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Am J Physiol Cell Physiol 295: C1417-C1426, 2008. First published September 24, 2008; doi:10.1152/ajpcell.00341.2008
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RECEPTORS AND SIGNAL TRANSDUCTION

The monomeric G proteins AGS1 and Rhes selectively influence G{alpha}i-dependent signaling to modulate N-type (CaV2.2) calcium channels

Ashish Thapliyal,* Roger A. Bannister,* Christopher Hanks, and Brett A. Adams

Department of Biology, Utah State University, Logan, Utah

Submitted 1 July 2008 ; accepted in final form 17 September 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Activator of G protein Signaling 1 (AGS1) and Ras homologue enriched in striatum (Rhes) define a new group of Ras-like monomeric G proteins whose signaling properties and physiological roles are just beginning to be understood. Previous results suggest that AGS1 and Rhes exhibit distinct preferences for heterotrimeric G proteins, with AGS1 selectively influencing G{alpha}i and Rhes selectively influencing G{alpha}s. Here, we demonstrate that AGS1 and Rhes trigger nearly identical modulation of N-type Ca2+ channels (CaV2.2) by selectively altering G{alpha}i-dependent signaling. Whole-cell currents were recorded from HEK293 cells expressing CaV2.2 and G{alpha}i- or G{alpha}s-coupled receptors. AGS1 and Rhes reduced basal current densities and triggered tonic voltage-dependent (VD) inhibition of CaV2.2. Additionally, each protein attenuated agonist-initiated channel inhibition through G{alpha}i-coupled receptors without reducing channel inhibition through a G{alpha}s-coupled receptor. The above effects of AGS1 and Rhes were blocked by pertussis toxin (PTX) or by expression of a Gβ{gamma}-sequestering peptide (masGRK3ct). Transfection with HRas, KRas2, Rap1A-G12V, Rap2B, Rheb2, or Gem failed to duplicate the effects of AGS1 and Rhes on CaV2.2. Our data provide the first demonstration that AGS1 and Rhes exhibit similar if not identical signaling properties since both trigger tonic Gβ{gamma} signaling and both attenuate receptor-initiated signaling by the Gβ{gamma} subunits of PTX-sensitive G proteins. These results are consistent with the possibility that AGS1 and Rhes modulate Ca2+ influx through CaV2.2 channels under more physiological conditions and thereby influence Ca2+-dependent events such as neurosecretion.

voltage-gated calcium channels; Ras-like; Dexras1; Dexras2; RASD1; RASD2

ACTIVATOR OF G PROTEIN SIGNALING 1 (AGS1; Dexras1; RASD1) and Ras homologue enriched in striatum (Rhes; Dexras2; RASD2) define a distinct group of Ras-like monomeric G proteins (2). At the amino acid level, AGS1 is ~35% identical to each of the major Ras subfamilies (8) and ~62% identical to Rhes (13). AGS1 is expressed at high concentrations in brain and at lower concentrations in heart, liver, kidney, skeletal muscle, pancreas, and placenta (14, 23, 37). Rhes is expressed prominently in the striatum and other brain regions, and also in thyroid and pancreatic islets (4, 32, 39). Both proteins are under hormonal control, with AGS1 being upregulated by dexamethasone and estradiol (3, 23, 37) and Rhes being upregulated by thyroid hormones (13, 38). Although their physiological functions remain to be fully elucidated, AGS1 has been implicated in photic and nonphotic responsiveness of the circadian clock (7), whereas Rhes has been implicated in striatal dopaminergic signaling (12, 18) and motor coordination (33).

Several signaling properties of AGS1 have been identified. In yeast, AGS1 activated the pheromone response pathway independently of G protein-coupled receptors (GPCRs) and interacted with G{alpha}i but not with G{alpha}s or G{alpha}16 (9). In vitro, AGS1 functioned as a guanine nucleotide exchange factor (GEF) for purified G{alpha}i monomers (8). In Xenopus oocytes and in COS-7 cells, AGS1 increased the basal activities of GIRK ion channels and of the MAP kinase ERK1/2, and attenuated activation of these effectors by G{alpha}i-coupled GPCRs (8, 16, 35). In HEK293 cells, AGS1 blocked ERK1/2 activation and the sensitization of adenylyl cyclase 1 (AC1) without reducing G{alpha}i-mediated inhibition of AC1 through G{alpha}i-coupled GPCRs (28). All of the above effects of AGS1 were prevented by pertussis toxin (PTX) or by expression of Gβ{gamma}-sequestering peptides. Together these results indicate that AGS1 promotes tonic Gβ{gamma}-dependent signaling and interferes with the ability of GPCRs to trigger Gβ{gamma}-dependent signaling by PTX-sensitive heterotrimeric G proteins.

Much less is known about Rhes. On the basis of their sequence similarity, AGS1 and Rhes should be expected to have very similar signaling properties. It is therefore surprising that, on the one hand, Rhes failed to increase basal ERK1/2 activity in COS-7 cells and failed to attenuate serum response element (SRE)-dependent activation of a reporter gene following activation of G{alpha}i-coupled M2 muscarinic acetylcholine receptors in PC12 cells (39). On the other hand, Rhes significantly attenuated the cAMP-dependent activation of a reporter gene in PC12 cells following activation of thyroid stimulating hormone (TSH) or β2-adrenergic (β2AR) receptors. Because the preceding receptors are known to couple to G{alpha}s, the latter results were taken to indicate that Rhes selectively interferes with G{alpha}s-mediated signal transduction (39). Rhes has been shown to influence dopaminergic signaling in striatum (12, 18), but it remains unclear whether Rhes interacts with G{alpha}s-coupled D1 receptors or G{alpha}i-coupled D2 receptors. In contrast to AGS1, previous studies have not reported that Rhes triggers tonic Gβ{gamma} signaling. Altogether, results presented thus far have suggested that AGS1 and Rhes have distinct signaling properties.

