A splice variant of the G protein ß3-subunit implicated in disease states does not modulate ion channels

Victor Ruiz-Velasco and Stephen R. Ikeda

Laboratory of Molecular Physiology, Guthrie Research Institute, Sayre, Pennsylvania 18840


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
A single-nucleotide polymorphism (C825T) in the GNB3 gene produces an alternative splice variant of the heterotrimeric G protein ß3 subunit (Gß3). Translation of the alternatively spliced mRNA results in a protein product, Gß3-s, in which 41 amino acids are deleted from Gß3. Interestingly, previous studies indicate that the C825T allele occurs with a high frequency in patients with certain vascular disorders. However, little information is available regarding the functional role Gß3-s might play in ion channel modulation. To examine this aspect, Gß3 or Gß3-s, along with either G{gamma}2 or G{gamma}5, were expressed in rat sympathetic neurons by nuclear microinjection of vector encoding the desired protein. In contrast to Gß3, expression of Gß3-s did not modulate N-type Ca2+ or G protein-gated inwardly rectifying K+ channels. In addition, Gß3-s did not appear to complex with a pertussis toxin-insensitive mutant of G{alpha}i2 or couple to natively expressed {alpha}2-adrenergic receptors. Finally, fluorescence resonance energy transfer (FRET) measurements indicated that enhanced yellow fluorescent protein (EYFP)-labeled Gß3-s does not form a Gß{gamma} heterodimer when coexpressed with enhanced cyan fluorescent protein (ECFP)-labeled G{gamma}2. Therefore, when expressed in sympathetic neurons, Gß3-s appears to lack biological activity-hence pathological conditions in patients carrying the homozygous C825T allele may result from a functional knockout of Gß3.

Gß3-s; signal transduction; ion channel modulation; FRET; N-type calcium channels


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
HETEROTRIMERIC G PROTEINS (G{alpha}ß{gamma}) mediate signal transduction pathways that modulate neuronal excitability as well as cardiac rate. Neurotransmitter binding to G protein-coupled receptors (GPCR) results in the dissociation of G{alpha}ß{gamma} into G{alpha} and {gamma} subunits. Both of these moieties modulate a number of intracellular effectors such as N- and P/Q-type Ca2+ channels, G protein-gated inwardly rectifying K+ (GIRK) channels, phospholipases, G protein-coupled receptor kinases (GRK), and adenylyl cyclases (11).

Of the five Gß subunits cloned thus far, Gß1–4 share an 80–90% sequence identity and contain ~341 amino acids (15). The heterotrimeric G protein crystal structure reveals that Gß subunits contain two distinct structural motifs (27, 51, 54): the first is an {alpha}-helical structure comprising the amino terminus, and the second is a seven-bladed propeller structure. Each of the seven blades comprises a WD repeat [normally bounded by a tryptophan (W) and an aspartate (D) residues] that fold into ß-sheets. G{alpha} subunits and several effector proteins share several common interacting regions along the Gß propeller structure (14, 30, 34).

The GNB3 gene coding for human Gß3 is located on chromosome 12p13 and comprised of 11 exons and 10 introns (38). Recently, the discovery of a C825T polymorphism in exon 10 of GNB3 was reported to result in expression of a novel Gß3 splice variant, termed Gß3-s (45). The C825T exchange does not change the encoded amino acid, serine, but alters a splice junction resulting in the deletion of 123 nucleotides in exon 9. As a result, Gß3-s is shorter than Gß3 by 41 amino acids and is predicted to contain only 6 WD repeats.

The effect the C825T polymorphism has on Gß3-s function is incompletely understood, and whether the presence of this polymorphism conveys certain pathophysiological conditions remains controversial. On the one hand, numerous studies demonstrate a strong correlation between the presence of the GNB3 825T allele and diabetes (8, 37), obesity (18, 46, 47), hypertension (4, 13, 17, 44, 45, 48) and other vascular disorders (2, 3, 22, 23, 35, 55). At the cellular level, Gß3-s has been proposed to lead to enhanced signal transduction pathways that are coupled to pertussis toxin (PTX)-sensitive G{alpha}i subunits (52, 53). On the other hand, a lack of association between the incidence of this allele and hypertension (9, 28, 50), obesity (7, 50), and diabetes (5, 57) has been documented. Interestingly, there is little direct evidence demonstrating that Gß3-s forms a functional Gß{gamma} heterodimer (i.e., Gß{gamma}) and, consequently, a G{alpha}ß{gamma} heterotrimer. To address this issue, we took advantage of the expression system employed in our laboratory (i.e., heterologous expression in sympathetic neurons) that is amenable to electrophysiological and optical measurements of G protein signaling pathways (21, 24, 25, 3941). Gß3-s, along with G{gamma} subunits, were heterologously expressed in superior cervical ganglion (SCG) neurons to determine whether the expressed subunits would modulate ion channels, form functional heterotrimers, or couple to GPCRs. Fluorescence resonance energy transfer (FRET) measurements were also employed to examine the interaction of enhanced yellow fluorescent protein (EYFP)-labeled Gß3-s with enhanced cyan fluorescent protein (ECFP)-labeled G{gamma}2. As a positive control, parallel experiments were undertaken with wild-type Gß3.


