0022-3565/08/3253-869–874$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 325, No. 3 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 137968/3337100 JPET 325:869–874, 2008 Printed in U.S.A.

Pharmacological Assessment of M1 Muscarinic Receptor-Gq/11 Protein Coupling in Membranes Prepared from Postmortem Human Brain Tissue

Hasib Salah-Uddin, David R. Thomas, Ceri H. Davies, Jim J. Hagan, Martyn D. Wood, Jeannette M. Watson, and R. A. John Challiss Department of Cell Physiology and Pharmacology, Henry Wellcome Building, University of Leicester, Leicester, United Kingdom (H.S.-U., R.A.J.C.); and Psychiatry Centre for Excellence in Drug Discovery, GlaxoSmithKline, Harlow, Essex, United Kingdom (D.R.T., C.H.D., J.J.H., M.D.W., J.M.W.) Downloaded from Received February 12, 2008; accepted February 26, 2008

ABSTRACT ␣ Using a selective G q/11 protein antibody capture guanosine 1-methylpyridine] and AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4- 5Ј-O-(3-[35S]thio)triphosphate ([35S]GTP␥S) binding approach, oxo-1-butyl]-piperidine hydrogen chloride), increased [35S]- jpet.aspetjournals.org ␥ ␣ it has been possible to perform a quantitative pharmacolog- GTP S-G q/11 binding, but with reduced intrinsic activities, induc- Ϯ Ϯ ical examination of the functional activity of the M1 musca- ing maximal responses that were 42 1 and 44 2% of the rinic (mAChR) in membranes prepared -M-induced response, respectively. These data indi- from human postmortem cerebral cortex. Oxotremorine-M cate that the M1 receptor is the predominant mAChR subtype Ն 35 ␥ ␣ ␣ caused a 2-fold increase in [ S]GTP S-G q/11 binding with a coupling to the G q/11 G protein in these brain regions and that it Ϯ pEC50 of 6.06 0.16 in Brodmann’s areas 23 and 25 that is possible to quantify the potency and intrinsic activity of full and was almost completely inhibited by preincubation of membranes partial M1 mAChR receptor agonists in postmortem human brain ␣ 35 ␥ at ASPET Journals on January 20, 2020 with the M1 mAChR subtype-selective antagonist muscarinic using a selective G q/11 protein antibody capture [ S]GTP S toxin-7. In addition, the orthosteric and allosteric agonists, xa- binding assay. nomeline [3(3-hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-

Muscarinic acetylcholine receptors (mAChRs) are widely e.g., N-type voltage-gated Ca2ϩ channels (Caulfield and Birds- expressed throughout the CNS. Five mAChR subtypes have all, 1998). been cloned (M1,M2,M3,M4, and M5) that can be subdivided A variety of experimental approaches, including the use of into two distinct classes based on sequence homologies and knockout mice (Hamilton et al., 1997; Anagnostaras et al., signal transduction properties. The M1 mAChR (together 2003; Wess, 2004), brain lesioning, and pharmacological with the M and M receptor subtypes) preferentially couples 3 5 blockade (see Bartus, 2000), have implicated M1 mAChR to Gq/11 proteins and stimulation of the inositol 1,4,5- signaling in learning and memory processes (Levey, 1996; 2ϩ trisphosphate/Ca /diacylglycerol/protein kinase C signal Hasselmo, 2006), and, as such, it is thought that selective transduction cascade (Caulfield and Birdsall, 1998). This activation of the M1 mAChR subtype will provide an effica- mAChR subtype also activates the extracellular signal-regu- cious treatment for a variety of neurological and psychiatric lated kinase pathway (Berkeley et al., 2001) and modulates disorders, including Alzheimer’s disease (Levey, 1996; Terry the activity of a diverse group of ion channels, including and Buccafusco, 2003) and schizophrenia (Raedler et al., N-methyl-D-aspartate receptors and the M-current (Marino 2007), in which cognitive dysfunction is a prominent feature et al., 1998; Delmas and Brown, 2005). In contrast, M and 2 of the disease. M4 mAChRs signal through Gi/o proteins to inhibit adenylate cyclase activity and modulate a variety of other ion channels, As the potency and intrinsic activity of agonists at specific receptor subtypes can differ dramatically between experi- mental systems (recombinant versus native tissue) because Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. of differences in, for example, receptor reserve (Watson et al., doi:10.1124/jpet.108.137968. 2000; Nelson and Challiss, 2007), it is extremely important to

