Monomers and oligomers of the M2 muscarinic cholinergic receptor purified from Sf9 cells

G protein-coupled receptors are known to form oligomers. To probe the nature of such aggregates, as well as the role and prevalence of monomers, epitope-tagged forms of the M(2) muscarinic receptor have been isolated as oligomers and monomers from Sf9 cells. Membranes from cells coexpressing the c-Myc- and FLAG-tagged receptor were solubilized in digitonin-cholate, and the receptor was purified by successive passage through DEAE-Sepharose, the affinity resin 3-(2'-aminobenzhydryloxy)tropane (ABT)-Sepharose, and hydroxyapatite. Coimmunoprecipitation of the two epitopes indicated the presence of oligomers at each stage of the purification up to but not including the fraction eluted specifically from ABT-Sepharose. The affinity-purified receptor therefore appeared to be monomeric. The failure to detect coimmunoprecipitation was not due to an ineffective antibody, nor did the conditions of purification appear to promote disaggregation. Receptor at all stages of purification bound N-[(3)H]methylscopolamine and [(3)H]quinuclidinylbenzilate with high affinity, but the capacity of receptors that were not retained on ABT-Sepharose was only 4% of that expected from densitometry of western blots probed with an anti-M(2) antibody. Similarly low activity was found with oligomers isolated by successive passage of coexpressed receptor on anti-c-Myc and anti-FLAG immunoaffinity columns. M(2) muscarinic receptors therefore appear to coexist as active monomers and largely or wholly inactive oligomers in solubilized extracts of Sf9 cells. A different pattern emerged when coinfected cells were treated with quinuclidinylbenzilate prior to solubilization, in that ABT-purified receptors from those cells exhibited coimmunoprecipitation. Treatment with the antagonist therefore led to oligomers in which at least some of the constituent sites were active and were retained by ABT-Sepharose.     

Characterization of M2 muscarinic receptor activation of different G protein subtypes

The M2 muscarinic acetylcholine receptor (mAChR) expressed in insect cells (Spodoptera frugiperda, Sf9) using the baculovirus system formed active functional complexes with coexpressed Gi as well as with Go proteins, while no complexes could be detected with internal G proteins. Comparison of the abilities of different muscarinic agonists and partial agonists to increase [35S]GTPgammaS binding revealed no significant differences between M2/Gi and M2/Go complexes neither with respect to affinities nor efficacies of the ligands studied. Coexpression with either G protein caused constitutive activity of the receptor amounting up to 66% of stimulable [35S]GTPgammaS binding. Muscarinic antagonists, like atropine, scopolamine and N-methylscopolamine, behaved as inverse antagonists with potencies in good agreement with their binding affinities to the receptor. The results implicate that the functional reconstitution of M2 muscarinic receptor with either Gi or Go proteins in insect cells provides a valuable tool for screening of potencies as well as efficacies of agonists, partial agonists and inverse agonists at this receptor.     

Expression of Functional Human Muscarinic M2 Receptors in Different Insect Cell Lines

Abstract

Human M2 receptors were expressed using the baculovirus expression system in three different insect cell lines: Sf9, Sf21 and High5. The level of expression was slightly increased in Sf21 cells versus Sf9 cells. In contrast, High5 cells were not able to produce more recombinant protein than Sf9. We also show that in both Spodoptera frugiperda cell lines a peak of expression was reached after 6 days of infection, whereas in High5 cells, the maximum of expression occurred after 3 days. Immunodetection of m2 muscarinic receptor clearly shows that the expressed protein undergoes significant proteolysis in both the Sf9 and High5 cells, whereas in the Sf21 cells this phenomenon was less detectable. Additionally, we show that in all three cell lines, the expressed recombinant receptor was functional in that it was able to stimulate GTPγS binding in the presence of exogenous G-proteins. Analysis of the population of G-proteins (Gαi₁ Gα_o and Gβcommon) in Sf21 and High5 cells is provided.     