CaV2 channels are inhibited by the Gβ{gamma} subunits of heterotrimeric G proteins, and such inhibition is voltage dependent (VD), meaning that it is transiently relieved by membrane depolarization (19, 21). Potentially, any protein that functions as a GEF or that modifies signal processing through heterotrimeric G proteins could alter CaV2 channel activity and thereby influence downstream Ca2+-dependent events such as neurosecretion. Relatively little is known about modulation of voltage-gated Ca2+ channels (CaV) by monomeric G proteins. In this work, we demonstrate that AGS1 and Rhes produce similar modulation of N-type (CaV2.2) channels. Both proteins trigger tonic, VD inhibition of CaV2.2 and both attenuate channel inhibition through GPCRs that couple to PTX-sensitive G proteins. Furthermore, both monomeric G proteins fail to attenuate channel inhibition through G{alpha}s-coupled β2-adrenergic receptors. Our findings indicate that AGS1 and Rhes have similar, if not identical, signaling properties, and they additionally suggest that AGS1 and Rhes may modulate physiological Ca2+ signaling by CaV2 family channels.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and transfection. Human embryonic kidney (HEK293) cells were obtained from the American Type Culture Collection (Manassas, VA) and propagated in culture medium containing 90% DMEM (GIBCO-BRL; cat. no. 11995), 10% defined fetal bovine serum (HyClone Laboratories, Logan, UT), and 50 µg/ml gentamycin. Cells were replated every few days onto 60-mm culture dishes at ~20% confluence. CaPO4 precipitation (CellPhect kit, GE Healthcare) was used to transfect recently plated cells. The transfection mixture included expression plasmids encoding N-type Ca2+ channel subunits (CaV2.2, {alpha}2{delta}1b, and β3) at 1.25 µg each per dish. Transfections also included a plasmid encoding the M2 muscarinic acetylcholine receptor (0.025 µg/dish), the {kappa}-opioid receptor ({kappa}OR; 0.1 µg/dish) or the β2-adrenergic receptor (0.625 µg/dish). The quantities of transfected receptor plasmid were empirically determined to support maximal agonist-dependent channel inhibition. Plasmids encoding AGS1, Rhes, or other monomeric G proteins (Gem, HRas, KRas2, Rap1A-G12V, Rap2B, or Rheb2) were included at 1.25 µg/dish.

To control for expression of wild-type AGS1 or Rhes, we omitted the plasmids encoding these proteins from the transfection mixture or substituted plasmids encoding inactive AGS1 mutants. In the {Delta}CAAX mutant of AGS1, the final four amino acids (Cys-Val-Iso-Ser) of the carboxy terminus have been deleted; these residues comprise a conserved CAAX motif essential for plasma membrane localization (8, 17, 23, 37). In AGS1-G31V, glycine 31, located within the conserved P-loop and critical for GTP binding and hydrolysis, is substituted by valine (9, 35).

In selected experiments, the transfection mixture included (at 1.25 µg per dish) a plasmid encoding the carboxy terminal region (amino acids 495-688) of G protein-coupled receptor kinase 3 (Grk3ct) fused in-frame at its amino terminus to a membrane-associating myristic acid attachment sequence (MGSSKSK) to increase its association with the plasma membrane; this construct is termed masGRK3ct. Transfections also included a plasmid (pEGFP-C3) encoding enhanced green fluorescence protein (at 0.125 µg/dish) as an expression marker. The day following transfection, cells were briefly trypsinized, replated onto 12-mm round glass coverslips, and incubated overnight at 37°C. Electrophysiological recordings were performed 24–36 h later. Successfully transfected cells were visually identified by their green fluorescence under UV illumination. Recordings were obtained exclusively from green cells.

DNA constructs. Complementary DNA encoding rabbit brain CaV2.2 (GenBank accession number D14157) was in the expression vector pKCRH2. Human {kappa}OR (AF498922 [GenBank] ), human β2AR (J02960 [GenBank] ), human wild-type AGS1 (AF498923 [GenBank] ), AGS1-G31V, human Rap1A-G12V constitutively active mutant (AF493912 [GenBank] ), human HRas (AF493916 [GenBank] ), human KRas2 (AF493917 [GenBank] ), human Rap2B (AF493915 [GenBank] ), human Rheb2 (AF493921 [GenBank] ), human Rhes (BC013419 [GenBank] ), and bovine masGRK3ct (NM_174500 [GenBank] ) were in pcDNA3.1+ or pcDNA3.1– (Invitrogen, Carlsbad, CA). Rat brain {alpha}2{delta}1b (M86621 [GenBank] ) was in pMT2 (Genetics Institute, Cambridge, MA). Rabbit brain β3 (X64300 [GenBank] ) was in pcDNA3 (Invitrogen); rat brain β2a (M80545 [GenBank] ) was in p91023(b). Human M2 muscarinic acetylcholine receptor (X15264 [GenBank] ) was in pRK5. Mouse Gem (NM_010276 [GenBank] ) was in pRc-CMV (Invitrogen). Jellyfish enhanced green fluorescent protein (EGFP) was in pEGFP-C3 (U55763 [GenBank] ; Clontech, Cambridge, UK).

{Delta}CAAX was constructed by using high-fidelity PCR to amplify the entire coding sequence of AGS1, excluding the final four codons. The primers included restriction sites that enabled unidirectional cloning of the amplicon into pcDNA3.1 or pIRES2-EGFP. Untagged AGS1-G31V was constructed using high-fidelity PCR to amplify the coding sequence of His-tagged AGS1-G31V. The PCR product was ligated into pcDNA3.1–. Both AGS1 mutants were fully sequenced to confirm the absence of unintended mutations.