ABBREVIATIONS: mAChR, muscarinic acetylcholine receptor; CNS, central nervous system; GTP␥S, guanosine 5Ј-O-(3-thiotriphosphate); SPA, scintillation proximity assay; AC-42, 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride; , 3(3-hexyloxy- 1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine; MT-7, muscarinic toxin-7; NEM, N-ethylmaleimide. 869 870 Salah-Uddin et al.

be able to assess the pharmacological activity of putative Polytron in 10 volumes of 10 mM HEPES, pH 7.4, containing 1 mM drugs in regions of the human brain in which they are hy- EGTA, 1 mM dithiothreitol, 10% sucrose, and complete protease pothesized to alter neuronal function to treat disease. To inhibitor cocktail. The resultant homogenate was diluted 10-fold and date, the majority of studies that have examined mAChR centrifuged at 1000g for 10 min at 4°C, the supernatant was saved, function in human brain tissue have relied on the following: and the pellet was rehomogenized and centrifuged as above. The combined supernatants were then centrifuged at 11,000g for 20 min 1) antagonist radioligand binding studies where agonist dis- at 4°C. The resulting pellet was rehomogenized in 40 volumes of 10 placement curves have been performed in the absence and mM HEPES, pH 7.4, containing 1 mM EGTA, 1 mM dithiothreitol, presence of GTP (Ladner et al., 1995); 2) assessment of and 1 mM MgCl (buffer A), and centrifuged at 27,000g for 20 min at 35 2 [ S]GTP␥S binding as an index of total G protein activation 4°C. The resulting pellet was resuspended in buffer A at a protein (Gonza´lez-Maeso et al., 2000; Scarr et al., 2006); or 3) non- concentration of 1 mg/ml, and aliquots were snap-frozen in liquid membrane-based assays of more distal signaling events nitrogen and stored at Ϫ80°C. (Garro et al., 2001). N-Ethylmaleimide and Pertussis Toxin Pretreatments. A We have sought to improve on these approaches by devel- frozen aliquot of rat or human cortical membranes was diluted to the appropriate protein concentration in assay buffer containing N-eth- oping a more specific and direct measurement of M1 mAChR function. In this respect, G protein-coupled receptor function ylmaleimide (up to 10 mM; final concentration) and incubated on ice is most directly assessed by measuring the ability of the for 60 min before running the assay. Pertussis toxin pretreatment of membranes was performed as described by Hudson and Johnson receptor to facilitate G protein [35S]GTP␥S for GDP exchange 35 (1980). ␥ Downloaded from (Hilf et al., 1989). By combining [ S]GTP S binding with an [35S]GTP␥S Binding/Immunoprecipitation Assay. [35S]GTP␥S- immunocapture step in which G␣ subtype-selective antibod- ␣ Gq/11 immunospecific binding as a measure of M1 mAChR activation 35 ies are used to recover specific G␣ isoform-[ S]GTP␥S com- was determined using a method modified from that described by Akam plexes, it is possible to enrich the response for specific recep- et al. (2001). In brief, frozen membrane aliquots were diluted in assay