Expression of functional M2 muscarinic acetylcholine receptor in Escherichia coli

The M2 muscarinic acetylcholine receptor mutant (M2 mutant), with a lack of glycosylation sites, a deletion in the central part of the third inner loop, and the addition of a six histidine tag at the C-terminus, was fused to maltose binding protein (MBP) at its N-terminus and expressed in Escherichia coli. The expression level was 0.2 nmol receptor per 100 ml culture, as assessed as [3H]L-quinuclidinyl benzilate ([3H]QNB) binding activity, when the BL 21 strain was cultured at 37 degrees C to a late growth phase and the expression was induced by isopropyl beta-thiogalactoside at 20 degrees C. No [3H]QNB binding activity was detected when it was not fused to MBP or when expression was induced at 37 degrees C instead of 20 degrees C. The MBP-M2 mutant expressed in E. coli showed the same ligand binding activity as the M2 mutant expressed in the Sporodoptera frugiperda (Sf9)/baculovirus system, as assessed as displacement of [(3)H]QNB with carbamylcholine and atropine. The MBP-M2 mutant was solubilized, purified with Co2+-immobilized Chelating Sepharose gel and SP-Sepharose, and then reconstituted into lipid vesicles with G protein Go or Gi1 in the presence or absence of cholesterol. The reconstituted vesicles showed GTP-sensitive high affinity binding for carbamylcholine and carbamylcholine-stimulated [35S]GTP gamma S binding activity in the presence of GDP. The proportion of high affinity sites for carbamylcholine and the extent of carbamylcholine-stimulated [(35)S]GTP gamma S binding were the same as those observed for the M2 mutant expressed in Sf9 cells and were not affected by the presence or absence of cholesterol. These results indicate that the MBP-M2 mutant expressed in E. coli has the same ability to interact with and activate G proteins as the M2 mutant expressed in Sf9, and that cholesterol is not essential for the function of the M2 muscarinic receptor.     

Constitutive oligomerization of human D2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9) and in HEK293 cells. Analysis using co-immunoprecipitation and time-resolved fluorescence resonance energy transfer.

Human D2Long (D2L) and D2Short (D2S) dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus (HIV) or a FLAG epitope tag. The receptors were then expressed in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer (FRET). [3H]Spiperone labelled D2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D2L or D2S receptors, with a pKd value of approximately 10. Co-immunoprecipitation using antibodies specific for the tags showed constitutive homo-oligomerization of D2L and D2S receptors in Sf9 cells. When the FLAG-tagged D2S and HIV-tagged D2L receptors were co-expressed, co-immunoprecipitation showed that the two isoforms can also form hetero-oligomers in Sf9 cells. Time-resolved FRET with europium and XL665-labelled antibodies was applied to whole Sf9 cells and to membranes from Sf9 cells expressing epitope-tagged D2 receptors. In both cases, constitutive homo-oligomers were revealed for D2L and D2S isoforms. Time-resolved FRET also revealed constitutive homo-oligomers in HEK293 cells expressing FLAG-tagged D2S receptors. The D2 receptor ligands dopamine, R-(-)propylnorapomorphine, and raclopride did not affect oligomerization of D2L and D2S in Sf9 and HEK293 cells. Human D2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can be detected by different approaches, and D2 oligomerization in these cells is not regulated by ligands.     

Recombinant expression of a selective blocker of M(1) muscarinic receptors

Mamba venoms contain peptides with high selectivity for muscarinic receptors. Due to the limited availability of the M(1) muscarinic receptor-selective MT7 or m1-toxin 1, the peptide was expressed in Sf9 cells using a synthetic cDNA and purified. The isolated peptide had over four orders of magnitude higher affinity for the M(1) compared to M(2)-M(5) muscarinic receptors. The peptide strongly inhibited Ca(2+) mobilisation through recombinant and endogenously expressed M(1) receptors, having no effect on the function of the other subtypes. The MT7 peptide provides a unique tool for identification and functional characterisation of M(1) receptors in cells and tissues.     