Electrophysiological recordings. Large-bore patch pipettes were pulled from 100-µl borosilicate glass micropipettes (VWR 53432-921) and filled with a solution containing (in mM) 155 CsCl, 10 Cs2EGTA, 4 MgATP, 0.32 LiGTP, and 10 HEPES, with pH adjusted to 7.4 by use of CsOH. Aliquots of the pipette solutions were stored at –80°C, kept on ice after thawing and filtered at 0.22 µm immediately before use. Filled pipettes had resistances of 1.0–1.5 M{Omega}. Pipette tips were coated with paraffin to reduce capacitance. The bath solution contained (in mM) 145 NaCl, 40 CaCl2, 2 KCl, and 10 HEPES, with pH adjusted to 7.4 with NaOH. Stock solutions of carbachol (CCh), dynorphin A and isoproterenol (Iso) were dissolved directly in the bath solution. Agonists were applied by bulk exchange of the bath, which was continuously perfused at a rate of 2–3 ml/min throughout recordings. Agonist application was complete within 1–3 s whereas washout typically required ~30 s. Experiments were performed at room temperature (20–24°C). Bath temperature was monitored by using a miniature thermocouple placed in the chamber outlet; temperature did not vary more than ± 1.0°C during agonist application and washout. CCh, Iso, and other chemicals were purchased from Sigma (St. Louis, MO). Dynorphin A was purchased from Calbiochem (La Jolla, CA). All reagents were the highest grade available.

After forming a gigaohm seal in the cell-attached configuration, residual pipette capacitance was compensated using the analog circuit of the amplifier. Currents were recorded using the whole cell, ruptured-patch configuration. The direct current resistance of the whole cell configuration was routinely >1 G{Omega}. The steady holding potential was –90 mV. No corrections were made for liquid junction potentials. Currents were filtered at 2–10 kHz by using the built-in Bessel filter (four-pole low-pass) of the amplifier (Axopatch 200B, Axon Instruments, Foster City, CA) and sampled at 10–50 kHz by use of a Digidata 1200 or 1320 analog-to-digital board installed in a Gateway Pentium computer. The pCLAMP 8.0 software programs Clampex and Clampfit were used for data acquisition and analysis, respectively. Figures were made using Microcal Origin (version 6.0).

Linear cell capacitance (C) was determined by integrating the area under the whole cell capacity transient, which was evoked by a 10-ms voltage-clamp step from –90 to –80 mV. The average value of C was 27 ± 1 pF (means ± SE; n = 245 cells). To minimize voltage errors, the time constant for decay of the whole cell capacity transient ({tau}) was reduced as much as possible by using the analog compensation circuit of the amplifier. Series resistance (RS) was calculated as the product of {tau} and 1/C. The average values of {tau} and RS, measured before electronic compensation, were 110 ± 4 µs and 4.4 ± 0.2 M{Omega}, respectively. After compensation these values were 85 ± 3 µs and 3.4 ± 0.1 M{Omega}, respectively. Maximal Ca2+ current amplitude was 939 ± 59 pA. The average maximum voltage error was 3.1 ± 0.2 mV.

Currents were corrected for linear capacitance and leakage currents by –P/4 or –P/6 subtraction. Ca2+ current amplitudes were measured at the time of peak inward current. Statistical comparisons were performed by an unpaired, two-tailed t-test (when comparing two groups) or by one-way ANOVA (when comparing three or more groups). A probability value (P value) <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
AGS1 and Rhes reduce basal densities of CaV2.2 currents. Figure 1A illustrates representative whole cell Ca2+ currents recorded from HEK293 cells transfected with CaV2.2 channels and M2 muscarinic acetylcholine receptors (M2R), or these proteins together with AGS1 or Rhes (Fig. 1B). In these experiments, we initially recorded "basal" Ca2+ currents from cells not previously exposed to CCh, the acetylcholine receptor agonist used later in this study. In one set of experiments, we found that basal currents (evoked at +30 mV) had a density of 26 ± 5 pA/pF (n = 25) in AGS1-transfected cells but significantly higher (44 ± 8 pA/pF; n = 23) in matched controls (P < 0.05). Basal current density was similarly reduced by Rhes (14 ± 1 pA/pF, n = 16) compared with that (33 ± 11 pA/pF, n = 9) in matched control cells (P < 0.05). Overall, AGS1 and Rhes reduced basal current density in these experiments by 41 and 58%, respectively. It is important to note that expression of CaV2.2 in HEK293 cells varies among transfections (cf. Table 1). Nevertheless, over the course of numerous independent transfections we consistently observed lower current densities in AGS1- and Rhes-transfected cells relative to their matched controls.


Figure 1
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  		Fig. 1. AGS1 and Rhes reduce the density of basal CaV2.2 currents and shift activation to more positive potentials. A: representative whole cell Ca2+ currents recorded from a control cell. The illustrated currents were evoked by steps from the steady holding potential (–90 mV) to test potentials between –10 and +80 mV. Linear cell capacitance (C) = 25 pF; series access resistance (RS) = 2.5 M{Omega}; maximal voltage error (VE) = 2.7 mV. B: representative currents from AGS1- and Rhes-transfected cells. AGS1 cell: C = 35 pF; RS = 2.9 M{Omega}; VE = 1.4 mV. Rhes cell: C = 23 pF; RS = 3.2 M{Omega}; VE = 1.0 mV. Scale bar represents 15 pA/pF and 5 ms. C: average current-voltage (I-V) relationships for 25 AGS1-expressing (bullet) cells and 23 matched control cells ({circ}). D: average I-V relationships for 16 Rhes-expressing cells ({blacktriangleup}) and 9 matched control cells ({circ}). ICa, calcium current. Error bars represent ± SE. Currents were normalized to cell capacitance.

 

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  		Table 1. Other monomeric G proteins fail to modulate CaV2.2

 
In control experiments, AGS1 and Rhes were omitted or cells were transfected with {Delta}CAAX or AGS1-G31V, two previously characterized, inactive AGS1 mutants (9, 17, 28, 35). Basal current densities were 44 ± 9 pA/pF (n = 16) in {Delta}CAAX-transfected cells and 50 ± 12 pA/pF (n = 18) in G31V-transfected cells, indistinguishable (P = 0.93) from current density in untransfected controls.