tor subtypes (Milligan, 2003). In this respect, DeLapp et al. buffer (10 mM HEPES, 100 mM NaCl, 10 mM MgCl2, pH 7.4) to (1999) have described a method that incorporates [35S]- generate a final protein concentration of 75 ␮g of protein per 50 ␮l, ␥ ␣ which was preincubated with 0.1 ␮M GDP for 5 min on ice. Membranes GTP S binding coupled with immunocapture using G jpet.aspetjournals.org q/11 ␮ ␮ antibodies and anti-IgG scintillation proximity assay (SPA) (75 g) were then added to buffer (40 l) containing agonists and/or ␮ beads, which provides sufficient signal/noise to study mAChR- antagonists for 20 min at room temperature, after which 10 lof [35S]GTP␥S was added (to generate an approximate 1 nM final concen- G protein coupling not only in human embryonic kidney cells q/11 tration), and the mixture was incubated for a further 5 min at 30°C. The expressing recombinant receptors but also in rat striatal mem- experiment was then terminated by the addition of 1 ml of ice-cold branes, a method that has subsequently been exploited by Por- assay buffer and immediate transfer to an ice bath. Cell membranes ter et al. (2002) to demonstrate that oxotremorine-M stimulates were recovered from the reaction mixture by centrifugation (20,000g,6 ␣ 35 ␥ G q/11-[ S]GTP S binding via activation of the M1 mAChR min, 4°C). Membrane pellets were then solubilized by the addition of 50 at ASPET Journals on January 20, 2020 subtype in mouse cortex and hippocampus. We now report ␮l of ice-cold solubilization buffer comprised of 100 mM Tris/HCl, 200 mM NaCl, 1 mM EDTA, and 1.25% Igepal CA-630, pH 7.4, containing how this method has been modified to investigate M1 mAChR- ␣ 0.2% SDS. Once the pellet had been completely solubilized by vortex stimulated Gq/11 -GDP/GTP exchange in postmortem human cerebral cortex. mixing, an equal volume of solubilization buffer without SDS was added to each tube. Solubilized protein was precleared with normal rabbit serum (1:100 dilution) and 30 ␮l of protein A-Sepharose bead Materials and Methods suspension [3% (w/v) in 10 mM Tris/HCl, 10 mM EDTA, pH 8.0] for 60 min at 4°C. The protein A-Sepharose beads and any insoluble material Materials. [35S]GTP␥S (1000–1200 Ci/mmol), anti-rabbit-IgG- were collected by centrifugation (20,000g, 6 min, 4°C), and 100 ␮lofthe coated SPA beads (RPNQ0016), and protein-A-Sepharose CL-4B supernatant transferred to a fresh Eppendorf tube containing G␣ pro- were obtained from GE Healthcare (Chalfont St. Giles, UK). Com- tein antiserum (1:100 dilution). Samples were vortex-mixed and rotated plete protease inhibitor cocktail was purchased from Roche Applied for 60 min at 4°C. Seventy microliters of protein A-Sepharose bead Science (Burgess Hill, UK). All other chemicals and reagents were suspension was added to each sample tube, and the samples were obtained from Sigma-Aldrich (Poole, UK). Sprague-Dawley rats were vortex-mixed and rotated for 90 min at 4°C. Protein A-Sepharose beads ␣ purchased from Charles River UK Ltd. (Margate, UK). The Gq/11 were then pelleted (20,000g, 6 min, 4°C), and the supernatant was antiserum was generated [against the C-terminal sequence removed by aspiration. The beads were washed three times with 500 ␮l (C)LQLNLKEYNLV] as described previously (Akam et al., 2001). of solubilization buffer (minus SDS), and after the final wash, the AC-42 (Spalding et al., 2002) and xanomeline (Shannon et al., 1994) recovered beads were mixed with scintillation cocktail and counted. were synthesized by GlaxoSmithKline (Harlow, UK). Muscarinic Nonspecific binding was determined in the presence of 10 ␮M GTP␥S. 35 ␥ 35 ␥ ␣ toxin-7 (MT-7) was purchased from Peptides International Inc. [ S]GTP S Binding/Immunocapture Assay. [ S]GTP S-Gq/11 (Louisville, KY) and prepared for use according to the manufac- binding using a 96-well SPA-based method was performed using a turer’s instructions. method modified from DeLapp et al. (1999). Frozen membranes were Human Tissue. Normal control brain samples (Brodmann’s diluted in immunocapture assay buffer (20 mM HEPES, 100 mM

