mediators of GIRK channel inhibition either to Galpha subunits of the Galpha q family or to Gbeta gamma dimers containing Gbeta 5 (12). The next series of experiments was designed to determine the role of these subunits in acute receptor-mediated inhibition of GIRK channels.

First, we examined TRH-induced inhibition of GIRK channel currents in cells transfected with either Galpha q* or Gbeta 5gamma 2, as shown in Fig. 3. Consistent with the results presented above, expression of both Gbeta 5gamma 2 and Galpha q* inhibited basal GIRK channel currents. When challenged with TRH, however, GIRK channel currents were further diminished in Gbeta 5gamma 2-transfected cells, but not in Galpha q*-transfected cells. When inhibition was expressed as percent of control (Fig. 3, inset), it was clear that TRH inhibited GIRK channel currents equally effectively in control and Gbeta 5gamma 2-expressing cells. These data are consistent with the possibility that TRH receptor-mediated GIRK channel inhibition was occluded by Galpha q*, but not by Gbeta 5gamma 2.


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  		Fig. 3.   Effect of Galpha q* and Gbeta 5gamma 2 on TRH-induced inhibition of GIRK channel currents. The effect of TRH was tested in control cells (n = 41) and in cells transfected with either Gbeta 5gamma 2 (n = 8) or Galpha q* (n = 9). Averaged data (means ± S.E.) show basal conductance (black bars) and conductance in the presence of TRH (gray bars). Inset, TRH-induced inhibition of conductance (percent of control conductance; means ± S.E.). Asterisks denote statistically significant differences from control cells (p < 0.05 by ANOVA with post hoc Bonferroni's test). Inhibition of GIRK channel currents was preserved in cells expressing Gbeta 5gamma 2, but not in cells expressing Galpha q*.

It is important to note that, although TRH was without effect on GIRK channel currents in Galpha q*-expressing cells, the basal GIRK channel currents were essentially eliminated even before TRH application. Thus, although the results are consistent with the interpretation that Galpha q* occluded receptor inhibition, it is also possible that chronic overexpression of Galpha q* inhibited GIRK channel currents through a separate mechanism and that TRH was without effect simply because there was no residual basal GIRK channel current to inhibit. Therefore, we performed additional experiments in which cells were transfected with minigene constructs designed to interfere with signaling via either Galpha q or Gbeta gamma subunits (Fig. 4).


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  		Fig. 4.   Receptor-mediated GIRK channel inhibition involves Galpha q. A, the effect of TRH on GIRK channel conductance in representative cells transfected with the Galpha q sinks PLCbeta 1-ct (left panel) and RGS2 (right panel). Note that TRH had little effect on conductance in cells expressing either of these constructs, which interfere with signaling via Galpha q family subunits. B, the effect of TRH on GIRK channel conductance in cells expressing the Gbeta 5 sinks RGS6 and RGS11, which inhibit signaling by Gbeta 5-containing Gbeta gamma dimers (21). TRH caused a strong inhibition of GIRK channel conductance in these cells. C, summary data showing inhibition of GIRK channel conductance by TRH (percent of control conductance; means ± S.E.) in cells transfected with constructs that disrupt signaling by Galpha q family subunits (i.e. Galpha q sinks) or Gbeta gamma dimers (i.e. Gbeta gamma sinks). Asterisks denote statistically significant differences from control cells (p < 0.05 by ANOVA with post hoc Bonferroni's test; the number of cells in each group is indicated in parentheses). Note that inhibition of GIRK channel currents by TRH was significantly diminished by the Galpha q sinks, but not by the Gbeta gamma sinks, consistent with a primary role for Galpha q in mediating the effects of TRH.