Agonist-induced phosphorylation and desensitization of human m2 muscarinic cholinergic receptors in Sf9 insect cells.

The human m1 (hm1) and m2 (hm2) muscarinic cholinergic receptors (mAChR) expressed in Sf9 insect cells using recombinant baculovirus were tested for their ability to undergo agonist-dependent phosphorylation and desensitization. The muscarinic agonist carbachol induced phosphorylation of the hm2 mAChR in the Sf9 cells incubated with 32P(i) to an extent of 4-5 mol of phosphate/mol of receptor. In contrast, no phosphorylation of the hm1 mAChR was observed. The hm2 mAChR stimulated [35S]GTP gamma S binding to, and GTPase activity of, the insect cell G-proteins. These receptor-mediated activities were reduced by 50% in membranes prepared from agonist-treated cells compared to control, suggesting that the agonist-induced phosphorylation of the hm2 mAChR resulted in desensitization of the receptors. No role for protein kinase C or cyclic nucleotide-dependent kinases in receptor phosphorylation and desensitization was suggested from studies using agents known to modulate the activity of these enzymes. However, pertussis toxin was found to completely eliminate the interaction of the hm2 receptors with the insect cell G-proteins, but did not perturb the ability of carbachol to induce agonist-dependent phosphorylation of the receptors. These results suggested that G-proteins and/or G-protein-activated signalling were not necessary for the agonist-induced phosphorylation of the receptors. Overall, the data indicated that the human m2 (but not the human m1) mAChR expressed in Sf9 insect cells undergo phosphorylation and desensitization in an agonist-dependent, G-protein-independent fashion by an endogenous insect cell kinase. The results demonstrated that a human G-protein-linked receptor is regulated in insect cells in a manner that is similar to that involving members of the G-protein receptor-kinase family.     

Reconstitution reveals additional roles for N- and C-terminal domains of g(alpha) in muscarinic receptor coupling

The molecular basis for selectivity of M1 and M2 muscarinic receptor coupling to heterotrimeric G proteins has been studied using receptors expressed in Sf9 cell membranes and reconstituted with purified chimeric G(alpha) subunits containing different regions of Gi1alpha and Gq(alpha). The abilities of G protein heterotrimers containing chimeric alpha subunits to stabilize the high-affinity state of the receptors for agonist and to undergo receptor stimulated guanine nucleotide exchange was compared with G protein heterotrimers containing either native Gi1alpha or Gq(alpha). The data confirm the importance of the proper context of the C-terminus of Galpha by demonstrating that the C-terminus of Gi1alpha, when placed in the context of Gq(alpha), prevents coupling to muscarinic M1 receptors, while the C-terminus of Gq(alpha), when placed in the context of Gi1alpha, prevents coupling to muscarinic M2 receptors. However, C-terminal amino acids of Gq(alpha) placed in the context of Gi1alpha were not sufficient to allow M1 receptor coupling, nor were C-terminal amino acids of Gi1alpha placed in the context of Gq(alpha) sufficient for M2 receptor coupling. The unique six amino acid N-terminal extension of Gq(alpha) when added to the N-terminus of Gi1alpha neither prevented M2 receptor coupling nor permitted M1 receptor coupling. A Gi1alpha-based chimera containing both N- and C-terminal regions of Gq(alpha) gained the ability to productively couple M1 receptors suggesting that the proper context of both N- and C-termini is required for muscarinic receptor coupling.     