areas 23 and 25) from two tissue donors were obtained from the NaCl, 10 mM MgCl2, pH 7.4) to generate a protein concentration of NeuroResource tissue bank at the Institute of Neurology (Univer- 25 ␮g/60 ␮l and incubated on ice for 5 min with 0.1 ␮M GDP before sity College London, UK) with the appropriate ethical consent. addition to the assay reaction. Experimental reactions were per- Death-to-snap freezing time for donor 1 was 16 h. This individual formed in a final volume of 100 ␮l in 96-well Costar plates. Buffer (20 was female, 68 years old, and died from a colorectal metastatic ␮l) containing the selected agonist and/or antagonist concentrations tumor. Death-to-snap-freezing time for donor 2 was 26 h. This were added to each well followed by membranes (60 ␮l), and the individual was also female, 93 years old, and died from a chest resultant mixture was incubated at 25°C for 20 min. After this time, infection. Neither donor displayed clinical or neuropathological [35S]GTP␥S (20 ␮l; 0.5 nM final concentration) was added to each evidence of neurological disease. well, and incubations continued for a further 60 min at 25°C. The Membrane Preparation. Human cerebrocortical tissue from reaction was terminated by the addition of 20 ␮l of ice-cold 0.27% each individual donor or rat frontal cortex was homogenized using a Igepal CA-630, and membranes were allowed to solubilize on ice for Human Brain M1 mACh Receptor-Gq/11 Coupling 871

30 min. For immunocapture of [35S]GTP␥S-G␣ proteins, 10 ␮lof ␣ no pretreatment Gq/11 antiserum was added to each well to generate a final antibody dilution of 1:300, and plates were incubated at 4°C for 60 min with +NEM (0.1 mM) shaking. Anti-IgG-coated PVT-SPA beads (50 ␮l) were then added to +NEM (1 mM) each well, and plates were shaken for an additional 30 min at 4°C. Plates were then centrifuged at 1000g for 5 min and radioactivity +NEM (10 mM) determined using a Wallac MicroBeta counter. [3H]NMS Radioligand Binding. Cerebrocortical membranes 4000

were diluted in binding buffer (50 mM HEPES, 110 mM NaCl, 5.4 S γ γ γ γ mM KCl, 1.8 mM CaCl2, 1 mM MgSO4, 25 mM glucose, and 58 mM 3000 sucrose, pH 7.4) to generate a final protein concentration of 25 ␮g/ml. The membrane preparation (400 ␮l) was added to wells in a deep- S]-GTP

well 96-well block placed on ice. For each membrane preparation, 35 2000 total [3H]NMS binding, nonspecific binding (10 ␮M ), and

the component displaced by the selective M1 mAChR antagonist, 1000 MT-7 (100 nM; Adem and Karlsson, 1997), were determined. Pre- (c.p.m.) binding liminary experiments demonstrated that maximal blockade of the specific [ M mAChR or the total mAChR population at 4°C was achieved 0 1 -9 -8 -7 -6 -5 -4 -3 within 30 min of addition to membranes of 100 nM MT-7 or 10 ␮M atropine, respectively (data not shown). Therefore, after adding buff- [oxotremorine-M] (log M) Downloaded from er/atropine/MT-7, plates were incubated for 30 min on ice before Fig. 1. Effect of NEM (0–10 mM) pretreatment on oxotremorine-M- 3 ␣ 35 ␥ addition of a saturating concentration of [ H]NMS (5 nM) and incu- induced Gq/11 -[ S]GTP S binding, using the rat cortex SPA-based im- 3 35 ␥ ␣ bation at 37°C for 30 min. Bound and free [ H]NMS was separated by munocapture assay. Data are shown as [ S]GTP S bound to Gq/11 in filtration through a GF/B filter-mat (presoaked with 0.05% polyeth- response to increasing concentrations of oxotremorine-M. A representa- tive experiment is shown (performed in duplicate). Similar data were ylenimine) using a 96-port cell harvester and rapid washing with 5 ϫ obtained for n ϭ 3 membrane preparations. 1-ml volume of ice-cold binding buffer. [3H]NMS retained on the jpet.aspetjournals.org filters was quantified by liquid scintillation counting. 35 ␥ Data Analysis. Concentration-response curves were fitted using improvement in signal/noise by reducing basal [ S]GTP S- ␣ sigmoidal nonlinear regression analysis with variable slope using Gq/11 binding, but this effect was not as great, or as repro- GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA). Inhi- ducible, as that produced by NEM pretreatment (data not

bition constants (Ki) were determined from antagonist inhibition shown). Thus, all subsequent human native tissue assays curves constructed in the presence of a fixed agonist concentration, were performed in the presence of NEM. using the equation of Cheng and Prusoff (1973). One-way analysis of [35S]GTP␥S Binding Studies in Human Cortex. In