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

cDNA constructs.
Human Gß3 cDNA, kindly provided by Dr. M. I. Simon (California Institute of Technology, Pasadena, CA), was subcloned into the mammalian expression vector, pCI (Promega, Madison, WI). The Gß3-s cDNA plasmid was constructed by deleting the sequence coding for the 41 amino acids absent in Gß3-s (45) using the PCR. Four primers were designed to amplify regions that flank the excised sequence. The NH2 terminus 5' primer consisted of TTG AAT TCC ACC ATG GGG GAG ATG GAG CAA C and was cut with the EcoR1 restriction enzyme. The NH2 terminus 3' primer was C AGC TCG GGG GAC ACC ACG TGC GCA TTA A and was cut with the FspI restriction enzyme. The COOH terminus 5' primer was T TAA GGC GCC AAG CTC TGG GAT GTG and was cut with the SfoI restriction enzyme. Finally, the COOH terminus 3' primer was C CTC AAA ATC TGG AAC TGA TCT AGA CGC C and was cut with the XbaI restriction enzyme. The remaining ends were then blunt-ligated. Deletion of the 123 bases was confirmed by automated DNA sequencing (ABI 310; Perkin-Elmer, Foster City, CA). Plasmids coding for human G{gamma}2 and G{gamma}5 (both subcloned into pCI) were prepared using anion exchange columns (Qiagen, Chatsworth, CA). Human GIRK1 and GIRK4 (Kir 3.1 and 3.4) in pcDNA3.1 (Invitrogen, Carlsbad, CA) were kindly supplied by Dr. D. E. Logothetis (Mt. Sinai Medical Center, New York, NY). Enhanced yellow fluorescent protein (EYFP)-Gß fusion constructs were made by ligating Gß3 or Gß3-s coding regions into the multiple cloning site of pEYFP-C1 (Clontech Laboratories, Palo Alto, CA). The resulting plasmids were predicted to code for EYFP-Gß proteins separated by a 17-residue linker (SGLRSRAQASNSAVDGT) region. The ECFP-G{gamma}2 clone was constructed by linking the enhanced cyan fluorescent protein (pECFP-C1, Clontech Laboratories) vector to the NH2 terminus of G{gamma}2 cDNA. The linker sequence was TCC GGA, encoding for S and G, respectively. Site-directed mutagenesis of the mouse G{alpha}i2 subunit was accomplished using the PCR as previously described (24). A C->G mutation at fourth amino acid from the carboxy terminus (G{alpha}i2, C352G) was introduced to convey a PTX-insensitive phenotype. Following subcloning into pCI, the entire coding region was sequenced to confirm introduction of the mutation and the integrity of the PCR product. All plasmids were stored in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).

Neuron isolation and cDNA plasmid microinjection.
Single neurons from adult rat superior cervical ganglion (SCG) were prepared using the method described previously (40). The experiments carried out were approved by Institutional Animal Care and Use Committee (IACUC). Male Wistar rats (175–225 g) were anesthetized with CO2 and then decapitated using a laboratory guillotine. Cell isolation was then carried out (40).

Plasmid microinjection in neurons was performed with an Eppendorf 5246 microinjector and 5171 micromanipulator (Madison, WI) 3–5 h after cells were plated. Plasmids were injected at a final concentration of 10 to 100 ng/µl. The plasmid coding for the jellyfish green fluorescent protein (pEGFP-N1, 5 ng/µl; Clontech Laboratories) was coinjected with the plasmids and served as a "marker" for cells receiving a successful injection. Neurons were identified 12–18 h later using an inverted microscope (Diaphot 300; Nikon) equipped with an epifluorescence unit (DM510 filter cube; Nikon).

Electrophysiology and data analysis.
Ca2+ and GIRK currents were recorded using the whole-cell variant of the patch-clamp technique. Patch pipettes were pulled from borosilicate glass capillaries (Corning 7052; Garner Glass, Claremont, CA) on a Flaming-Brown (P-97) micropipette puller (Sutter Instrument, San Rafael, CA), coated with Sylgard (Dow Corning, Midland, MI), and fire polished. Whole-cell currents were acquired with a patch-clamp amplifier (Axopatch 1-C; Axon Instruments, Foster City, CA), analog filtered at 5–10 kHz (-3 dB; 4-pole Bessel), and digitized using custom-designed software (S4) on a PowerPC computer (Power Computing, Austin, TX) equipped with a 16-bit analog-to-digital converter board (model ITC16; Instrutech, Elmont, NY). Cell membrane capacitance and series resistance (80–85%) were electronically compensated. Experiments were performed at room temperature (21–24°C). Data and statistical analysis were performed with Igor (Lake Oswego, OR) and GB-Stat PPC (Silver Spring, MD) software packages, respectively, employing one-way analysis of variance (ANOVA) followed by the Newman-Keuls test. P < 0.05 was considered statistically significant. Graphs and current traces were produced with Igor and Canvas (Deneba Software, Miami, FL) software packages.

The pipette solution for Ca2+ currents contained (in mM) 120 N-methyl-D-glucamine, 20 tetraethylammonium hydroxide (TEA-OH), 11 EGTA, 10 HEPES, 10 sucrose, 1 CaCl2, 4 Mg-ATP, 0.3 Na2-ATP, and 14 Tris creatine phosphate. The pH was adjusted to 7.2 with methanesulfonic acid and HCl (20 mM), and the osmolality was 299–308 mosmol/kg. The external Ca2+ current solution consisted of (in mM) 145 TEA-OH, 10 HEPES, 15 glucose, 10 CaCl2, and 0.0003 tetrodotoxin (TTX). The pH was adjusted to 7.4 with methanesulfonic acid, and the osmolality was 319–327 mosmol/kg. The pipette solution for GIRK currents contained (in mM) 135 KCl, 11 EGTA, 1 CaCl2, 2 MgCl2, 10 HEPES, 4 Mg-ATP, and 0.3 Na2-ATP. The pH was adjusted to 7.2 with KOH and the osmolality was 305–308 mosmol/kg. The GIRK external solution consisted of (in mM) 130 NaCl, 5.4 KCl, 10 HEPES, 10 CaCl2, 0.8 MgCl2, 15 glucose, 15 sucrose, and 0.0003 TTX. The pH was adjusted to 7.4 with NaOH, and the osmolality was 326 mosmol/kg.