We chose to use two different inhibitors of Galpha q signaling: RGS2 and a GFP-tagged C-terminal construct of PLCbeta 1 (18). Both of these constructs selectively bind activated Galpha q-like proteins, and their overexpression interferes specifically with receptor signaling mediated by Galpha q (18, 29, 30). Although each of these constructs exhibits GTPase activity, the inhibition of signaling appears to be independent of that activity and due rather to their ability to compete with effectors for Galpha q binding (i.e. as "sinks" or "effector antagonists") (30). We tested the effect of TRH on GIRK channel currents in cells transfected with these two different inhibitors of Galpha q signaling. As is evident in the sample records provided in Fig. 4A, the inhibition of GIRK channel currents by TRH was diminished by PLCbeta 1-ct or RGS2. Indeed, unlike the control condition, in which GIRK channel current inhibition was >55% in the overwhelming majority of cells (38 of 41 cells tested; 93%), inhibition by TRH was >55% in only 3 of 25 of cells transfected with PLCbeta 1-ct (12%) and in just 3 of 16 cells transfected with RGS2 (19%). This was also borne out in summary data from these cells, where the averaged percent inhibition by TRH was significantly reduced by both constructs (Fig. 4C). These data indicate that inhibition of GIRK channels mediated by the TRH receptor involves signaling by Galpha q.

In contrast to the abrogating effects of the minigene inhibitors of Galpha q signaling, we found that receptor-mediated GIRK channel inhibition was largely preserved in cells transfected with a number of different Gbeta gamma buffers. First, we overexpressed two RGS proteins, RGS6 and RGS11, which, by virtue of their Ggamma -like domains that bind specifically to Gbeta 5 (19, 20), can interfere with signaling mediated by Gbeta 5-containing Gbeta gamma pairs (21). As shown in Fig. 4B, TRH produced a strong inhibition of GIRK channel currents in cells transfected with either of the Ggamma -like domain-containing RGS proteins; the averaged percent inhibition in cells transfected with RGS6 and RGS11 was not different from that in control cells (Fig. 4C). To rule out possible involvement of other Gbeta gamma dimers in receptor-mediated GIRK channel inhibition (e.g. by their ability to activate PLC) (8), we tested two additional, relatively nonselective Gbeta gamma sinks, wild-type Galpha t and beta ARK-ct. Neither had any effect on TRH-induced inhibition of GIRK channel currents (Fig. 4C). Attesting to the efficacy of Gbeta gamma sequestration by Galpha t and beta ARK-ct, the basal GIRK channel conductance was significantly reduced in Galpha t- and beta ARK-ct-expressing cells (7.1 ± 0.7 nanosiemens in control cells versus 3.7 ± 0.6 and 2.2 ± 0.4 nanosiemens in Galpha t- and beta ARK-ct-expressing cells, respectively; both p < 0.05) (see also Ref. 12); and moreover, expression of each completely blocked GIRK channel activation by 5-HT1A receptors, a known Gbeta gamma -mediated effect (data not shown). Thus, these data indicate that GIRK channel inhibition by TRH-R1 is due, at least in large part, to Galpha q rather than Gbeta gamma signaling.

Receptor-mediated GIRK Channel Inhibition Involves PLC-- In light of the above results that suggest a primary role for Galpha q signaling in receptor-mediated GIRK channel inhibition, we tested if activation of PLC, a well known Galpha q effector, might also play a role. As illustrated in Fig. 5, TRH-induced inhibition of GIRK channels was markedly diminished in cells pretreated with the PLC inhibitor U73122, but not with the control compound U73343 (both at 1 µM for at least 30 min.). The inhibition by TRH was >55% in only three cells and averaged just 35.7 ± 4.4% (n = 19) during U73122 exposure, significantly less than the inhibition recorded in the presence of U73343 (75.2 ± 3.9%, n = 18) or in control cells that were not pretreated with either compound (72.6 ± 2.0%, n = 41).