Recovery of oligomers and cooperativity when monomers of the M2 muscarinic cholinergic receptor are reconstituted into phospholipid vesicles

FLAG- and HA-tagged M2 muscarinic receptors from coinfected Sf9 cells have been purified in digitonin-cholate and reconstituted into phospholipid vesicles. The purified receptor was predominantly monomeric: it showed no detectable coimmunoprecipitation; it migrated as a monomer during electrophoresis before or after cross-linking with bis(sulfosuccinimidyl)suberate; and it bound agonists and antagonists in a manner indicative of identical and mutually independent sites. Receptor cross-linked after reconstitution or after reconstitution and subsequent solubilization in digitonin-cholate migrated almost exclusively as a tetramer. The binding properties of the reconstituted receptor mimicked those reported previously for cardiac muscarinic receptors. The apparent capacity for N-[3H]methylscopolamine (NMS) was only 60% of that for [3H]quinuclidinylbenzilate (QNB), yet binding at saturating concentrations of [3H]QNB was inhibited fully and in a noncompetitive manner at comparatively low concentrations of unlabeled NMS. Reconstitution of the receptor with a saturating quantity of functional G proteins led to the appearance of three classes of sites for the agonist oxotremorine-M in assays with [3H]QNB; GMP-PNP caused an apparent interconversion from highest to lowest affinity and the concomitant emergence of a fourth class of intermediate affinity. All of the data can be described quantitatively in terms of cooperativity among four interacting sites, presumably within a tetramer; the effect of GMP-PNP can be accommodated as a shift in the distribution of tetramers between two states that differ in their cooperative properties. Monomers of the M2 receptor therefore can be assembled into tetramers with binding properties that closely resemble those of the muscarinic receptor in myocardial preparations.     

Affinity profiles of various muscarinic antagonists for cloned human muscarinic acetylcholine receptor (mAChR) subtypes and mAChRs in rat heart and submandibular gland.

A family of five subtypes of muscarinic acetylcholine receptors (mAChR) has been identified based on their molecular structures and second signal transduction pathways. In the present study, we examined the antagonist binding profiles of 9 muscarinic antagonists (atropine, 4-DAMP, pirenzepine, oxybutynin, tiquizium, timepidium, propiverine, darifenacin and zamifenacin) for human muscarinic acetylcholine receptor subtypes (m1, m2, m3, m4 and m5) produced by using a baculovirus infection system in Sf9 insect cells, and rat tissue membrane preparations (heart and submandibular gland). In a scopolamine methyl chloride [N-methyl-3H]- ([3H]NMS) binding assay, pirenzepine and timepidium displayed the highest affinities for the m1 and m2 subtypes, respectively, and both zamifenacin and darifenacin had the highest affinities for the m3 subtype, although the selectivities among the five subtypes were less than 10-fold. Propiverine showed a slightly higher affinity for the m5 subtype, whereas none of the drugs used in this study was uniquely selective for the m4 subtype. The binding affinities of muscarinic antagonists for rat heart and submandibular gland strong correlated with those for human cloned m2 and m3 subtypes, respectively. These data suggest that [3H]NMS binding studies using rat heart and submandibular gland might be useful methods which predict the affinities of test drugs for human muscarinic M2 and M3 receptor subtypes.     

Betel quid extract promotes oral cancer cell migration by activating a muscarinic M4 receptor-mediated signaling cascade involving SFKs and ERK1/2