As illustrated in Fig. 1, C and D, the average current-voltage (I-V) relationship exhibited a reduced peak and a shift of 5–10 mV to more depolarized potentials in AGS1- and Rhes-expressing cells. These effects are consistent with the idea that AGS1 and Rhes trigger VD inhibition of CaV2.2, a prediction based on the previously demonstrated ability of AGS1 to function as a GEF for G{alpha}i (8). To investigate this possibility further, we examined currents for the presence of kinetic slowing, a hallmark of VD inhibition. A single exponential function was fit to the activating phase of basal CaV2.2 currents (evoked at +30 mV) to obtain time constants for activation ({tau}act). This analysis was restricted to cells in which membrane charging was relatively fast (i.e., the whole cell capacity transient exhibited a time constant of 0.1 ms or less). We found that {tau}act was 2.14 ± 0.17 ms (n = 8) in control cells, compared with 3.11 ± 0.38 ms (n = 9) in AGS1-expressing cells and 3.00 ± 0.18 ms (n = 4) in Rhes-expressing cells (P < 0.04). These data indicate that AGS1 and Rhes triggered slight but significant kinetic slowing of CaV2.2.

AGS1 and Rhes trigger VD inhibition of CaV2.2. We next used a prepulse protocol (Fig. 2A) to further test for the presence of VD inhibition. The protocol selected results in maximal prepulse facilitation of CaV2.2 in our system (26). The facilitating effect of the prepulse (PP) to +100 mV was quantified as the ratio of currents evoked by the first (P1) and second test pulses (P2) to various potentials. Despite using a PP of minimum effective duration (10 ms), the PP induced some voltage-dependent inactivation of CaV2.2 and the average ratio of currents evoked by the test pulses (P2/P1) was therefore <1.0 in control cells.


Figure 2
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  		Fig. 2. AGS1 and Rhes trigger voltage-dependent (VD) inhibition of basal CaV2.2 currents. A: representative currents from control, AGS1-transfected, and Rhes-transfected cells. The voltage protocol is diagrammed at top. Prepulse (PP) and first and second test pulses (P1 and P2, respectively) were each 10 ms in duration; PP and P2 were separated by 5 ms. Scale bars correspond to 5 ms and 500 pA (Control), 100 pA (AGS1), and 150 pA (Rhes). Control cell: C = 56 pF; RS = 5.4 M{Omega}; VE = 5.1 mV. AGS1 cell: C = 51 pF; RS = 4.7 M{Omega}; VE = 1.4 mV. Rhes cell: C = 30 pF; RS = 2.6 M{Omega}; VE = 1.4 mV. B: P2/P1 ratios at various test potentials. Voltage protocol as in A, except P1 and P2 were covaried between +20 and +60 mV, with PP constant at +100 mV. Symbols represent means ± SE of 8–17 cells. C: VD inhibition is mediated by the Gβ{gamma} subunits of pertussis toxin (PTX)-sensitive G proteins. Error bars represent ± SE. Cells were exposed to PTX (1 µg/ml) in media for at least 4 h prior to recordings. Grk3ct is the masGrk3ct construct; G31V and {Delta}CAAX are inactive mutants of AGS1; all are described in MATERIALS AND METHODS.

 
Basal CaV2.2 currents exhibited significant prepulse facilitation in both AGS1- and Rhes-transfected cells. As illustrated in Fig. 2A, the current evoked by P2 was smaller than that evoked by P1 in control cells, whereas the opposite was true in both AGS1- and Rhes-transfected cells. Figure 2B plots average P2/P1 ratios for test pulses ranging from +20 through +60 mV. Over this voltage range, P2/P1 ratios were significantly higher for AGS1- and Rhes-transfected cells. At a test potential of +30 mV, the P2/P1 ratio was 1.30 ± 0.06 (n = 16) in AGS1-transfected cells and 1.43 ± 0.07 (n = 16) in Rhes-transfected cells, compared with 0.85 ± 0.04 (n = 8) in control cells (P < 0.05). In cells transfected with {Delta}CAAX or AGS1-G31V, the average P2/P1 ratios were 0.80 ± 0.02 (n = 16) and 0.78 ± 0.03 (n = 7), respectively, similar to the ratio in control cells (P < 0.05). Thus, in these particular experiments, AGS1 and Rhes increased the P2/P1 ratio by ~53 and ~68%, respectively.

Because the PP induces voltage-dependent inactivation as well as facilitation of CaV2.2, we compared the densities of P2-evoked currents in AGS1- and Rhes-expressing cells with P2-evoked currents in matching controls. With this approach, the PP is assumed to induce equivalent inactivation in experimental and control cells. In one experiment, the density of P2 current was 12 ± 1 pA/pF (n = 18) in AGS1-expressing cells and 17 ± 3 pA/pF (n = 17) in matching controls (P = 0.13). In Rhes-expressing cells, the density of P2 current was 17 ± 3 pA/pF (n = 17) compared with 27 ± 9 pA/pF (n = 9) in matching controls (P = 0.17). These comparisons suggest that AGS1 and Rhes did not significantly reduce the densities of P2 currents, consistent with the interpretation that the PP restores current density to very near control levels, and that most of the decrease in basal P1 current density (Fig. 1) is attributable to tonic VD inhibition.