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  		Fig. 5.   Receptor- and Galpha q*-mediated GIRK channel inhibition involves PLC. A, the effect of TRH on GIRK channel currents in representative cells pretreated (1 µM, >30 min) with the PLC inhibitor U73122 (left panel) or its inactive analog, U73343 (right panel). Note that TRH had little effect in the continued presence of U73122, whereas it strongly inhibited GIRK channel conductance during exposure to the control U73343 compound. B, averaged TRH-induced inhibition (percent of control conductance; means ± S.E.) in control cells and in cells treated with U73122 and U73343. C, averaged conductance (means ± S.E.) in control cells treated with U73122 (1 µM, ~36-48 h) compared with that in Galpha q*-transfected cells that were untreated or treated with U73122 or U73343. GIRK channel conductance was smaller in all cells transfected with Galpha q*, but this effect was significantly diminished in cells treated with the PLC inhibitor U73122 (i.e. the current reduction was not as great). *, statistically significant difference from control cells; Dagger , statistically significant difference different from Galpha q*-transfected cells (p < 0.05 by ANOVA with post hoc Bonferroni's test; the number of cells in each group is indicated in parentheses).

We also tested if PLC activation contributes to the sustained inhibition of GIRK channels obtained by expression of constitutively active Galpha q* (Fig. 5C). Throughout the period following transfection with Galpha q* (or empty vector), cells were incubated continuously with U73122 at 1 µM (~36-48 h). Note that we observed an apparent nonspecific inhibition of GIRK channels in control cells chronically treated with U73122 (see also Ref. 17). Thus, the averaged conductance in those U73122-treated cells was smaller than that seen in untreated control cells (3.1 ± 0.5 versus 9.2 ± 0.6 nanosiemens, respectively; p < 0.05) (compare Figs. 5C and 2C, first bars). Nevertheless, as shown in Fig. 5C, the averaged conductance was further reduced in all cells transfected with Galpha q*; and importantly, the reduction associated with expression of Galpha q* was significantly attenuated in cells treated with the PLC inhibitor U73122. As a control, inhibition of GIRK channel currents by Galpha q* was unaffected by identical treatment with the inactive analog, U73343. Thus, activation of PLC by TRH and Galpha q* contributes, at least in part, to GIRK channel inhibition.

Lowering Plasma Membrane PIP2 Levels Diminishes Receptor-mediated GIRK Channel Inhibition-- It has recently been established that the membrane phospholipid PI(4,5)P2 is an important coactivator of GIRK channels (9, 10, 31), and it has been suggested that PLC-mediated decreases in PI(4,5)P2 levels might account for GIRK channel inhibition following receptor activation in atrial cells (15-17). Since we found that TRH-induced inhibition of GIRK channel currents also appears to involve PLC, we tested the possibility that GIRK channel inhibition might be sensitive to membrane PI(4,5)P2 levels.

To this end, we used two minigenes that deplete membrane PI(4,5)P2 levels via different mechanisms (23). The first was a GFP-tagged fusion protein that includes the pleckstrin homology domain of PLCdelta , a construct that sequesters PI(4,5)P2; as a control for PLCdelta -PH, we used a different GFP-tagged pleckstrin homology domain derived from AKT (AKT-PH), which interacts with a separate isoform of the membrane phospholipid PI(3,4)P2. The second construct was a GFP fusion protein that included a constitutively active yeast 5'-PI-PTase that was targeted to the plasma membrane by incorporation of a myristoylation-palmitoylation sequence. When tested within 24 h of transfection, both the PLCdelta -PH and 5'-PI-PTase constructs are localized to the plasma membrane, and both lower membrane PI(4,5)P2 levels (23). As shown in the example records of Fig. 6, TRH caused very little inhibition of GIRK channel currents in cells expressing either the membrane-targeted 5'-PI-PTase (Fig. 6A, upper panel) or PLCdelta -PH (middle panel) construct, but was highly effective in cells expressing the control AKT-PH construct (lower panel). Indeed, whereas GIRK channel inhibition by TRH was >55% in nearly all control cells (38 of 41 cells tested; 93%) and AKT-PH-transfected cells (9 of 11 cells tested; 82%), TRH inhibition was >55% in only one 5'-PI-PTase-transfected cell (of 12 cells tested; 8%) and in just four PLCdelta -PH-transfected cells (of 15 cells tested; 27%). These differences were also reflected in summary data presented in Fig. 6B, in which the averaged TRH-induced inhibition of GIRK channel currents was significantly diminished in cells expressing the two inhibitors of PIP2 compared with either control cells or cells expressing AKT-PH.