Betel quid (BQ) is a widely accepted etiological factor for oral squamous cell carcinoma (OSCC) in Southeast Asia, but how BQ chewing leads to oral carcinogenesis remains to be elucidated. We have previously demonstrated that the activation of Src family kinases (SFKs) is critical for BQ-induced oral cancer cell motility. Here we investigate whether this biological effect is mediated by specific membrane receptors in oral cancer cells. We found that BQ-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and cell migration could be inhibited by atropine, suggesting the involvement of the muscarinic receptor family. The enhanced activities of ERK1/2 and cell migration were significantly counteracted by PD102807, the selective antagonist of muscarinic M4 receptor. Moreover, cold BQ extract effectively competed with a known ligand, [³H]-N-methyl scopolamine, for binding to muscarinic M4 receptor in vitro, thereby implying that BQ could activate motility-promoting signaling pathways through direct interaction with the receptor. The requirement of muscarinic M4 receptor for BQ-induced oral cancer cell migration was demonstrated by knockdown of the receptor using RNA interference (RNAi). Remarkably, ectopic expression of muscarinic M4 receptor in two oral cancer cell lines, Ca9-22 and SCC-9, further augmented BQ-induced cell migration by 83% and 99%, respectively. Finally, we verified that BQ-induced oral cancer cell migration was mediated through a muscarinic M4 receptor → SFKs → ERK1/2 signaling pathway. Thus, our findings have identified a novel signaling cascade mediating BQ-induced oral cancer cell motility, which could be a therapeutic target for BQ-related oral malignancies.     

Expression of human muscarinic cholinergic receptors in tobacco

We expressed human m1, m2 and chimeric muscarinic cholinergic receptors (MAChR) in tobacco plants and in cultured BY2 tobacco cells using Agrobacterium-mediated transformation. The membranes of most transgenic plants and calli bound muscarinic ligands with appropriate affinities, kinetics and pharmacologic specificity, as determined by direct and competitive binding measurements using the muscarinic ligand [3H]quinuclidinyl benzylate (QNB). Membranes of untransformed plants and calli or those transformed with vector alone did not bind [3H]QNB. Preliminary experiments did not suggest regulation of endogenous plant G protein signalling pathways by the recombinant receptors. Membranes from one callus clone expressed m1 MAChR at the level of 2.0–2.5 pmol [3H]QNB bound per mg membrane protein, more than the number of m1 MAChR in mammalian brain and comparable to that expressed in Sf9 insect cells using baculovirus vectors. This work demonstrates high level expression of active G protein-coupled receptors in plants, such that signaling might be genetically reconstituted by co-expression of appropriate G proteins and effectors.     

Regulation of Oligomeric Organization of the Serotonin 5-Hydroxytryptamine 2C (5-HT2C) Receptor Observed by Spatial Intensity Distribution Analysis

The questions of whether G protein-coupled receptors exist as monomers, dimers, and/or oligomers and if these species interconvert in a ligand-dependent manner are among the most contentious current issues in biology. When employing spatial intensity distribution analysis to laser scanning confocal microscope images of cells stably expressing either a plasma membrane-associated form of monomeric enhanced green fluorescent protein (eGFP) or a tandem version of this fluorophore, the eGFP tandem was identified as a dimer. Similar studies on cells stably expressing an eGFP-tagged form of the epidermal growth factor receptor demonstrated that, although largely a monomer in the basal state, this receptor rapidly became predominantly dimeric upon the addition of its ligand epidermal growth factor. In cells induced to express an eGFP-tagged form of the serotonin 5-hydroxytryptamine 2C (5-HT_2C) receptor, global analysis of construct quantal brightness was consistent with the predominant form of the receptor being dimeric. However, detailed spatial intensity distribution analysis demonstrated the presence of multiple forms ranging from monomers to higher-order oligomers. Furthermore, treatment with chemically distinct 5-HT_2C receptor antagonists resulted in a time-dependent change in the quaternary organization to one in which there was a preponderance of receptor monomers. This antagonist-mediated effect was reversible, because washout of the ligand resulted in the regeneration of many of the oligomeric forms of the receptor.     