Previous studies have established that VD inhibition of CaV2 channels is mediated by the Gβ{gamma} subunits of heterotrimeric G proteins (19, 21). To confirm that Gβ{gamma} underlies AGS1- and Rhes-triggered prepulse facilitation of CaV2.2, we coexpressed a membrane-associating peptide (masGRK3ct) that effectively sequesters Gβ{gamma} subunits and thereby interferes with Gβ{gamma}-dependent events (22, 24, 27). Transfection with masGRK3ct appeared to prevent the decrease in basal current density observed in AGS1- and Rhes-transfected cells. For example, in one experiment current density was 12 ± 3 pA/pF (n = 8) in AGS1-expressing cells but considerably higher (47 ± 15 pA/pF; n = 12) in cells transfected with both AGS1 and masGRK3ct (P = 0.08). By contrast, transfection with masGRK3ct alone did not reduce current density (35 ± 13 pA/pF; n = 9 without masGRK3ct vs. 38 ± 9 pA/pF; n = 15 with masGRK3ct). More importantly, masGRK3ct completely blocked the ability of AGS1 and Rhes to elevate P2/P1 ratios (Fig. 2C). Thus P2/P1 was reduced to 0.89 ± 0.04 (n = 14; P < 0.05) in cells transfected with both AGS1 and masGRK3ct and was reduced to 0.92 ± 0.03 (n = 9; P < 0.05) in cells transfected with both Rhes and masGRK3ct. These data confirm that AGS1 and Rhes trigger Gβ{gamma}-mediated inhibition of CaV2.2.

To determine which class of heterotrimeric G proteins was responsible for the above effects, we pretreated cells with PTX (1 µg/ml) for 4–12 h before experiments. PTX also appeared to prevent the decrease in basal current density. In one experiment, the density of P1 currents was reduced to 14 ± 3 pA/pF (n = 12) in AGS1-transfected cells, compared with 37 ± 10 pA/pF (n = 7) in their matching controls (P = 0.01). In contrast, current density in the same AGS1-transfected cells that had been pretreated with PTX was 39 ± 14 pA/pF (n = 8), significantly different from the untreated, matching AGS1 cells (P = 0.04). As summarized in Fig. 2C, PTX prevented both AGS1 and Rhes from triggering prepulse facilitation of CaV2.2. The P2/P1 ratio was 1.19 ± 0.08 (n = 8) in AGS1-expressing cells but only 0.91 ± 0.06 (n = 4) in PTX-treated, matching AGS1-transfected cells (P = 0.04). Similar results were obtained for Rhes-transfected cells, where the P2/P1 ratio was 0.94 ± 0.04 (n = 8) in PTX-treated cells and 1.43 ± 0.07 (n = 16) in untreated cells (P < 0.001). In conjunction with the masGRK3ct data presented above, these results demonstrate, for the first time, that Rhes evokes tonic signaling by the Gβ{gamma} subunits of PTX-sensitive G proteins.

Effects of AGS1 and Rhes do not reflect altered voltage-dependent inactivation of CaV2.2. We also examined whether the effects of AGS1 and Rhes involved changes in the voltage-dependent inactivation of CaV2.2. Open-state inactivation of CaV2.2 was measured during long (350 ms) depolarizations to +40 mV. Currents evoked by this step inactivated with a single-exponential time constant of 110 ± 9 ms (n = 9) in control cells, 117 ± 19 ms (n = 4) in Rhes cells and 103 ± 9 ms (n = 4) in AGS1 cells (P = 0.43), suggesting that AGS1 and Rhes do not alter open-state inactivation. Closed-state inactivation was measured by using a two-pulse protocol (35) consisting of paired, 15-ms test pulses to +30 mV separated by a variable (10–45 ms) interval at 0 mV, which was the most depolarized potential that did not evoke significant ionic current under our experimental conditions. As assessed by this protocol, closed-state inactivation did not differ between Rhes-expressing and control cells (data not shown). To further investigate this issue, we expressed the β2a subunit in place of β3, since previous studies (30) indicate that β2a eliminates closed-state inactivation of CaV2.2. As expected, coexpression of β2a greatly diminished closed-state inactivation of CaV2.2 (not shown). However, β2a failed to alter the actions of AGS1 and Rhes on CaV2.2. For example, the average P2/P1 ratio was 1.33 ± 0.08 (n = 4) in AGS1-transfected β2a cells and 1.2 ± 0.02 (n = 9) in Rhes-transfected β2a cells, compared with 1.0 ± 0.01 (n = 10) in matching β2a control cells. Additionally, in β2a cells the inhibition of CaV2.2 by 50 µM CCh was attenuated from 75 ± 3% (n = 5) to 26 ± 11% (n = 4) by AGS1 and from 77 ± 1% (n = 4) to 28 ± 3% (n = 6) by Rhes, comparable to the level of attenuation in β3-expressing cells (Fig. 6). Finally, the presence of β2a did not prevent AGS1 and Rhes from reducing basal current densities. Thus, in β2a-expressing cells, basal current density was 5 ± 1 pA/pF (n = 4) in AGS1 cells and 21 ± 5 pA/pF (n = 6) in matching controls (P = 0.03); similarly, basal current density was 14 ± 6 pA/pF (n = 9) in Rhes/β2a cells and 40 ± 11 pA/pF (n = 6) in matching controls cells (P = 0.04). Although these experiments are not exhaustive, they suggest that AGS1 and Rhes do not alter inactivation of CaV2.2, and they additionally indicate that the effects of AGS1 and Rhes are independent of closed-state inactivation and the identity of the β subunit.


Figure 6
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  		Fig. 6. Summary of results obtained with M2 muscarinic acetylcholine receptors (M2R) or β2-adrenergic receptors (β2AR). All experiments with β2AR were performed on PTX-pretreated cells (1 µg/ml for 4–12 h). CCh and Iso were applied at 50 and 1 µM, respectively. The voltage protocols were identical to those used for Figs. 3 and 5. Error bars represent ± SE; number of cells is in parentheses.