Norepinephrine (NE) bitartrate (Sigma Chemical) was prepared as a stock solution (10 mM) in H2O and diluted to 10 µM just prior to use. NE was applied to the neuron under study with a custom-designed gravity-fed microperfusion system. PTX (List Biological Laboratories, Campbell, CA) was prepared in H2O and added to the culture medium (12–20 h) at a concentration of 500 ng/ml.

Fluorescence resonance energy transfer analysis.
FRET was employed to detect the interaction between heterologously expressed ECFP-G{gamma}2 (donor) with either EYFP-Gß3 or EYFP-Gß3-s (acceptor) fusion proteins. Neurons expressing both fusion proteins were identified 12–20 h later using an inverted microscope (Diaphot 300; Nikon) equipped with a x40 objective (0.6 NA). FRET excitation was carried out with a filter cube containing a 440 ± 10.5-nm excitation filter and a 455-nm (long pass) dichroic beam splitter. FRET emission, expressed in volts (V), was detected with a dual photometer unit (model 814 PMT; Photon Technology International, Lawrenceville, NJ) equipped with emission filters at 480 ± 10 nm (for donor, ECFP) and 535 ± 13 nm (for acceptor, EYFP) and a 455-nm (long pass) dichroic beam splitter. An EYFP filter set with an excitation filter at 500 ± 12 nm, a dichroic beam splitter of 525 nm (long pass), and an emission filter at 545 ± 17 nm were used to estimate EYFP expression levels. Statistical analysis of FRET data was done by paired Student’s t-test.

Fluorescence images of EYFP-Gß3- and EYFP-Gß3-s-expressing neurons were obtained by employing an EYFP filter cube set (model EF-4 yellow GFP BP HYQ; Nikon) containing an excitation filter at 500 ± 10 nm, a dichroic beam splitter of 515 nm (long pass), and an emission filter at 535 ± 15 nm. Fluorescence images were acquired with a MagnaFire cooled CCD camera and software (Optronics, Goleta, CA) and processed with the Adobe Photoshop software package (Adobe Systems, Seattle, WA).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

Heterologous expression of tagged and untagged Gß3 and Gß3-s in rat sympathetic neurons.
The goal of this set of experiments was to determine whether expression of Gß3 and Gß3-s with either G{gamma}2 or G{gamma}5 subunits would modulate N-type Ca2+ channels. In rat SCG neurons, most GPCR modulate N-type Ca2+ channels in a voltage-dependent (VD) and membrane-delimited manner. For instance, activation of {alpha}2-adrenergic receptors ({alpha}2-AR) results in kinetic slowing of the rising Ca2+ currents during a depolarizing test pulse. Kinetic slowing is believed to result from a VD relief of block during the application of the depolarizing test pulse. Another consequence of VD relief of inhibition is a phenomenon referred to as "facilitation." Facilitation is determined by evoking Ca2+ currents with two identical test pulses (usually to +10 mV) separated by a large depolarizing conditioning pulse (+80 mV) as illustrated at the bottom of Fig. 2A. The ratio of the postpulse to prepulse current amplitude (measured isochronally 10 ms after the start of the test pulse) is termed facilitation. The VD inhibition of N-type Ca2+ currents has been shown to be mediated by Gß{gamma} (2021).



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Fig. 2. Effect of heterologous expression of ß3, ß3-s, EYFP-ß3, or EYFP-ß3-s with either {gamma}2 or {gamma}5 on basal facilitation and norepinephrine (NE)-mediated inhibition of Ca2+ currents in SCG neurons. Superimposed Ca2+ current traces evoked with the "double pulse" voltage protocol (bottom of A) in the absence (bottom traces) and presence (top traces) of 10 µM NE for control (A), ß3{gamma}5- (B), and ß3-s{gamma}5-expressing neurons (C). Currents were evoked every 10 s. Dashed lines refer to the zero current level. D: summary graphs of mean (±SE) basal facilitation (left) and Ca2+ current inhibition (right) for neurons expressing ß3, EYFP-ß3, ß3-s, and EYFP-ß3-s with {gamma}2 or {gamma}5. Facilitation was calculated as the ratio of Ca2+ current amplitude determined from the test pulse (+10 mV) occurring after (postpulse) and before (prepulse) the +80-mV conditioning pulse. **P < 0.01 vs. control. Numbers in parentheses indicate the number of experiments.

 
Figure 1 shows phase and fluorescence images of two neurons expressing EYFP-Gß3/G{gamma}5 (Fig. 1A) and EYFP-Gß3-s/G{gamma}5 (Fig. 1B). Both fluorescence images were acquired 12–14 h following microinjection of the constructs. Both Gß and G{gamma} plasmids were coinjected at a final concentration of 100 ng/µl. These results demonstrate successful expression of both EYFP-tagged subunits and are similar to those previously observed in SCG neurons expressing EYFP-Gß1 (41).



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Fig. 1. Heterologous expression of enhanced yellow fluorescent protein (EYFP)-labeled ß3{gamma}5 (A) and EYFP-ß3-s{gamma}5 (B) in adult rat superior cervical ganglion (SCG) neurons: fluorescence (left) and phase images (right) of neurons expressing EYFP-ß3{gamma}5 and EYFP-ß3-s{gamma}5. Fluorescence images were taken with a filter set specific for EYFP (500 ± 10 nm excitation and 535 ± 10 nm emission). Fluorescence images shown are pseudocolored.