A specific multi-nutrient formulation enhances M1 muscarinic acetylcholine receptor responses in vitro

Recent evidence indicates that supplementation with a specific combination of nutrients may affect cell membrane synthesis and composition. To investigate whether such nutrients may also modify the physical properties of membranes, and affect membrane-bound processes involved in signal transduction pathways, we studied the effects of nutrient supplementation on G protein-coupled receptor activation in vitro. In particular, we investigated muscarinic receptors, which are important for the progression of memory deterioration and pathology of Alzheimer's disease. Nerve growth factor differentiated pheochromocytoma cells that were supplemented with specific combinations of nutrients showed enhanced responses to muscarinic receptor agonists in a membrane potential assay. The largest effects were obtained with a combination of nutrients known as Fortasyn? Connect, comprising docosahexaenoic acid, eicosapentaenoic acid, uridine monophosphate as a uridine source, choline, vitamin B6, vitamin B12, folic acid, phospholipids, vitamin C, vitamin E, and selenium. In subsequent experiments, it was shown that the effects of supplementation could not be attributed to single nutrients. In addition, it was shown that the agonist-induced response and the supplement-induced enhancement of the response were blocked with the muscarinic receptor antagonists atropine, telenzepine, and AF-DX 384. In order to determine whether the effects of Fortasyn? Connect supplementation were receptor subtype specific, we investigated binding properties and activation of human muscarinic M1, M2 and M4 receptors in stably transfected Chinese hamster ovary cells after supplementation. Multi-nutrient supplementation did not change M1 receptor density in plasma membranes. However, M1 receptor-mediated G protein activation was significantly enhanced. In contrast, supplementation of M2- or M4-expressing cells did not affect receptor signaling. Taken together, these results indicate that a specific combination of nutrients acts synergistically in enhancing muscarinic M1 receptor responses, probably by facilitating receptor-mediated G protein activation.     

Inhibition of muscarinic receptor-mediated Ca27#x002B; mobilization by phorbol 12-myristate 13-acetate in chick embryo cells

Summary A muscarinic cholinergic receptor is present on undifferentiated cells of the chick embryo. Stimulation of the muscarinic receptor with muscarinic agonists triggers intracellular Ca^2#x002B; mobilization. Here, we investigate the effect of phorbol 12-myristate 13-acetate (PMA) on the muscarinic receptor-mediated Ca^2#x002B; mobilization, which is monitored in cell suspensions of chick embryos of stage 24 by chlorotetracycline fluorescence. PMA inhibits the Ca^2#x002B; mobilization in a time-dependent and concentration-dependent manner without changing the ED₅₀ of acetylcholine. The concentration of PMA that gives halfmaximal inhibition is 3.1×10^–9 M PMA.     

The hsp90 cochaperone p23 is the limiting component of the multiprotein hsp90/hsp70-based chaperone system in vivo where it acts to stabilize the client protein: hsp90 complex

A variety of signaling proteins form heterocomplexes with and are regulated by the heat shock protein chaperone hsp90. These complexes are formed by a multiprotein machinery, including hsp90 and hsp70 as essential and abundant components and Hop, hsp40, and p23 as non-essential cochaperones that are present in much lower abundance in cells. Overexpression of signaling proteins can overwhelm the capacity of this machinery to properly assemble heterocomplexes with hsp90. Here, we show that the limiting component of this assembly machinery in vitro in reticulocyte lysate and in vivo in Sf9 cells is p23. Only a fraction of glucocorticoid receptors (GR) overexpressed in Sf9 cells are in heterocomplex with hsp90 and have steroid binding activity, with the majority of the receptors present as both insoluble and cytosolic GR aggregates. Coexpression of p23 with the GR increases the proportion of cytosolic receptors that are in stable GR.hsp90 heterocomplexes with steroid binding activity, a strictly hsp90-dependent activity for the GR. Coexpression of p23 eliminates the insoluble GR aggregates and shifts the cytosolic receptor from very large aggregates without steroid binding activity to approximately 600-kDa heterocomplexes with steroid binding activity. These data lead us to conclude that p23 acts in vivo to stabilize hsp90 binding to client protein.     