variance was used to investigate whether maximal responses to preliminary experiments using the immunoprecipitation as- at ASPET Journals on January 20, 2020 different compounds or assay conditions were significantly different say format, NEM (10 mM) pretreatment markedly increased from each other. Where appropriate, Student’s t test was used to the degree of oxotremorine-M-induced [35S]GTP␥S-G ␣ ascertain significance at a 95% confidence level. q/11 binding in human cortex (Fig. 2). Subsequent experiments revealed that a 3- to 4-fold increase in maximal response Results could be achieved (ϪNEM, 139 Ϯ 9%; ϩNEM, 491 Ϯ 13% increase over basal; n ϭ 3 tissue preparations from two 35 [ S]GTP␥S Binding Studies in Rat Cerebral Cortex. donors; p Ͻ 0.01), with no significant change in the oxotremo- Preliminary experiments, using membranes prepared from rine-M EC50 value. These studies demonstrated that mea- adult rat frontal cerebral cortex, were performed to predict 35 ␥ ␣ surement of mAChR agonist-induced [ S]GTP S-Gq/11 optimal assay conditions for subsequent postmortem human binding using the immunoprecipitation methodology gives cortical studies. In these experiments, oxotremorine-M- rise to a high signal/noise ratio in human cortex. However, as 35 ␥ ␣ induced stimulation of [ S]GTP S-Gq/11 coupling was previously mentioned, this format is low throughput (and greatest when membranes were preincubated with a low (0.1 ␮ M) concentration of GDP, and agonist was added before 300 35 ␥ 35 ␥ ␣ no pre-treatment adding [ S]GTP S to initiate the [ S]GTP S-Gq/11 binding reaction (data not shown). Preincubating rat cortical mem- 250 NEM-treated branes with different concentrations of the irreversible alky- 200 lating agent, N-ethylmaleimide (NEM; 0–10 mM), reduced S binding γ γ γ basal levels of [35S]GTP␥S binding, resulting in a marked γ 150 35 ␥ ␣ increase in the -fold change in [ S]GTP S-Gq/11 binding 100 stimulated by oxotremorine-M, the largest increase in signal S]-GTP 50 being observed at 10 mM NEM, irrespective of whether im- 35 [

munoprecipitation or immunocapture methods were used to (% increase-over-basal) 0 measure agonist activity (Fig. 1). In agreement with previous work by Flynn and Potter (1985) and Horva´th et al. (1986) -9 -8 -7 -6 -5 -4 [oxotremorine-M] (log M) indicating that NEM treatment does not alter M1 mAChR agonist affinity, we have shown in Chinese hamster ova- ␣ Fig. 2. Effect of NEM pretreatment on oxotremorine-M-induced Gq/11 - ry-m1 membranes that NEM pretreatment (at concentra- [35S]GTP␥S binding in human cerebral cortex membranes. Data are shown as the G ␣-[35S]GTP␥S binding response to increasing concen- tions up to and including 10 mM) did not alter M mAChR- q/11 1 trations of oxotremorine-M. A representative experiment (performed in oxotremorine-M affinity (data not shown). Pretreatment with triplicate and repeated on two occasions with similar results) using ␣ the G i/o-inactivating agent pertussis toxin also produced an membranes from one donor. 872 Salah-Uddin et al. uses comparatively large amounts of tissue; 75 ␮g of mem- brane protein per data point), and so a series of studies were oxotremorine-M performed to determine whether the higher throughput im- 100 xanomeline munocapture methodology would generate a similarly robust AC-42 data set with an adequate signal/noise ratio. This was proven 80 to be the case in that oxotremorine-M increased [35S]GTP␥S- ␣ Ϯ 60 Gq/11 binding by 2-fold with a pEC50 of 6.06 0.16 (Fig. 3). The selective M mAChR antagonist, MT-7, inhibited the 1 40 oxotremorine-M-stimulated response with a pKi value % maximal (9.28 Ϯ 0.33) comparable with its binding affinity for human 20 M1 mAChRs (Adem and Karlsson, 1997). Furthermore, 100 nM MT-7 almost completely abolished the oxotremorine-M- 0 oxotremorine-M response oxotremorine-M 35 ␥ ␣ stimulated [ S]GTP S-Gq/11 binding (Fig. 3), suggesting -10 -9 -8 -7 -6 -5 -4 that the M1 mAChR subtype is principally responsible for the observed response to oxotremorine-M. [compound] (log M) In a subsequent series of experiments, the pharmacological Fig. 4. Effect of increasing concentrations of oxotremorine-M, xanome- ␣ 35 ␥ properties of the M1 mAChR-selective orthosteric agonist, line, and AC-42 on Gq/11 -[ S]GTP S binding to human cerebral cortex membranes measured using the immunocapture assay. Data are repre- xanomeline (Shannon et al., 1994), was compared with those Downloaded from sented as means Ϯ S.E.M. for n ϭ 3 different membrane preparations of the M1 mAChR-selective allosteric agonist, AC-42 (Spald- from two donors, each performed in triplicate. ing et al., 2002; Langmead et al., 2006). Both compounds behaved as partial agonists compared with oxotremorine-M, agonist Ϯ Ϯ 70 with intrinsic activities of 42 1 and 44 2% and pEC50sof agonist + MT-7 6.92 Ϯ 0.09 and 6.46 Ϯ 0.21, respectively (Fig. 4). Maximal 60