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  		Fig. 6.   Receptor-mediated GIRK channel inhibition requires permissive levels of PIP2. A, the effect of TRH on GIRK channel conductance in representative cells transfected with 5'-PI-PTase (upper panel) and PLCdelta -PH (middle panel), two constructs that deplete membrane PI(4,5)P2, as well as a control AKT-PH construct that does not bind to PI(4,5)P2 (lower panel). Note that TRH had little effect on conductance in cells expressing either of the constructs that lower membrane PI(4,5)P2 levels, but was highly effective in the cell expressing the control construct that does not bind PI(4,5)P2. B, summary data quantifying the effects of TRH on GIRK channel currents in cells transfected with the indicated constructs. Note that relative to the control, inhibition of GIRK channel currents by TRH was significantly diminished by the two constructs that lower membrane PI(4,5)P2 levels (5'-PI-PTase and PLCdelta -PH), but not by the control construct (AKT-PH). Cells expressing AKT-PH, 5'-PI-PTase, and PLCdelta -PH were recorded within 24 h of transfection (23). Asterisks denote statistically significant differences from control cells (p < 0.05 by ANOVA with post hoc Bonferroni's test; the number of cells in each group is indicated in parentheses).

It should be pointed out that the basal GIRK channel conductance tended to be somewhat lower in cells transfected with 5'-PI-PTase or PLCdelta -PH compared with control cells (~4 versus ~7 nanosiemens), as expected given the demonstrated facilitating effects of PI(4,5)P2 on GIRK channels (9-11). In many cells, however, a substantial basal GIRK channel conductance was retained (e.g. see records in Fig. 6A, upper and middle panels), suggesting that differences in initial current amplitudes could not account for the diminished effect of TRH. It is also noteworthy that PLCdelta -PH can bind IP3 (32), a product of PLC-mediated hydrolysis. Although it is therefore conceivable that buffering IP3 could be responsible for inhibitory effects of the PLCdelta -PH construct (but see below), this could not account for the effects of 5'-PI-PTase. Thus, depletion of membrane PI(4,5)P2 levels appears to disrupt receptor-mediated inhibition of GIRK channel currents.

Receptor-mediated Inhibition of GIRK Channel Currents Is Independent of PKC or IP3-- The data presented up to this point indicate a role for PLC and its substrate, PI(4,5)P2, in GIRK channel inhibition by TRH receptors. Therefore, we tested if signaling pathways typically activated as a result of PLC-mediated PI(4,5)P2 hydrolysis are involved in GIRK channel current inhibition. We were particularly interested in investigating a role for PKC since it has earlier been implicated in inhibition of recombinant GIRK channels (Ref. 33, but see Ref. 34). As illustrated in Fig. 7A, neither the PKC-activating phorbol ester PDBu nor its inactive analog, 4alpha -PDBu, had any effect on GIRK channel currents (both applied via the perfusate at 1 µM). Because some PKC isozymes are insensitive to phorbol esters, we also tested if a competitive inhibitor of ATP binding to the catalytic site of PKC, the bisindolylmaleimide compound GF 109203X, could interfere with TRH-induced inhibition of GIRK channel currents. As depicted in the records from the representative cell shown in Fig. 7B (left panel), TRH was highly effective at inhibiting GIRK channel currents in cells pretreated with bisindolylmaleimide (5 µM for >= 30 min). Indeed, TRH inhibited 81.1 ± 2.6% of the initial GIRK channel conductance in bisindolylmaleimide-treated cells (n = 8), clearly not less than the TRH-induced inhibition in control cells (~73%). In control experiments using HEK293 cells expressing mouse TREK-1 channels, we found that PDBu strongly inhibited TREK-1 currents and that bisindolylmaleimide blocked those effects of PDBu, attesting to the efficacy of both PDBu and bisindolylmaleimide compounds (data not shown). These data argue against a role for PKC in TRH receptor-mediated inhibition of GIRK channel currents.