Subtype-specific regulation of the expression and function of muscarinic acetylcholine receptors in embryonic chicken retinal cells

We examined the effect of long-term agonist exposure on muscarinic acetylcholine receptor expression and function in embryonic chicken retinal cells. Long-term carbachol exposure induced a time- and concentration-dependent decrease in M2, M3 and M4 muscarinic receptor numbers. Kinetic analyses revealed a first-order process with similar rate constants for all three subtypes. Both the maximal decrease and the agonist potency for regulation of M3 were significantly higher than those for M2 and M4. Upon agonist removal, M2 and M4 numbers returned to control values, but M3 recovery after 24 h was no higher than 40%. Agonist treatment did not alter the levels of receptor mRNAs. Receptor inactivation with a covalent alkylating antagonist demonstrated that the partial M3 protein recovery was not due to a decreased intrinsic basal rate of synthesis, suggesting that it is induced by agonist treatment. Prolonged carbachol exposure induced concomitant decreases in muscarinic-mediated inhibition of cyclic AMP accumulation which were completely reversed after agonist removal. Sustained receptor activation also promoted significant decreases in muscarinic receptor-stimulated phosphoinositide turnover, which were only partially reversed after agonist removal. These data demonstrate subtype-specific regulation of the expression and function of muscarinic receptors in the retina.     

Influence of MT7 toxin on the oligomerization state of the M1 muscarinic receptor1

Background information. The idea that GPCRs (G‐protein‐coupled receptors) may exist as homo‐ or hetero‐oligomers, although still controversial, is now widely accepted. Nevertheless, the functional roles of oligomerization are still unclear and gaining greater insight into the mechanisms underlying the dynamics of GPCR assembly and, in particular, assessing the effect of ligands on this process seems important. We chose to focus our present study on the effect of MT7 (muscarinic toxin 7), a highly selective allosteric peptide ligand, on the oligomerization state of the hM₁ (human M₁ muscarinic acetylcholine receptor subtype). Results. We analysed the hM₁ oligomerization state in membrane preparations or in live cells and observed the effect of MT7 via four complementary techniques: native‐PAGE electrophoresis analysed by both Western blotting and autoradiography on solubilized membrane preparations of CHO‐M1 cells (Chinese‐hamster ovary cells expressing muscarinic M₁ receptors); FRET (fluorescence resonance energy transfer) experiments on cells expressing differently tagged M₁ receptors using either an acceptor photobleaching approach or a novel fluorescence emission anisotropy technique; and, finally, by BRET (bioluminescence resonance energy transfer) assays. Our results reveal that MT7 seems to protect the M₁ receptor from the dissociating effect of the detergent and induces an increase in the FRET and BRET signals, highlighting its ability to affect the dimeric form of the receptor. Conclusions. Our results suggest that MT7 binds to a dimeric form of hM₁ receptor, favouring the stability of this receptor state at the cellular level, probably by inducing some conformational rearrangements of the pre‐existing muscarinic receptor homodimers.     

Quaternary structure of the chloride channel ClC-2

The chloride channel ClC-2 is thought to be essential for chloride homeostasis in neurons and critical for chloride secretion by the developing respiratory tract. In the present work, we investigated the quaternary structure of ClC-2 required to mediate chloride conduction. We found using chemical cross-linking and a novel PAGE system that tagged ClC-2 expressed in Sf9 cells exists as oligomers. Fusion of membranes from Sf9 cells expressing this protein confers double-barreled channel activity, with each pore exhibiting a unitary conductance of 32 pS. Polyhistidine-tagged ClC-2 from Sf9 cells can be purified as monomers, dimers, and tetramers. Purified, reconstituted ClC-2 monomers do not possess channel function whereas both purified ClC-2 dimers and tetramers do mediate chloride flux. In planar bilayers, reconstitution of dimeric ClC-2 leads to the appearance of a single, anion selective 32 pS pore, and tetrameric ClC-2 confers double-barreled channel activity similar to that observed in Sf9 membranes. These reconstitution studies suggest that a ClC-2 dimer is the minimum functional structure and that ClC-2 tetramers likely mediate double-barreled channel function.