 
AGS1 and Rhes attenuate agonist-initiated inhibition of CaV2.2 through G{alpha}i-coupled receptors. We next examined the effects of AGS1 and Rhes on receptor-mediated signaling. Cells were transfected with CaV2.2 and G{alpha}i-coupled M2R, and calcium currents were evoked by steps to +30 mV. A receptor-saturating concentration (50 µM) of CCh was applied through bath perfusion. As shown in Fig. 3A, CCh reversibly inhibited CaV2.2 currents by 79 ± 4% (n = 7) in control cells. Currents recorded from {Delta}CAAX- or AGS1-G31V-transfected cells were similarly inhibited, by 64 ± 3% (n = 16) and 71 ± 6% (n = 8), respectively (data not shown). In contrast, CCh evoked significantly less inhibition in AGS1-transfected (20 ± 6%; n = 7) and in Rhes-transfected (11 ± 4%; n = 14) cells (Fig. 3, B and C). On average, AGS1 and Rhes attenuated the M2R-mediated inhibition of CaV2.2 by ~75 and 86%, respectively (Fig. 6).


Figure 3
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  		Fig. 3. AGS1 and Rhes attenuate inhibition of CaV2.2 through G{alpha}i-coupled muscarinic acetylcholine receptors (M2R). A: carbachol (CCh; 50 µM) generates robust inhibition of CaV2.2 in control cells. Left: currents recorded directly before (a) CCh application and during (b) maximal CCh-induced inhibition. Right: plot of current amplitudes vs. time in the same cell. Currents were evoked at 0.5 Hz by steps from –90 mV to +30 mV. Horizontal line indicates CCh application. Control cell: C = 33 pF; RS = 3.6 M{Omega}; VE = 4.7 mV. Scale represents 350 pA and 5 ms. B: AGS1 cell: C = 33 pF; RS = 3.0 M{Omega}; VE = 1.4 mV. Scale represents 150 pA and 5 ms. C: Rhes cell: C = 30 pF; RS = 2.6 M{Omega}; VE = 1.4 mV. Scale represents 150 pA and 5 ms.

 
Figure 4A plots the attenuating effects of AGS1 and Rhes on CCh-initiated inhibition of CaV2.2 at various test potentials. AGS1 and Rhes produced similar attenuation of inhibition at test potentials ranging from +10 to +70 mV. Figure 4B reveals that AGS1 attenuated M2R-initiated inhibition of CaV2.2 over a wide range of CCh concentrations. The concentration of CCh needed to produce half-maximal inhibition (IC50) of CaV2.2 currents was 10-fold higher in AGS1 cells (67 nM) than in control cells (6.7 nM).


Figure 4
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  		Fig. 4. Effects of AGS1 and Rhes on CCh-dependent channel inhibition. A: attenuation of agonist-dependent inhibition at various test potentials. Currents were recorded from cells transfected with CaV2.2 and M2R (control) or these plus AGS1 or Rhes. Currents were evoked at 0.33 Hz by 25-ms depolarizations from –90 to –10 through +80 mV, in 10 mV increments; this protocol was applied to each cell before application of 50 µM CCh and again during maximal, CCh-induced inhibition. Symbols represent means ± SE for 10 control cells ({circ}), 7 AGS1-expressing cells (bullet), and 6 Rhes-expressing cells ({blacktriangleup}). B: effect of AGS1 on the dose-response relationship. Currents were recorded from cells transfected with CaV2.2 and M2R, or these plus AGS1. Data for control ({circ}) and AGS1-transfected cells (bullet). Symbols represent means ± SE for 3–7 cells in each group. Currents were evoked at 0.5 Hz by steps from –90 to +30 mV. Percent inhibition by CCh was calculated, for each CCh concentration, as [1 – (current amplitude during maximal CCh-induced inhibition/current amplitude just prior to CCh application)] x 100%. Continuous lines represent the fits of mean values to a nonlinear dose-response equation, with maximal inhibition of 74% (control) and 23% (AGS1), slopes of 0.75 (control) and 1.17 (AGS1), and IC50 values of 6.7 nM (control) and 67 nM (AGS1).

 
To examine whether the effects of AGS1 were receptor specific, we performed similar experiments using the G{alpha}i-coupled {kappa}OR in place of the M2R. The agonist, dynorphin A, was applied at a receptor-saturating concentration (100 nM). Dynorphin A triggered robust and reversible inhibition (67 ± 2%; n = 4) of CaV2.2 currents (evoked at +30 mV) in control cells but significantly less inhibition (30 ± 7%; n = 10) in cells transfected with AGS1 (P < 0.05). As anticipated, PTX pretreatment (1 µg/ml for 4–12 h) abolished dynorphin A-mediated inhibition of CaV2.2 (n = 6; data not shown). These results indicate that the effects of AGS1 are not restricted to M2R.

Other monomeric G proteins fail to replicate the effects of AGS1 and Rhes. We tested whether other monomeric G proteins could modulate CaV2.2. As summarized in Table 1, none of the transfected proteins significantly altered P2/P1 ratio, with the exception of HRas, which produced a small but significant decrease in P2/P1 ratio. CCh-initiated inhibition of CaV2.2 was also slightly attenuated in HRas-transfected cells, but this parameter was unaffected in cells transfected with Rap1A-G12V, Rap2B, Rheb2, Gem, or KRas2. In agreement with previous studies (1), basal current density was significantly reduced in Gem-transfected cells.

Rhes fails to attenuate signaling through the G{alpha}s-coupled β2AR receptor. AGS1 was previously shown to interact with G{alpha}i, but not with G{alpha}s, in yeast (9). By contrast, Rhes was previously reported to antagonize G{alpha}s-dependent signaling, but not G{alpha}i-dependent signaling, in mammalian cells (39). To further characterize the preferences of AGS1 and Rhes for heterotrimeric G proteins, we determined their abilities to attenuate agonist-initiated inhibition of CaV2.2 through the β2AR. Although β2AR couple primarily to G{alpha}s, they can also couple to G{alpha}i (10). Therefore, all experiments involving β2AR were performed on PTX-pretreated cells (1 µg/ml for 4–12 h) to eliminate potential contributions from G{alpha}i.