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  		Fig. 7.   Receptor-mediated GIRK channel inhibition is independent of PKC activation and intracellular IP3. A, the effect on GIRK channel conductance of a PKC-activating phorbol ester (PDBu) and its inactive analog (4alpha -PDBu) in a representative cell (left panel). The compounds were applied in the bath (1 µM) as indicated; neither had any effect on GIRK channel currents, as is also apparent in averaged data from cells treated with the two phorbol ester compounds (percent of control conductance; means ± S.E.) (right panel). B, the effect of a bisindolylmaleimide inhibitor of PKC (GF 109203X) on TRH-induced inhibition of GIRK channel currents in a representative cell pretreated with 5 µM bisindolylmaleimide for >=  30 min (left panel). TRH caused a strong inhibition of GIRK channel currents in this bisindolylmaleimide-treated cell; averaged values for the magnitude of TRH-induced inhibition (percent of control conductance; means ± S.E.) indicated that it was equal in magnitude in control and bisindolylmaleimide-treated cells (right panel). C, the effect of IP3 (100 µM in the whole cell recording pipette) on GIRK channel conductance and on TRH-mediated inhibition of GIRK channel conductance in a representative cell (left panel). The conductance at all time points (Gt) was normalized (norm.) to the initial conductance (Gi) to illustrate the close correspondence of the current rundown and the inhibition by TRH in control and IP3-treated cells. Averaged data show the magnitude of TRH-induced inhibition (percent of control conductance; means ± S.E.) in these experiments and indicate that there was no difference between control cells and those recorded with IP3 in the pipette (right panel).

Activation of PLC also leads to production of IP3 and increases in intracellular calcium, either of which could conceivably lead to GIRK channel inhibition. It is unlikely that changes in intracellular calcium contribute to the effects of TRH since our whole cell experiments were performed with intracellular calcium buffered to ~10-8 M by including 10 mM EGTA in the internal solution. To determine if IP3 could have effects independent of altering intracellular calcium, we introduced IP3 into cells by including it in the recording pipettes (100 µM). As illustrated in the example cells of Fig. 7C, we found that the time- and TRH-dependent decreases in GIRK channel current in IP3-dialyzed cells were not different from those in control cells recorded with pipettes that did not contain IP3. As is evident in the averaged data of Fig. 7C, the TRH-induced inhibition of GIRK channel currents in IP3-containing cells (75.5 ± 4.1%, n = 7) was essentially identical to that in control cells (i.e. ~73%). Thus, intracellular perfusion with IP3 neither mimicked nor occluded the TRH receptor-mediated inhibition of GIRK channel currents.

Receptor-mediated GIRK Channel Inhibition Involves a Readily Diffusible Messenger-- Our experiments provide no evidence to implicate the major signaling pathways downstream of PLC (either PKC or IP3/Ca2+) in receptor-mediated GIRK channel inhibition. To test whether other diffusible messengers might be involved, we recorded the effects of bath-applied TRH on GIRK channel activity in cell-attached patches. In this recording configuration, the agonist does not have direct access to channels recorded within the patch. Therefore, any actions of the agonist must be transmitted by some intermediary that can move between the channels inside the patch and the receptors outside the patch, either via the cytosol or within the plane of the membrane.

Cell-attached patch recordings of K+ channels were obtained in G1/4 cells either from the cell line stably expressing the TRH-R1 and 5-HT1A receptors (Fig. 8A) or from the parental G1/4 cell line, which does not express these receptors (Fig. 8B). Single channel currents were clearly inwardly rectifying (data not shown), with a unitary conductance of ~40 picosiemens (39.0 ± 1.4 picosiemens, n = 5) and a mean open time of ~1 ms (0.9 ± 0.1 ms, n = 9) (Fig. 8C), as expected for GIRK1/4 channels (35). Moreover, when exposed to extracellular 5-HT in an inside-out patch configuration, the channels were vigorously activated by adding GTP to the inside face of the patch (data not shown). These are defining properties of GIRK channels (1, 2, 8).