 
Figure 2A shows superimposed Ca2+ currents evoked with the double-pulse voltage protocol (shown at the bottom of Fig. 2A) before and after application of 10 µM NE in an uninjected neuron. Prior to NE exposure, the prepulse Ca2+ current (lower trace) rose rapidly and reached a plateau within 5–10 ms. Furthermore, the postpulse current (lower trace) was slightly affected by the conditioning pulse (to +80 mV). On the other hand, application of NE caused the rising phase to be slow and biphasic. In addition, the amplitude of the postpulse current was greater than the prepulse current due to the VD relief of inhibition during the test pulse. In the absence of NE, the mean basal facilitation was 1.32 ± 0.04 (n = 11), and the NE-mediated Ca2+ current inhibition was 47.8 ± 2.3% (Fig. 2D).

Figure 2B shows Ca2+ current traces from a cell expressing Gß3/G{gamma}5. Under these conditions, basal facilitation was significantly increased, and there was an enhanced kinetic slowing, similar to that observed when NE was applied to uninjected neurons. The mean basal facilitation (2.74 ± 0.15, n = 5) was significantly greater (P < 0.01) than uninjected neurons (Fig. 2D). In addition, compared with uninjected neurons, the NE-mediated Ca2+ current inhibition (16.9 ± 1.7%) was significantly less (P < 0.01). Similar results were found with cells expressing EYFP-Gß3/G{gamma}2 and EYFP-Gß3/G{gamma}5 (Fig. 2D). Basal facilitation in cells expressing EYFP-Gß3 was significantly greater than in neurons expressing EYFP-Gß3-s but less than that of untagged wild-type Gß3. Unlike Gß3/G{gamma}5, expression of Gß3-s/G{gamma}5 did not lead to tonic inhibition of Ca2+ currents (Fig. 2C). The mean basal facilitation and NE-mediated Ca2+ current inhibition were 1.26 ± 0.04 (n = 5) and 51.5 ± 4.8%, respectively (Fig. 2D). The NE-mediated Ca2+ current inhibition observed following NE exposure is presumably the result of liberated endogenous {gamma} subunits. Expression of EYFP-Gß3-s/G{gamma}5 and EYFP-Gß3-s/G{gamma}2 produced similar results (Fig. 2D).

GIRK channels are activated by EYFP-Gß3 and not by EYFP-Gß3-s.
Given the lack of N-type Ca2+ channel modulation following Gß3-s/G{gamma} expression, experiments were undertaken to determine whether Gß3-s could modulate an additional "target" for Gß{gamma} subunits, GIRK-type K+ channels. GIRK channels are inwardly rectifying K+ channels that are activated by Gß{gamma} (11). Previous studies from our laboratory and others have shown that overexpression of Gß1–4 with several G{gamma} subunits results in tonic GIRK channel activation (29, 41, 42). Although rat SCG neurons do not possess endogenous GIRK channels, heterologously expressed GIRK channels are functional and couple to native GPCRs (39). In the present study, GIRK currents were recorded every 10 s by applying a 200-ms voltage ramp from -120 to -40 mV from a holding potential of -60 mV. Basal and NE-stimulated peak GIRK currents were calculated by digitally subtracting current traces obtained before and after NE exposure, respectively, from those obtained after application of 1 mM Ba2+ (an efficient GIRK channel blocker). Maximal inward currents normally occurred between -115 and -105 mV.

Figure 3A depicts peak GIRK current as a function of time in a neuron expressing GIRK1 and 4 channels. Application of 10 µM NE (solid bar) produced an ~1 nA increase in peak inward current. Prior to NE application, a small basal GIRK current was recorded (~50 pA) as indicated by sensitivity to application of 1 mM Ba2+ (solid bar). Figure 3, B and C, illustrates peak GIRK current vs. time for neurons expressing GIRK1 and 4 with EYFP-Gß3/G{gamma}2 (Fig. 3B) and EYFP-Gß3-s/G{gamma}2 (Fig. 3C), respectively. Expression of EYFP-Gß3/G{gamma}2 resulted in a large (~1 nA) tonic activation of GIRK currents as demonstrated by subsequent blockade by exposure to 1 mM external Ba2+ (solid bar). Conversely, expression of EYFP-Gß3-s/G{gamma}2 failed to produce basal activation of GIRK channels. Application of NE to either set of neurons resulted in agonist-mediated GIRK channel activation. Figure 3D summarizes basal and NE-activated GIRK currents in cells expressing EYFP-Gß3/G{gamma}2 and EYFP-Gß3-s/G{gamma}2. Mean basal GIRK current was similar in cells expressing GIRK1 and 4, GIRK1 and 4 + Gß3-s/G{gamma}2 and GIRK1 and 4 + Gß3-s/G{gamma}5, but significantly greater in GIRK1 and 4 + EYFP-Gß3/G{gamma}2- and GIRK1 and 4 + Gß3-s/G{gamma}5-expressing neurons (P < 0.01). The NE-activated GIRK current was not statistically different among the five groups tested. The scatter in the magnitude of NE-activated GIRK currents presumably arises from varying expression levels of GIRK1 and 4 channels. At present, it is difficult to confirm this assumption, as methods to quantify protein expression levels in single neurons are lacking.



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Fig. 3. G protein-gated inwardly rectifying K+ (GIRK) channel activation in rat SCG neurons expressing EYFP-ß3{gamma}2, EYFP-ß3-s{gamma}2, EYFP-ß3{gamma}5, and EYFP-ß3-s{gamma}5 on GIRK channel activation. Time course of basal and NE-activated GIRK channel currents in control (GIRK1 and 4 only) (A), GIRK1 and 4 + EYFP-ß3{gamma}2 (B), and GIRK1 and 4 + EYFP-ß3-s{gamma}2-expressing neurons (C). Currents were evoked by 200-ms voltage ramps from -120 to -40 mV from a holding potential of -60 mV applied every 10 s. Solid horizontal bars indicate application of 10 µM NE or 1 mM Ba2+. D: summary graph showing the mean (±SE) basal (left) and NE-activated (right) peak GIRK currents recorded before and after NE application. **P < 0.01 vs. control. Numbers in parentheses indicate the number of experiments.