responses evoked by xanomeline and AC-42 were signifi- jpet.aspetjournals.org 50 cantly less than that induced by oxotremorine-M (p Ͻ 0.05). 35 ␥ ␣ MT-7 (100 nM) abolished the [ S]GTP S-Gq/11 binding re- 40 sponses to EC80 concentrations of xanomeline and AC-42 and 30 reduced that induced by oxotremorine-M by ϳ90% (Fig. 5). [3H]NMS Radioligand Binding Studies in Human 20

Cortex. To estimate the relative proportions of M1 versus 10 Increase-over-basal (%)

M -M mAChR subtypes expressed in human cortical mem- at ASPET Journals on January 20, 2020 2 5 0 branes, [3H]NMS binding studies were performed at a satu- e 3 n 42 -M rating concentration of [ H]NMS (5 nM) in the absence and li - e e presence of the broad-spectrum mAChR antagonist, atropine AC rin mo (to define M1-toM5-specific binding), or the selective M1 xanom tre o x mAChR antagonist, MT-7 (to define the M1 mAChR binding o component). Applying this methodology, we were able to Fig. 5. Inhibition of mAChR agonist-stimulated G ␣-[35S]GTP␥S bind- demonstrate a total mAChR expression (B ) of 1043 Ϯ 98 q/11 max ing to human cerebral cortex membranes by the M1 mAChR-specific fmol/mg protein in the human cortical tissue used in these toxin, MT-7. Membranes were preincubated with MT-7 (100 nM) for 15 studies, of which 36 Ϯ 2% (372 Ϯ 36 fmol/mg protein) was min before addition of xanomeline (300 nM), AC-42 (10 ␮M), or oxotremo- ␮ ␣ 35 ␥ rine-M (10 M) and assessment of Gq/11 -[ S]GTP S binding using the contributed by the M1 mAChR subtype (Fig. 6). immunocapture assay. Data are presented as means Ϯ S.E.M. for n ϭ 3 different membrane preparations from two donors, each point performed in triplicate. oxotremorine-M MT-7 100 Discussion

80 There is a wealth of evidence to support the concept that selective activation of M1 mAChRs should lead to cognitive 60 benefit in a number of neurological and psychiatric disorders 40 (Levey, 1996; Terry and Buccafusco, 2003; Raedler et al., % maximal 2007). Therefore, the M mAChR subtype represents an at- 20 1 tractive CNS drug target. The use of cell systems expressing 0

oxotremorine-M response near-homogeneous populations of recombinant receptors has

-12 -11 -10 -9 -8 -7 -6 -5 -4 greatly facilitated the identification of potential ligands for [compound] (log M) this receptor. However, it is increasingly clear that these systems may not always accurately reflect the functional ␣ 35 ␥ Fig. 3. Effect of MT-7 on oxotremorine-M-stimulated Gq/11 -[ S]GTP S binding, using the SPA-based immunocapture assay. Data show a con- pharmacology of agonists, and, as such, quantitative phar- centration-response curve for oxo-M, where no addition ϭ 0% (basal, macological evaluation in native tissue systems is required 687 Ϯ 52 dpm) and oxotremorine-M (100 ␮M) ϭ 100% (ϩoxo-M, 1393 Ϯ to provide real insight into how molecules progressing into 256 dpm). Concentration-dependent inhibition of the response to a sub- clinical development probably modulate CNS activity and maximal concentration of oxotremorine-M (10 ␮M) by MT-7 is also shown. Data points are presented as means Ϯ S.E.M. for n ϭ 3 different thereby treat both psychiatric and neurological disorders. In membrane preparations from two donors, each performed in triplicate. this respect, although studies in rodent tissue go some way to Human Brain M1 mACh Receptor-Gq/11 Coupling 873