As illustrated in Fig. 5, application of the β-adrenergic receptor agonist Iso (1 µM) triggered significant inhibition of CaV2.2 current. Inhibition by Iso was irreversible under the conditions of our experiments; however, nearly full recovery could be achieved when the washout solution contained 1 µM propranolol, a β-blocker (data not shown). Upon application of 1 µM Iso, currents were inhibited by 49 ± 2% (n = 14) in control cells, 53 ± 3% (n = 9) in AGS1-transfected cells, and 56 ± 3% (n = 6) in Rhes-transfected cells (P = 0.15). These results indicate that AGS1 and Rhes cannot attenuate Gβ{gamma}-mediated signaling through the G{alpha}s-coupled β2AR under conditions in which G{alpha}i-dependent signaling is blocked.


Figure 5
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  		Fig. 5. Rhes and AGS1 do not attenuate inhibition of CaV2.2 through G{alpha}s-coupled β2-adrenergic receptors (β2AR). A, Left: currents recorded (a) directly before and (b) during maximal inhibition by 1 µM isoproterenol (Iso). Right: plot of current amplitudes vs. time in the same cell. Currents were evoked at 0.5 Hz by steps from –90 to +30 mV. Horizontal line indicates Iso application. C = 91 pF; RS = 5.0 M{Omega}; VE = 6.2 mV. Scale represents 500 pA and 5 ms. B: AGS1 cell: C = 23 pF, RS = 5.0 M{Omega}; VE = 2.3 mV. Scale represents 150 pA and 5 ms. C: Rhes cell. C = 26 pF, RS = 3.0 M{Omega}; VE = 1.3 mV. Scale represents 200 pA and 5 ms.

 
Figure 6 summarizes results obtained from cells expressing M2R or β2AR. Coexpression of masGRK3ct prevented inhibition of CaV2.2 through either receptor, confirming that channel inhibition required Gβ{gamma} signaling in both cases. We also found that Iso triggered smaller maximal inhibition than CCh, consistent with a recent report that G{alpha}s releases its Gβ{gamma} subunits less readily than G{alpha}i (11). Taken altogether, these data support previous evidence (41) that G{alpha}s-coupled receptors can trigger VD and Gβ{gamma}-dependent inhibition of CaV2.2. More interestingly, these results strongly suggest that Rhes cannot attenuate signaling by the Gβ{gamma} subunits of G{alpha}s.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we show that AGS1 and Rhes trigger tonic VD inhibition of N-type Ca2+ channels (CaV2.2) and attenuate Gβ{gamma}-dependent inhibition of CaV2.2 through GPCRs coupled to PTX-sensitive G proteins. Our findings raise the interesting possibility that AGS1 and Rhes influence the activity of CaV2.2 channels under more physiological circumstances. Importantly, our results also indicate that AGS1 and Rhes have very similar, if not identical, signaling properties.

AGS1 and Rhes reduced the basal density of P1 currents by ~40–60%. In addition, AGS1 and Rhes shifted the voltage dependence of current activation to slightly more positive potentials (Fig. 1, C and D) and induced slight but significant kinetic slowing. Altogether, these effects and our experiments with masGrk3ct strongly suggest that AGS1 and Rhes triggered tonic, VD inhibition of CaV2.2 (Fig. 2, A and B). The additional finding that P2 current density were not significantly different between AGS1- or Rhes-expressing cells and their respective controls suggests that the PP restores current density to near control levels. Thus the predominant effect of AGS1 and Rhes appears to be tonic VD inhibition of basal CaV2.2 currents. However, our present experiments do not allow us to exclude the possibility that AGS1 and Rhes also decreased channel expression, similar to the previously reported action of certain RGK proteins (1). We note, however, that any effects of AGS1 and Rhes on channel expression are likely to be small, judging from the P2 current data.

Coexpression of masGRK3ct, or pretreatment with PTX, completely prevented AGS1 and Rhes from increasing P2/P1 ratios (Fig. 2C). These findings are significant because they clearly demonstrate that Rhes promotes tonic signaling by the Gβ{gamma} subunits of PTX-sensitive G proteins. This is a novel finding for Rhes, whereas the ability of AGS1 to promote tonic Gβ{gamma} signaling was previously described (8, 9, 16, 28, 35).

AGS1 and Rhes attenuated agonist-initiated inhibition of CaV2.2 by G{alpha}i-coupled GPCRs (Fig. 3). In contrast, AGS1 and Rhes failed to attenuate signaling by the G{alpha}s-coupled β2AR (Fig. 5). These results are consistent with previous reports that AGS1 interferes with agonist-dependent signaling through G{alpha}i-coupled GPCRs (9, 16, 28, 34). However, our finding that Rhes attenuates signaling by G{alpha}i-coupled GPCRs differs significantly from previous communications. In particular, a previous study (39) found that Rhes failed to attenuate signaling through the G{alpha}i-coupled M2R but observed instead that Rhes attenuated signaling through the TSH receptor and the β2AR. Since both TSH and β2AR are known to couple (at least partially) to G{alpha}s, these observations were interpreted to indicate that Rhes specifically attenuates G{alpha}s-mediated signal transduction. However, PTX was not used to eliminate signaling by G{alpha}i coupled to TSH and β2AR (39). In contrast, our present experiments employed PTX in all experiments with the β2AR (Fig. 5 and 6). In addition, the aforementioned study (39) used an SRE-activated reporter gene to monitor signaling through the G{alpha}i-coupled M2R. This assay was relatively indirect since it involved, at minimum, the sequential activation of the M2R, its associated heterotrimeric G protein(s), one or more downstream effector(s) of the G protein(s) and production of diffusible second messenger(s) that activated the SRE. In comparison, our present experiments employed a considerably more direct assay, since it only required activation of Gβ{gamma} signaling to directly inhibit CaV2.2 channels (19, 21). In conclusion, our present findings argue strongly against the possibility (39) that Rhes specifically attenuates signaling through G{alpha}s-coupled GPCRs, and our results instead clearly indicate that Rhes attenuates signaling through G{alpha}i-coupled GPCRs. On the basis of our present experiments, the signaling properties of Rhes appear very similar, if not identical, to those of AGS1.