 
Determination of heterotrimer formation between EYFP-Gß3/G{gamma}5 or EYFP-Gß3-s/G{gamma}5 and PTX-resistant G{alpha}i2 subunits.
Although overexpression of Gß3-s/ G{gamma} did not produce ion channel modulation, reconstitution experiments to determine whether Gß3-s/G{gamma} could form a heterotrimer with GDP-bound G{alpha} subunits were performed. To this end, the PTX-insensitive G{alpha} subunit, G{alpha}i2 (C352G) was heterologously coexpressed with either EYFP-Gß3/G{gamma}5 or EYFP-Gß3-s/G{gamma}5 in neurons pretreated with PTX to disrupt native G protein coupling (43). Reconstitution was assessed by exposing the neurons to 10 µM NE and testing for the recovery of receptor-mediated VD inhibition of N-type Ca2+ channels. For successful reconstitution to occur, it was critical to obtain a stoichiometric "balance" of all three expressed G protein subunits. In this type of experiment, three outcomes were possible as previously described (24, 41). The first outcome results from greater expression levels of {gamma} subunits than G{alpha}i2 (C352G) (Gß{gamma} > G{alpha}i2) leading to tonic Ca2+ channel modulation as indicated by enhanced basal facilitation. The second outcome involves greater expression levels of G{alpha}i2 (C352G) than Gß{gamma} (G{alpha}i2 > Gß{gamma}). Under this condition, excess GDP-bound G{alpha}i2 (C352G) acts as a "buffer" for native as well as exogenous Gß{gamma}, resulting in a loss of both the NE-mediated Ca2+ current inhibition and basal facilitation (values <= 1.0; see Refs. 21, 24, 41). The third outcome is a balanced expression of all three G protein subunits. Under these conditions, application of NE leads to Ca2+ current inhibition. In this study, stoichiometric balance in neurons coexpressing G{alpha}ß{gamma} subunits was operationally defined as basal facilitation values between 1.15 to 1.45.

Reconstitution was determined in a total of 21 neurons following coinjection of EYFP-Gß3, G{gamma}5, and G{alpha}i2 (C352G) at a ratio (weight) of ~1:1:1. Eight of the 21 cells had a mean basal facilitation of 1.07 ± 0.02 indicating excess GDP-bound G{alpha} subunits. As expected, NE-mediated Ca2+ current inhibition in this group was ablated (3.92 ± 1.94%). Of the remaining 13 cells, six neurons had basal facilitation values between 1.15 to 1.45, and the NE-mediated Ca2+ current inhibition was reconstituted. In the other seven cells, however, basal facilitation fell within the defined range, but the NE-mediated Ca2+ current inhibition was not recovered. These cells were not included in the analysis. Figure 4A shows superimposed Ca2+ currents of an uninjected cell before and after application of 10 µM NE. In this group of cells, the mean NE-mediated inhibition of Ca2+ currents was 46.2 ± 3.0%, and basal facilitation was 1.39 ± 0.03 (n = 7). Pretreatment of uninjected neurons with PTX attenuated the NE-mediated Ca2+ current inhibition (Fig. 4B). Mean NE-mediated Ca2+ current inhibition and basal facilitation were 9.0 ± 2.5% and 1.21 ± 0.03 (n = 8), respectively. Figure 4C shows superimposed Ca2+ currents from a successfully reconstituted PTX-treated cell expressing G{alpha}i2 (C352G) and EYFP-Gß3/G{gamma}5. Upon exposure to 10 µM NE, the Ca2+ current was inhibited ~45%. The mean NE-mediated Ca2+ current inhibition and basal facilitation were 29.4 ± 2.8% and 1.26 ± 0.04 (n = 6), respectively. Although coupling was partially reconstituted in these experiments, the coupling efficiency was not as robust as we have previously observed with other G protein subunit combinations (24). It is possible that the level of "balanced" G proteins expressed, the EYFP tag, and/or the intrinsic coupling efficiency of this heterotrimer combination were not optimal. In a separate control group, the cells were injected with G{alpha}i2 (C352G) cDNA and pretreated with PTX overnight. The NE-mediated Ca2+ current inhibition was 6.0 ± 2.1%, and the basal facilitation value was 1.15 ± 0.8 (n = 5).



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Fig. 4. Reconstitution of {alpha}2-adrenergic receptor ({alpha}2-AR) coupling to N-type Ca2+ channels in rat SCG neurons expressing pertussis toxin (PTX)-resistant G{alpha}i2 with EYFP-ß3/{gamma}5 or EYFP-ß3-s/{gamma}5. Superimposed Ca2+ current traces evoked with the "double pulse" voltage protocol (bottom of C) in the absence (bottom traces) and presence (top traces) of 10 µM NE for control (no PTX) (A), control (500 ng/ml PTX, overnight) (B), PTX-pretreated neuron expressing G{alpha}i2(C352G)/YFP-ß3/{gamma}5 (C), and PTX-pretreated neuron coexpressing G{alpha}i2(C352G)/YFP-ß3-s/{gamma}5 (D). Currents were evoked every 10 s. Dashed lines refer to the zero current level. E: summary graph of mean (±SE) NE-mediated Ca2+ current inhibition for uninjected, G{alpha}i2(C352G)/YFP-ß3/{gamma}5-, and G{alpha}i2(C352G)/YFP-ß3-s/{gamma}5-expressing neurons. NE-mediated inhibition was determined as described in MATERIALS AND METHODS. **P < 0.01 vs. PTX-treated control neurons. Numbers in parentheses indicate the number of experiments.

