The orthosteric agonist 2-chloro-5-hydroxyphenylglycine activates mGluR5 and mGluR1 with similar efficacy and potency
The efficacy, potency, and selectivity of the compound 2-Chloro-5-hydroxyphenylglycine (CHPG), a nominally selective agonist for metabotropic glutamate receptor 5 (mGluR5), were examined with select mGluRs by examining their ability to induce modulation of the native voltage dependent ion channels in isolated sympathetic neurons from the rat superior cervical ganglion (SCG). SCG neurons offer a null mGluR-background in which specific mGluR subtypes can be made to express via intranuclear cDNA injection.
Consistent with previous reports, CHPG strongly activated mGluR5b expressed in SCG neurons with an apparent EC50 around 60 μM. Surprisingly, CHPG also activated two mGluR1 splice variants with a similar potency as at mGluR5 when calcium current inhibition was used as an assay for receptor function. No effect of 1 mM CHPG was seen in cells expressing mGluR2 or mGluR4, suggesting that CHPG only activates group I mGluRs (mGluR1 and 5). CHPG was also able to induce modulation of M-type potassium current through mGluR1, but not as consistently as glutamate. Since this channel is modulated through a Gq-dependent pathway, these data indicate that CHPG may exhibit some biased agonist properties on mGluR1. Closer examination of the voltage-independent, Gq-mediated component of mGluR-induced calcium current modulation data confirmed that some biased agonism was evident, but the effect was weak and inconsistent.
These data contrast with the established literature which suggests that CHPG is a selective mGluR5 agonist. Instead, CHPG appears to act equally well as an agonist at mGluR1. While some weak biased agonism was observed with CHPG acting on mGluR1, but not mGluR5, favoring Gi/o signaling over Gq/11, this effect does not appear sufficient to fully explain the discrepancies in the literature.
KeywordsInhibition Ratio Superior Cervical Ganglion Full Agonist Current Inhibition Patch Clamp Amplifier
Metabotropic glutamate receptor
Superior cervical ganglion
Metabotropic glutamate receptors (mGluRs) are important mediators of learning and excitability [1, 2], sensory signal transduction , and central information processing that play a critical role in many physiological and pathological processes . The group I mGluRs, which includes mGluR1 and 5, exhibit widespread expression in the central nervous system where they are generally expressed postsynaptically in addition to their dendro-somatic localization, where they can initiate some forms of plasticity and regulate neuronal excitability, respectively [1, 2]. mGluR1 and 5 are similar in sequence, G protein coupling and in their responses to many pharmacological compounds. While their expression profiles in the brain are distinct, mGluR1 and 5 serve analogous roles in different regions. For example, hippocampal mGluR5 localized near the postsynaptic density can initiate a form of long term depression (mGluR-LTD) [1, 5, 6]. Likewise, postsynaptic mGluR1 in the cerebellum can also produce mGluR-LTD [1, 2, 7]. In both cases, initiation of plasticity requires coupling to Gq/11 proteins and post-synaptic localization, although the mechanistic details of each phenomena are distinct. Further, both mGluR1 and 5 can couple to modulation of voltage gated calcium and other ion channels in several neuronal cell types [8, 9, 10, 11], leading to changes in cell excitability.
Our understanding of the role of these receptors derives in large part from the pharmacological tools used to manipulate their function. In recent years, many highly selective compounds have been developed that target mGluRs at allosteric sites, separate from the endogenous glutamate ligand binding site. Fewer highly selective orthosteric compounds are available. One exception is the compound 2-Chloro-5-hydroxyphenylglycine (CHPG), an orthosteric ligand that has been used extensively as a selective mGluR5 agonist . At least one report indicates that CHPG is selective for mGluR5 over even the closely related mGluR1, making it the only known orthosteric agonist with such selectivity . However, the selectivity of CHPG for mGluR5 over mGluR1 has not been subsequently tested, to my knowledge.
Here, selectivity of CHPG was examined by testing its ability to activate specific mGluRs expressed by intranuclear cDNA injection in sympathetic neurons from the rat superior cervical ganglion (SCG), a primary neuronal cell with a null mGluR background . Modulation of the native voltage-dependent calcium currents in SCG neurons was used as an assay for heterologously expressed mGluRs. The ability of a range of concentrations of CHPG to activate mGluR5, mGluR2, mGluR4, and two splice variants of mGluR1 (a and b) was tested. Consistent with previous reports in the literature, we found that CHPG functioned as a full agonist at mGluR5 and failed to activate mGluR2, or mGluR4. Surprisingly however, CHPG also functioned as a full agonist at both mGluR1a and mGluR1b with similar potency as mGluR5.