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  		Fig. 8.   Receptor-mediated GIRK channel inhibition involves a readily diffusible messenger. A: the effect of TRH on GIRK channels recorded in a cell-attached patch of G1/4R cells stably expressing TRH-R1. Left panel, GIRK channel activity was quantified as NPo (in 1-s bins) and plotted as a function of time (lower trace); TRH was added to the perfusate for the indicated period. The patch holding potential is provided (upper trace). Note that bath-applied TRH caused a strong inhibition of channel activity within the patch. This was true even after the patch potential was adjusted (to Em -60 mV) to correct for the anticipated TRH-induced depolarization of cell membrane potential (30 mV). For clarity, periods of transient instability in the patch recordings were blanked (indicated by stars). Right panel, shown are the continuous records (~1 s) of GIRK channel activity under control conditions (at a patch potential of Em -30 mV) and in the presence of TRH (at a patch potential of Em -60 mV). Note that the membrane potential correction resulted in current amplitudes in TRH that approximated those in the control (see the dashed line). B: the effect of TRH on GIRK channels recorded in a cell-attached patch of G1/4 cells not expressing TRH-R1. Left panel, GIRK channel activity was quantified as NPo (in 1-s bins) and plotted as a function of time (lower trace); TRH was added to the perfusate for the indicated period. The patch holding potential is provided (upper trace), and periods of brief patch instabilities were blanked (stars). As expected, TRH had no effect in control cells that do not have TRH receptors. Right panel, shown are continuous records (~1 s) of GIRK channel activity under control conditions and in the presence of TRH (at a patch potential of Em -30 mV). C: left panel, shown is a schematic of the experimental configuration. To modulate channels contained within the patch, activation of receptors outside the patch must either produce an inhibitory mediator that can diffuse to the channels or induce the withdrawal of an activating substance from the patch. Middle panel, shown are the averaged data (percent inhibition of NPo by TRH; means ± S.E.) in patches from G1/4R cells (i.e. with TRH-R1; n = 10) and from control G1/4 cells (i.e. without TRH-R1; n = 6). TRH inhibited channel activity by ~68%, similar to the TRH-induced inhibition of whole cell GIRK channel currents. Right panel, mean channel open time (±S.E.) was determined before and during TRH application in those patches of G1/4R cells that showed a robust response to TRH (n = 8). Note that TRH caused a significant decrease in mean open time (determined by paired t test).

As illustrated in Fig. 8A (left panel), GIRK channel activity (i.e. NPo) in a cell-attached patch from a TRH-R1-expressing G1/4 cell was strongly diminished by bath-applied TRH. Based on our whole cell recordings, we expect that the TRH-induced decrease in basal GIRK channel currents would cause a membrane depolarization of ~30 mV. To correct for effects of this membrane depolarization on the patch potential, we adjusted the patch holding potential by -30 mV in the presence of TRH. This adjustment was apparently appropriate since the unitary current was approximately the same amplitude in the presence of TRH, after correcting for the membrane depolarization (see dashed line in sample records of Fig. 8A, right panel). Furthermore, since channel activity remained strongly inhibited even after this correction, the inhibition by TRH could not be attributed solely to a TRH-induced change in membrane potential. TRH inhibited GIRK channel activity by >55% in 8 of 10 cell-attached patches, with an average inhibition in all patches of ~68% (NPo decreased from 0.09 ± 0.02 to 0.03 ± 0.02, n = 10; p < 0.05) (Fig. 8C, middle panel), which is very similar to the magnitude of TRH-induced inhibition of whole cell GIRK channel currents. In those patches with a robust inhibition of channel activity by TRH (n = 8), the inhibition involved, at least in part, a decrease in mean channel open time (Fig. 8C, right panel). As expected, there was no effect of TRH on GIRK channel activity in cell-attached patches from control G1/4 cells that did not express TRH-R1, as shown in Fig. 8 (B and C, middle panel). These data therefore indicate that GIRK channel inhibition involves a readily diffusible messenger.

    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we examined dual regulation of GIRK channels using mammalian cell lines that express GIRK1/4 channels together with two G protein-coupled receptors that couple preferentially to distinct classes of Galpha subunit. Unlike GIRK channel activation by Galpha i/o-coupled receptors, which is mediated by Gbeta