 
With regard to the Gß3-s subunit, reconstitution could not be determined due to the inability of Gß3-s to tonically modulate N-type Ca2+ currents (Fig. 2, C and D). However, this type of experiment can be used to probe whether Gß3-s forms a heterotrimer with G{alpha} subunits. In the following experiments, EYFP-Gß3-s, G{gamma}5, and G{alpha}i2 (C352G) cDNA were coinjected at a ratio (weight) of ~10:10:1. The increased ratio of Gß{gamma}:G{alpha} cDNA should greatly favor the expression of Gß{gamma} over GDP-bound G{alpha} subunits. Under these conditions, the buffering effect of G{alpha}i2 (C352G) would be predicted to be overwhelmed by the higher protein levels of EYFP-Gß3-s/G{gamma}5 if heterotrimer formation occurred. Figure 4D shows superimposed Ca2+ currents of a neuron expressing EYFP-Gß3-s/G{gamma}5 and G{alpha}i2 (C352G) before and after NE application. The cell’s basal facilitation was 1.11, and the NE-mediated inhibition was ~11%. Of the nine cells tested, one neuron had a basal facilitation of 1.17, and the remainder were closer to 1.0. The mean basal facilitation and NE-mediated Ca2+ current inhibition were 1.05 ± 0.02 and 7.5 ± 3.1%, respectively (Fig. 4E). These results suggest that, in contrast to EYFP-Gß3, EYFP-Gß3-s was unable to form a heterotrimer with GDP-bound G{alpha}i2 (C352G) and G{gamma}5.

FRET analysis of EYFP-Gß3/CFP-G{gamma}2 and EYFP-Gß3-s/CFP-G{gamma}2.
To further investigate why Gß3-s was inactive in the electrophysiological experiments, FRET was employed to determine whether in vivo interaction between EYFP-Gß3-s and ECFP-G{gamma}2 occurred. FRET involves the transfer of energy from an excited fluorophore (CFP-G{gamma}2, donor) to a second fluorophore (YFP-Gß3, acceptor) in a nonradiative manner. For FRET to occur, both fluorophores must be in close proximity (10–100 Å) and in the proper orientation (36). Recently, we reported that expression of EYFP-Gß1 and ECFP-G{gamma}2 in SCG neurons resulted in FRET following the excitation of ECFP-G{gamma}2 (41). Therefore, in the present study, similar FRET measurements were undertaken with EYFP-ß3 and ECFP-{gamma}2, as a positive control. In this set of experiments, excitation (at 440 nm) of ECFP-G{gamma}2 was performed with the FRET filter. Excitation (at 500 nm) of EYFP-ß3-s and EYFP-ß3 was performed with the EYFP filter set. Emission at 480 and 535 nm for ECFP-G{gamma}2 and EYFP-Gß3-s (or EYFP-ß3), respectively, was determined with photomultiplier tubes. With an optimal pair of FRET fluorophores, the emission spectrum of the donor overlaps with the excitation, but not the emission spectrum, of the acceptor, and acceptor excitation does not result in donor excitation. A disadvantage of using ECFP-YFP for FRET measurements is the potential for spectral cross talk or bleed-over, i.e., leakage of donor fluorescence into the acceptor emission channel (535 nm) and acceptor fluorescence into the donor emission channel (480 nm).

Correction for spectral cross talk can be determined from cells expressing only donor or acceptor fluorophores (16). The FRET terminology used here is taken from previous studies (16, 41). Donor emission cross talk from ECFP-{gamma}2-expressing cells was determined by recording the emission at 480 nm (Dd) and 535 nm (Fd) resulting from excitation at 440 nm. The Fd/Dd ratio is a measure of cross talk due to donor emission leaking into the acceptor channel produced by excitation at the donor excitation wavelength. Figure 5A shows the Fd emission as a function of Dd emission in cells expressing ECFP-G{gamma}2 (i.e., donor cross talk). The data was fitted to a straight line, and the slope (Fd/Dd) of the line was 0.310 ± 0.001 (n = 19). The plot demonstrates that over a broad range of expression values, donor emission cross talk was constant. Acceptor excitation cross talk in EYFP-ß3-s- or EYFP-ß3-expressing neurons was determined similarly. Fa and Aa are measures of acceptor fluorescence produced by excitation at the donor and acceptor excitation wavelengths, respectively. Figure 5B shows a plot of the Fa emission as a function of Aa emission, in cells expressing either EYFP-Gß3 (solid circles) or EYFP-Gß3-s (open circles) alone. Both sets of data were fitted to a straight line, and the slopes were 0.246 ± 0.015 (n = 8) and 0.247 ± 0.072 (n = 9) for EYFP-Gß3- and EYFP-Gß3-s-expressing cells, respectively. Thus the contribution of EYFP excitation cross talk was constant over varying EYFP-Gß3 and EYFP-Gß3-s expression levels. With the filter sets used, other forms of cross talk were negligible.



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Fig. 5. Fluorescence resonance energy transfer (FRET) values in SCG neurons coexpressing EYFP-ß3/CFP-{gamma}2 and EYFP-ß3-s/CFP-{gamma}2. Emission recorded in SCG neurons expressing enhanced cyan fluorescent protein (ECFP)-labeled {gamma}2 (A), EYFP-ß3 (solid circles) and EYFP-ß3-s (open circles) (B), and EYFP-ß3/CFP-{gamma}2 (solid circles) and EYFP-ß3-s/CFP-{gamma}2 (open circles) (C). Emission values were determined as described in MATERIALS AND METHODS. Dashed and solid lines represent best fits to a straight line equation. D: summary graph of mean (±SE) FRET values for neurons expressing EYFP-ß3/CFP-{gamma}2 and EYFP-ß3-s/CFP-{gamma}2. **P < 0.01 (Student’s t-test). Numbers in parentheses indicate the number of experiments.