Cell isolation, DNA injection and Plasmids
A description of cell isolation and cDNA injection is found elsewhere . Animal protocols were approved by the university committee on animal resources (UCAR). Briefly, SCGs were removed from adult male Wistar rats (175–225 g) after CO2 euthanasia and decapitation, then incubated in Earle’s balanced salt solution (InVitrogen, Life Technologies Carlsbad, CA) containing 0.6 mg/ml trypsin (Worthington Biochemicals, Freehold, NJ) & 0.8 mg/ml collagenase D (Boehringer Mannheim Biochemicals, Indianapolis, IN) for 60 min at 35°C. Cells were transferred to minimum essential medium (InVitrogen/Gibco), plated on poly-l-lysine (Sigma Chemical Co., St. Louis, MO) coated culture dishes and incubated at 37°C for 2–4 hours before cDNA injection. Injected cells were incubated overnight at 37°C (95% air and 5% CO2; 100% humidity) and patch clamp experiments were performed the next day.
Injection of cDNA was performed with an Eppendorf 5247 microinjector and InjectMan NI 2 micromanipulator (Madison, WI) 3–5 hours following cell isolation. Injection electrodes were made with a Sutter P-97 horizontal electrode puller (Novato, CA) from thin-walled, borosilicate glass (World Precision Instruments, Sarasota, FL). Plasmids were stored at −20°C as a 0.4 - 1 μg/μl stock solution in TE buffer (10 mM TRIS, 1 mM EDTA, pH 8). All mGluR constructs were injected at 100–130 ng μl-1 (pCDNA3.1+; InVitrogen). The mGluR4 clone was provided by D. Hampson (University of Toronto, Toronto, Onatrio, Canada). All neurons were co-injected with “enhanced” green fluorescent protein cDNA (0.02 μg/μl; pEGFPN1; BD Biosciences-Clontech, Palo Alto, CA) for identification of successfully injected cells.
All constructs were sequence confirmed. PCR products were purified with Qiagen (Valencia, CA) silica membrane spin columns prior to restriction digestion and ligation. Midipreps were prepared using Qiagen anion exchange columns, and amplified in either Top10 or DH5α E. coli. (InVitrogen).
Electrophysiology and data analysis
Pipettes for patch-clamp experiments were made with a Sutter P-97 horizontal puller from 8250 glass (Garner Glass, Claremont, CA) and had resistances of 1–3 MΩ. Series resistances were 2.3 ± 0.2 MΩ (n = 31) prior to electronic compensation of 80%. Whole-cell patch-clamp recordings were made with an EPC-7 patch clamp amplifier (Heka Elektronik, Germany). Voltage protocol generation and data acquisition were performed using custom software (courtesy Stephen R. Ikeda, NIAAA, Rockville, MD) on a Macintosh G4 computer (Apple Computer, Cupertino, CA) with an InstruTech (Port Washington, NY; now Heka Elektronik) ITC-16 data acquisition board. Currents were low-pass filtered at 3 kHz using the 4-pole Bessel filter in the patch clamp amplifiers, digitized at 2–5 kHz and stored on the computer for later analysis. Experiments were performed at 21–24°C (room temperature). Patch-clamp data analysis was performed using the Igor Pro software package (Wavemetrics, Lake Oswego, OR).
The external (bath) recording solution contained (in mM): 155 tris hydroxymethyl aminomethane, 20 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 glucose, 10 CaCl2, and 0.0003 tetrodotoxin (TTX), pH 7.4. The internal (pipette) solution contained: 120 N-methyl-D-glucamine (NMG) methanesulfonate, 20 TEA, 11 EGTA, 10 HEPES, 10 sucrose, 1 CaCl2, 4 MgATP, 0.3 Na2GTP, and 14 tris creatine phosphate, pH 7.2. l-Glutamate (Sigma) was used as the agonist for mGluRs. CHPG was obtained from two sources: Tocris Bioscience (Ellisville, MO) and Ascent Scientific (Avonmouth, Bristol, UK). All drugs and control solutions were applied to cells using a custom, gravity-driven perfusion system positioned ~100 μm from the cell, allowing rapid solution exchange (≤ 250 ms). The degree of calcium current inhibition was calculated as the maximum current inhibition in the presence of drug compared to the last current measurement prior to drug application.
