Laboratory of Molecular Physiology, Guthrie Research Institute, Sayre, Pennsylvania 18840
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ABSTRACT |
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Gß3-s; signal transduction; ion channel modulation; FRET; N-type calcium channels
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INTRODUCTION |
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Of the five Gß subunits cloned thus far, Gß14 share an 8090% 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
-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
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 Gi 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
heterodimer (i.e., Gß
) and, consequently, a G
ß
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
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
2. As a positive control, parallel experiments were undertaken with wild-type Gß3.
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MATERIALS AND METHODS |
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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 (175225 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) 35 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 1218 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 510 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 (8085%) were electronically compensated. Experiments were performed at room temperature (2124°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 299308 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 319327 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 305308 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 (1220 h) at a concentration of 500 ng/ml.
Fluorescence resonance energy transfer analysis.
FRET was employed to detect the interaction between heterologously expressed ECFP-G2 (donor) with either EYFP-Gß3 or EYFP-Gß3-s (acceptor) fusion proteins. Neurons expressing both fusion proteins were identified 1220 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 Students 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).
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RESULTS |
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Figure 2B shows Ca2+ current traces from a cell expressing Gß3/G5. 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
2 and EYFP-Gß3/G
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
5, expression of Gß3-s/G
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 Gß
subunits. Expression of EYFP-Gß3-s/G
5 and EYFP-Gß3-s/G
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 expression, experiments were undertaken to determine whether Gß3-s could modulate an additional "target" for Gß
subunits, GIRK-type K+ channels. GIRK channels are inwardly rectifying K+ channels that are activated by Gß
(11). Previous studies from our laboratory and others have shown that overexpression of Gß14 with several G
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
2 (Fig. 3B) and EYFP-Gß3-s/G
2 (Fig. 3C), respectively. Expression of EYFP-Gß3/G
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
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
2 and EYFP-Gß3-s/G
2. Mean basal GIRK current was similar in cells expressing GIRK1 and 4, GIRK1 and 4 + Gß3-s/G
2 and GIRK1 and 4 + Gß3-s/G
5, but significantly greater in GIRK1 and 4 + EYFP-Gß3/G
2- and GIRK1 and 4 + Gß3-s/G
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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Reconstitution was determined in a total of 21 neurons following coinjection of EYFP-Gß3, G5, and G
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
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
i2 (C352G) and EYFP-Gß3/G
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
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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FRET analysis of EYFP-Gß3/CFP-G2 and EYFP-Gß3-s/CFP-G
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-G2 occurred. FRET involves the transfer of energy from an excited fluorophore (CFP-G
2, donor) to a second fluorophore (YFP-Gß3, acceptor) in a nonradiative manner. For FRET to occur, both fluorophores must be in close proximity (10100 Å) and in the proper orientation (36). Recently, we reported that expression of EYFP-Gß1 and ECFP-G
2 in SCG neurons resulted in FRET following the excitation of ECFP-G
2 (41). Therefore, in the present study, similar FRET measurements were undertaken with EYFP-ß3 and ECFP-
2, as a positive control. In this set of experiments, excitation (at 440 nm) of ECFP-G
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
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-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
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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DISCUSSION |
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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 Gi or G
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
2 or G
5 would result in basal inhibition of N-type Ca2+ channels. Previously, our laboratory has shown that overexpression of Gß1-ß4 with several G
subunits results in tonic Ca2+ current inhibition (40, 41). Figure 2 shows that overexpression of Gß3-s with either
-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
2 or G
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 Gß14 with several G results in tonic GIRK channel activation (29, 41, 42). However, the expression of Gß3-s/G
2 did not lead to basal activation of GIRK currents (Fig. 3). On the other hand, Gß3/G
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/G5 could form a heterotrimer with GDP-bound G
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
i2, EYFP-Gß3, and G
5 cDNA. Reconstitution was observed in 6 of 21 cells (Fig. 4, C and E). In the remaining neurons, expression levels of G
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
sequestering both native and expressed Gß
. 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
5 to G
i2 (C352G) cDNA, thus favoring conditions for greater expression levels of Gß
than G
. Under these circumstances, it was anticipated that if heterotrimer formation occurred between GDP-bound G
i2 and Gß3-s/G
5, then a saturation of G
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
5, G
retained the ability to sequester native Gß
subunits. These results suggest that Gß3-s is not capable of forming a heterotrimer with G
5 and PTX-resistant G
i2. The lack of heterotrimer formation also indicates that Gß3-s lacks the capacity to couple to
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
2-AR activation. Lastly, heterotrimer formation was only tested with PTX-resistant G
i2 (C352G) and G
5. Whether Gß3-s is able to form a heterotrimer with other G
and G
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, Gi2/ß3-s/ G
5 and G
i2/ß3/G
5 expressed in Sf9 insect cells, possessed similar functional properties as measured by [35S]GTP
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
-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 subunits. Interaction of proteins separated by distances of 10100 Å can be determined by FRET. Recently, we reported that coexpression of EYFP-Gß1 and ECFP-G
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
2 were in sufficiently close proximity to produce FRET. Similar FRET values were obtained in EYFP-Gß1/CFP-G
2-expressing cells (41). On the other hand, FRET could not be detected in cells expressing EYFP-Gß3-s/CFP-G
2 (Fig. 5D). Furthermore, the FRET values obtained with EYFP-Gß3-s/CFP-G
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
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
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 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
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.
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ACKNOWLEDGMENTS |
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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).
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FOOTNOTES |
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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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References |
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