 
Emission measurements were recorded next in neurons coexpressing donor and acceptor. Following excitation of the cells with the FRET filter, emission at 480 nm (Df) and 535 nm (Ff) was measured. When corrected for cross talk from both donor and acceptor, the Ff/Df ratio represents the measure of FRET between both fluorophores. Figure 5C shows the plot of Ff emission as a function of Df emission in neurons expressing EYFP-Gß3/CFP-G{gamma}2 (solid circles) and EYFP-Gß3-s/CFP-G{gamma}2 (open circles). The slopes of both fitted lines for cells expressing EYFP-Gß3/CFP-G{gamma}2 and EYFP-Gß3-s/CFP-G{gamma}2 were 0.345 ± 0.008 (n = 16) and 0.309 ± 0.002 (n = 16), respectively. A change in slope between both groups of cells can be observed. Figure 5D is a bar graph showing the mean FRET values for EYFP-Gß3/CFP-G{gamma}2- and EYFP-Gß3-s/CFP-G{gamma}2-expressing cells. Neurons expressing EYFP-Gß3/CFP-G{gamma}2 had a significantly greater (P < 0.01, Student’s t-test) FRET value than cells expressing EYFP-Gß3-s/CFP-G{gamma}2. In fact, the FRET values obtained in cells expressing wild-type EYFP-Gß3 are comparable to those measured previously in cells expressing EYFP-Gß1/CFP-G{gamma}2 (41). On the other hand, the values measured with EYFP-Gß3-s/CFP-G{gamma}2-expressing neurons were similar to those groups of cells where no FRET occurred (41). Therefore, the absence of FRET between EYFP-Gß3-s and ECFP-G{gamma}2 suggests that the coexpressed subunits do not interact in vivo.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
G proteins couple seven transmembrane receptors to several intracellular effector proteins, including ion channels, phospholipases, and adenylyl cyclases. A recent study described a C825T polymorphism in the GNB3 gene that codes for Gß3 (45). The GNB3 C825T allele was found to be associated with the expression of a splice variant, named Gß3-s. A result of this splicing event is the deletion of 124 base pairs, which leads to a shortened Gß3 by 41 amino acids, including one WD domain. Conflicting views regarding the presence of the C825T allele and its correlation with several vascular disorders exist. Nonetheless, the actual effect Gß3-s exerts on G protein signaling mechanisms remains unresolved. Various studies, which have shown an association between C825T and diseased states, have regarded Gß3-s as a functional protein with an enhanced activity (33, 52, 53). However, little direct evidence exists that demonstrates Gß3-s possesses modulatory activity.

Evidence available in the literature, which suggests Gß3-s is a functional and overactive G protein subunit, is rather indirect. Overall, there are three main observations that have led investigators to this conclusion. First, G protein signaling pathways in neutrophils and platelets were found to be enhanced in individuals carrying the allele compared with noncarriers. That is, neutrophils of C825T allele carriers had a significantly greater N-formyl-methionyl-leucyl-phenylalanine (fMLP)- and interleukin-8-induced chemotaxis (52, 53). Chemotaxis in all three genotypes was PTX sensitive, suggesting coupling through either G{alpha}i or G{alpha}o. Moreover, a significantly greater epinephrine-induced platelet aggregation in those persons carrying the C825T allele was also reported (33). Unfortunately, no direct evidence was provided to show Gß3-s caused both the enhanced chemotaxis and platelet aggregation. In the present study, we took advantage of our expression system to determine whether the heterologous expression of Gß3-s or EYFP-Gß3-s with G{gamma}2 or G{gamma}5 would result in basal inhibition of N-type Ca2+ channels. Previously, our laboratory has shown that overexpression of Gß1-ß4 with several G{gamma} subunits results in tonic Ca2+ current inhibition (40, 41). Figure 2 shows that overexpression of Gß3-s with either {gamma}-subunit did not produce tonic modulation of Ca2+ currents. Tagging Gß3-s with the EYFP fluorophore to the NH2 terminus provided evidence that expression was successful (Fig. 1B). On the other hand, expression of wild-type Gß3 or EYFP-Gß3 with G{gamma}2 or G{gamma}5 resulted in basal inhibition of Ca2+ currents (Fig. 2, B and D). Similar modulatory effects of Gß subunits on Ca2+ channels have been previously reported (1, 40, 56). Therefore in this expression system, Gß3-s or EYFP-Gß3-s does not modulate N-type Ca2+ channels.

The second observation, which attributes a functional characteristic to Gß3-s, involved a link between the C825T allele and the amplitude of inwardly rectifying currents in right atrial myocytes (12). Employing the patch-clamp technique, it was shown that patients with the TT genotype had a significantly higher background inward rectifier current (IKI) and a reduced carbachol-activated GIRK current amplitude when compared with CC and CT genotypes. These findings were proposed to result from tonic activation of GIRK channels by Gß3-s, such that carbachol-activated GIRK currents were diminished. Once again, no direct proof was presented to demonstrate that Gß3-s caused the tonic activation of IKI or GIRK currents. The ability of Gß3-s to basally activate GIRK channels in a sympathetic neuron preparation was tested in the present study. It is now widely accepted that overexpression of Gß1–4 with several G{gamma} results in tonic GIRK channel activation (29, 41, 42). However, the expression of Gß3-s/G{gamma}2 did not lead to basal activation of GIRK currents (Fig. 3). On the other hand, Gß3/G{gamma}2-expressing neurons exhibited tonic GIRK channel activation. Consequently, Gß3-s does not seem to activate GIRK channels.