Our findings that AGS1 and Rhes decrease basal current density, trigger kinetic slowing, and increase prepulse facilitation of CaV2.2 are most parsimoniously explained by assuming that AGS1 and Rhes are both capable of functioning as GEFs for G{alpha}i (2, 8). In that capacity, both proteins could trigger tonic signaling by Gβ{gamma} and thereby produce the above effects on CaV2.2 basal currents. However, it is more difficult to account for the observed ability of AGS1 and Rhes to attenuate receptor-initiated channel inhibition (2). On the basis of evidence that AGS1 interacts both with G{alpha}i monomers (8) and Gβ{gamma} dimers (20), it is conceivable that AGS1 and Rhes bind heterotrimeric G{alpha}iGβ{gamma} and induce a conformational rearrangement that both triggers tonic Gβ{gamma} signaling and simultaneously impedes further receptor-initiated Gβ{gamma} signaling. An additional, not necessarily exclusive, possibility is that AGS1 and Rhes evoke such substantial tonic Gβ{gamma} signaling that modulation of downstream Gβ{gamma} effectors has already approached saturation prior to receptor activation. Further experiments will be needed to more fully understand the signaling properties of AGS1 and Rhes.

We note that agonist-initiated inhibition of CaV2.2 was voltage dependent and blocked by masGRK3ct regardless of whether M2R or β2AR was involved (Fig. 6). This observation indicates that Gβ{gamma} signaling was required for channel inhibition in either case, supporting a previous demonstration that G{alpha}s-coupled GPCRs can mediate VD inhibition of native neuronal N-type Ca2+ channels (41).

Relatively little is known about modulation of CaV channels by monomeric G proteins. To briefly summarize some of the previous studies, Fitzgerald (15) found that all components of whole cell Ca2+ currents recorded from rat sensory neurons were enhanced by transfection with constitutively active Harvey-Ras (HRas). Wilk-Blaszczak et al. (40) provided evidence that Rac1, and possibly also CdC42, is required for the bradykinin-initiated inhibition of N-type currents in NG108–15 cells. More recently, monomeric G proteins of the Rem/Gem/Kir (RGK) family have been shown to inhibit L-, N- and P/Q-type channel activity through direct interactions with Ca2+ channel β subunits (1). However, in contrast to AGS1 and Rhes, transfection with RGK proteins results in virtual elimination of the calcium current, and RGK proteins do not appear to induce tonic VD inhibition nor to interfere with receptor-initiated modulation of CaV2.2 (6). Thus, although other monomeric G proteins can clearly influence CaV channels, they appear to do so using mechanisms distinct from those employed by AGS1 and Rhes.

Potential physiological significance. CaV2.2 channels play essential roles in numerous physiological processes, including neurosecretion (25), neuropathic pain transmission (32), and neurite outgrowth and sensory innervation (31). Our present findings suggest that AGS1 and Rhes could potentially influence these and other Ca2+-dependent events by altering basal N-type channel activity and/or by attenuating Gβ{gamma}-dependent inhibition of CaV2.2 through G{alpha}i-coupled GPCRs. Although the physiological roles of AGS1 and Rhes remain to be fully elucidated, previous studies have shown that AGS1 forms a ternary complex with neuronal nitric oxide synthase and the adaptor protein CAPON, and that AGS1 is activated by S-nitrosylation following N-methyl-D-aspartate (NMDA) receptor-stimulated synthesis of nitric oxide (14). More recently, AGS1 was found to associate with protein kinase C (29) and was also shown to bind the peripheral benzodiazepine receptor-associated protein (PAP7), which in turn binds the divalent metal transporter (DMT1), thereby implicating AGS1 in iron uptake and NMDA-mediated neurotoxicity (5). Intriguingly, AGS1 is expressed in a circadian rhythmic pattern in the suprachiasmatic nucleus (34), and AGS1 knockout mice display abnormalities in photic and nonphotic responsiveness of the circadian clock (7). Rhes has been shown to interact (39) with the Ras-binding domain of PI3K (p110a) and may thereby activate signaling by PI3K and Akt/PKB. Additional evidence implicates Rhes in striatal dopaminergic signaling (12, 18), and Rhes-null mice display a clear deficit in motor coordination (33), a striatum-dependent function. In summary, the available data argue that AGS1 and Rhes are important neuronal signaling proteins. Our new findings regarding their properties should help in clarifying the physiological significance and specific roles of AGS1 and Rhes in signal transduction.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by grants to B. A. Adams from the American Heart Association (Established Investigator Grant 0040067N), the Muscular Dystrophy Association (MDA3663), the Whitehall Foundation (2005-12-22-APL), and the Utah Agricultural Experimental Station (UAES Project grant no. 638), and by an AHA Predoctoral Fellowship (9910094Z) to R. A. Bannister.


   ACKNOWLEDGMENTS
 
We thank Drs. Ulises Meza and Katarina Stroffekova for comments on an early version of the manuscript and Drs. V. Watts and S. M. Lanier for providing His-tagged AGS1-G31V. Many of the expression plasmids used in this study were obtained from the Missouri University of Science and Technology cDNA Resource Center (www.cDNA.org).

Present address of R. A. Bannister: Department of Physiology and Biophysics, School of Medicine, University of Colorado-Denver, RC-1, North Tower, P18-7130, PO Box 6511, Mail Stop F8307, Aurora, CO 80045 (e-mail: Roger.Bannister@UCHSC.edu).


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. A. Adams, Dept. of Biology, Utah State Univ., 5305 Old Main Hill, Logan, UT 84322 (e-mail: brett{at}biology.usu.edu)

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.

* A. Thapliyal and R. A. Bannister contributed equally to this work. Back


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