CHPG activates mGluR5
Effect of CHPG on other mGluRs
Efficacy of CHPG on mGluR5 and mGluR1
CHPG shows weak biased agonist properties when acting on mGluR1 but not mGluR5
As noted previously , the contribution of the voltage dependent and voltage independent calcium current inhibitory pathways via group I mGluRs in SCG neurons is quite variable. Further, based on the M-current data (Figure 6), it was expected that any differences in Gq/11 activation by CHPG and Glu would be inconsistent. Therefore, to insure that any changes would be detectable, analysis was restricted to cells in which the Post/Pre inhibition ratio by 100 μM Glu was 0.35 or higher, because these cells had a clearly detectable Gq/11 component. Figure 7A shows sample current traces illustrating the voltage dependence of inhibition by 100 μM Glu (upper) and 1 mM CHPG (lower) in SCG neurons expressing either mGluR5b (left) or mGluR1a (right). Figure 7B shows a plot of the Post/Pre inhibition ratio for Glu and CHPG in each group for every cell in which the ratio was > 0.35. While there was substantial variability in the ratios from cell to cell, and in the differences in ratios between Glu and CHPG, the Post/Pre inhibition ratios for Glu and CHPG in mGluR5-expressing cells was statistically indistinguishable. A small but statistically significant difference was observed however, in mGluR1a expressing cells (paired T-test, p ≤ 0.05) when comparing the inhibition by CHPG to that by Glu by providing more evidence that CHPG is a poorer activator than Glu of the Gq/11 pathway in some cells. These data lend some support for the ability of CHPG to act as a biased agonist on mGluR1, but both the calcium current and M-current inhibition data demonstrate that CHPG is capable of activating Gq/11, even fairly strongly, in some cells.
The data presented here indicate that contrary to previous reports , the nominally selective agonist CHPG can activate mGluR1 with similar efficacy and potency as mGluR5. Consistent with the literature however, CHPG did not produce any detectable activation of mGluR2 or mGluR4. The effects of CHPG were examined using heterologous expression of each receptor in rat sympathetic neurons, an adult neuronal cell type with null-mGluR expression, and assayed using G protein mediated modulation of native ion channel currents as an assay for receptor signaling. Further, using M-current inhibition as an assay for receptor function, a pathway that depends only on Gq signaling, revealed that in some cells, CHPG agonism of mGluR1 appeared to show some biased agonism. Specifically, while some mGluR1-expressing cells showed similar M-current inhibition using CHPG or Glu as an agonist, others were strongly inhibited by Glu, but only very weakly by CHPG. These data provide some contrast to those obtained using calcium current inhibition as an assay, which proceeds through a combination of Gq/11 and pertussis toxin sensitive Gβγ activation . Examination of calcium current inhibition by CHPG in mGluR1 expressing cells revealed that the Gq/11-mediated, voltage independent inhibitory pathway was not as strongly activated by CHPG as Glu, supporting the hypothesis that CHPG is a poorer Gq/11 activator than Glu. However, the difference was not robust. Thus, while these data indicate that CHPG can effectively act as an agonist at mGluR1a and its splice variant mGluR1b, the overall balance of Gi/o/Gq/11 protein activation may be altered when CHPG rather than Glu is used as the agonist.
Given the effect of CHPG on mGluR1a, it was not surprising that the drug had similar effects on mGluR1b since both splice variants are identical in the N-terminal, ligand binding region. In fact, these proteins differ only in their extreme cytoplasmic C-termini, which is not expected to alter receptor pharmacology. Furthermore, there is no evidence that G protein activation differs in these splice variants, as both mGluR1a and 1b can produce qualitatively similar calcium current and M-current inhibition in SCG neurons . Effects of CHPG on both variants was tested primarily to confirm the rather surprising result of CHPG agonism on mGluR1, a result which has not been previously reported despite fairly widespread use of this drug for over a decade [12, 26, 27, 28, 29]. Indeed, the mGluR1a data shown in Figure 3B is combined data from SCG neurons expressing mGluR1a from two separate, but similar, plasmids. One is an untagged rat mGluR1a, and the other is an N-terminally myc-tagged mGluR1a, both in pCDNA3.1. The data were combined because CHPG acted identically on cells expressing both constructs (not shown). Both constructs (as well as mGluR1b, in pRK5) were tested for responses to CHPG, and sequence-verified. Finally, it should be noted that the results with the mGluR1 constructs were generated using CHPG from two separate sources (Tocris and Ascent, see Materials and Methods) with indistinguishable results (not shown).
The data presented here indicate that contrary to current dogma, the nominally selective mGluR5 agonist CHPG can act as an agonist for mGluR1 and 5 with similar efficacy and potency, although under some circumstances CHPG may be a poorer activator than Glu of Gq/11 via mGluR1 compared to mGluR5.
All experiments, analysis, writing and editing of the manuscript were performed by PJK.
Parts of this work were supported by NIH grant GM101023.
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