Although Gß3-s did not tonically modulate ion channels, reconstitution experiments were undertaken to determine whether Gß3-s/G{gamma}5 could form a heterotrimer with GDP-bound G{alpha} subunits. In this type of experiments, balanced expression of all three G protein subunits reconstitutes the NE-mediated VD inhibition of Ca2+ currents (24, 41). As a positive control, one group of neurons was coinjected with PTX-insensitive G{alpha}i2, EYFP-Gß3, and G{gamma}5 cDNA. Reconstitution was observed in 6 of 21 cells (Fig. 4, C and E). In the remaining neurons, expression levels of G{alpha}i2 (C352G) appeared to predominate based on lower basal facilitation values and decreased NE-mediated inhibition of Ca2+ currents. This is presumably a result of GDP-bound G{alpha} sequestering both native and expressed Gß{gamma}. On the other hand, reconstitution could not be measured in cells expressing EYFP-ß3-s, because of its inability to modulate Ca2+ channels (Fig. 2 and 4D). Nonetheless, one group of neurons was injected with a 10-fold higher ratio of EYFP-Gß3-s/G{gamma}5 to G{alpha}i2 (C352G) cDNA, thus favoring conditions for greater expression levels of Gß{gamma} than G{alpha}. Under these circumstances, it was anticipated that if heterotrimer formation occurred between GDP-bound G{alpha}i2 and Gß3-s/G{gamma}5, then a saturation of G{alpha}’s buffering effect would be observed. However, in this group of cells, basal facilitation values ranged from 0.95 to 1.17. Thus, despite putatively higher expression levels of EYFP-Gß3-s/G{gamma}5, G{alpha} retained the ability to sequester native Gß{gamma} subunits. These results suggest that Gß3-s is not capable of forming a heterotrimer with G{gamma}5 and PTX-resistant G{alpha}i2. The lack of heterotrimer formation also indicates that Gß3-s lacks the capacity to couple to {alpha}2-AR. It should be noted that heterotrimer formation between the three subunits cannot be completely ruled out, because this set of experiments examined N-type Ca2+ channel modulation following {alpha}2-AR activation. Lastly, heterotrimer formation was only tested with PTX-resistant G{alpha}i2 (C352G) and G{gamma}5. Whether Gß3-s is able to form a heterotrimer with other G{alpha} and G{gamma} subunits remains to be determined.

Finally, the third observation, which attributes Gß3-s to have a functional phenotype, was initially described by Siffert and colleagues (45). Western blot analysis showed that Gß3-s was predominantly expressed in cultured lymphoblasts and platelets from individuals carrying the C825T allele (either CT or CC genotype) but not in patients lacking the allele. Surprisingly, it was found that the heterotrimers, G{alpha}i2/ß3-s/ G{gamma}5 and G{alpha}i2/ß3/G{gamma}5 expressed in Sf9 insect cells, possessed similar functional properties as measured by [35S]GTP{gamma}S binding assays. However, two recent studies have reported that in the same expression system (i.e., Sf9 cells), the expressed Gß3-s could not be purified and thus not reconstituted with several {gamma}-subunits (31, 32). These differences may be a result of different assay conditions employed.

The lack of ion channel modulation and heterotrimer formation of Gß3-s-expressing neurons prompted us to employ FRET analysis to examine whether Gß3-s forms a dimer with G{gamma} subunits. Interaction of proteins separated by distances of 10–100 Å can be determined by FRET. Recently, we reported that coexpression of EYFP-Gß1 and ECFP-G{gamma}2 results in FRET (41). Moreover, tagging the Gß subunits did not impair modulation of N-type Ca2+ or GIRK channels. Figure 5 shows that coexpressed EYFP-Gß3/CFP-G{gamma}2 were in sufficiently close proximity to produce FRET. Similar FRET values were obtained in EYFP-Gß1/CFP-G{gamma}2-expressing cells (41). On the other hand, FRET could not be detected in cells expressing EYFP-Gß3-s/CFP-G{gamma}2 (Fig. 5D). Furthermore, the FRET values obtained with EYFP-Gß3-s/CFP-G{gamma}2-expressing neurons were nearly identical to cells expressing EYFP-Gß3-s alone. Therefore, it appears that coexpression of EYFP-Gß3-s and ECFP-G{gamma}2 does not result in heterodimer formation. The deletion of 41 amino acids from Gß3-s, coupled with the inability of the subunit to form Gß{gamma} dimers, may help explain the absence of modulation. Alternatively, the shortened Gß3-s subunit may fold differently from wild-type Gß3, causing a disruption of the proper orientation or distance necessary for FRET to occur between both fluorophores. Therefore, it might still be possible for dimer formation to occur in the absence of FRET. Thus Gß3-s dimer formation is not definitively ruled out by these experiments.

In conclusion, electrophysiological experiments demonstrate that heterologous expression of the Gß3 splice variant, Gß3-s, with G{gamma} subunits does not result in ion channel modulation in rat SCG neurons. FRET studies suggest that Gß3-s does not form a heterodimer with G{gamma} subunits. Hence, the pathophysiological effects of the C825T allele may result from a "functional knockout" of Gß3, i.e., something missing rather than something with too much activity.


    ACKNOWLEDGMENTS
 
We thank Marina M. King and Linda Olmstead for excellent technical assistance and Dr. D .E. Logothetis (Mount Sinai School of Medicine, New York, NY) for GIRK1 and GIRK4 cDNA clones.

This work was supported by National Institutes of Health Grant GM-56180 to S. R. Ikeda and MH-12288 (to V. Ruiz-Velasco).

Present address of S. R. Ikeda: NIH/NIAAA/DICBR/LMP, Park Building, Room 150, 12420 Parklawn Dr MSC 8115, Bethesda, MD 20892-8115 (E-mail: sikeda{at}mail.nih.gov).


    FOOTNOTES
 
Article published online before print. See web site for date of publication ( http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: V. Ruiz-Velasco, Laboratory of Molecular Physiology, One Guthrie Square, Guthrie Research Institute, Sayre, PA 18840 (E-mail: vruizvel{at}inet.guthrie.org).

10.1152/physiolgenomics.00057.2002.


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