1000 enough signal window to allow construction of concentration- response curves to investigate agonist pharmacology. Care 800 needs to be taken when considering NEM pretreatment in studies of other G protein-coupled receptor subtypes; thus, 600 protein) previous studies have shown that unlike the situation for the -1 400 M1 mAChR, agonist-receptor interactions may be affected (Flynn and Potter, 1985; Horva´th et al., 1986). Pertussis 200 toxin pretreatment of membranes (which ADP-ribosylates (fmol mg (fmol

Receptor expression Gi/o proteins) caused a similar unmasking effect, but the need 0 to preactivate the pertussis toxin to increase signal/noise made this a more time-consuming and, in our hands, less 1 5 M -M reproducible procedure. In theory, the specificity of the im- 2 M ␣ munocapture method should be determined by the G q/11 Fig. 6. [3H]NMS binding to human cerebral cortex membranes. The open specificity of the antibody used (in this case, a previously bar represents MT-7 (100 nM)-sensitive binding (i.e., estimates the M ␣ 1 characterized and highly selective G q/11 polyclonal anti- mAChR population). The closed bar represents total specific binding minus MT-7-sensitive binding (i.e., estimates the M mAChR popula- body; Akam et al., 2001; Selkirk et al., 2001); however, it is 2–5 ␣ 35 ␥ tion). Data are presented as means Ϯ S.E.M. for n ϭ 3 different mem- possible that basal (or agonist-stimulated) G i/o-[ S]GTP S brane preparations from two donors, each point performed in triplicate. binding is sufficiently high to cause a significant background Downloaded from excitation of the SPA beads that would account for the re- rectifying the problems associated with recombinant sys- duction in basal signal produced by NEM pretreatment. An ␣ 35 ␥ tems, it is clearly more favorable to be able to study pharma- alternative explanation is that basal G i/o-[ S]GTP S bind- cology in human brain tissue. To date, the majority of human ing and/or [35S]GTP␥S-for-GDP exchange is sufficiently high ␣ 35 brain tissue-based experiments have focused on expression to compete with activated G q/11 for the available [ S]- ␥ studies employing immunohistochemistry, autoradiography, GTP S and that NEM pretreatment, by eliminating this jpet.aspetjournals.org or radioligand binding assays to investigate whether receptor competition, increases the availability of [35S]GTP␥S for ␣ populations or downstream signaling components change G q/11 binding. Whatever the case, we are confident that this with respect to their absolute level or subcellular location treatment does not affect the pharmacology of M1 mAChRs in postmortem brain tissue from clinically defined patient because of the following: 1) both NEM and pertussis toxin groups (Crook et al., 2001; Lo´pez de Jesu´s et al., 2006; Scarr pretreatment did not significantly affect the concentration et al., 2006). Studies examining the downstream conse- dependence of agonist activation of M1 mAChRs (assessed

quences of neurotransmitter receptor activation have been using both the immunoprecipitation and immunocapture as- at ASPET Journals on January 20, 2020 fewer in number and have relied, for example, on GTP shift say formats), and 2) NEM-mediated uncoupling of M2 analysis, using radiolabeled antagonist binding to construct mAChRs from Gi/o proteins in rat brain membranes has agonist displacement curves in the absence and presence of previously been shown to have no significant effect on the 35 ␥ GTP (Ladner et al., 1995), assessment of [ S]GTP S binding affinity state of the M1 mAChR for or as an index of total G protein activation (Gonza´lez-Maeso et (Flynn and Potter, 1985). al., 2000), or evaluation of more distal signaling events, for A second important aspect of the current experimental example, through the addition of an exogenous substrate protocol was the use of a low concentration of GDP (0.1 ␮M) 3 ␣ 35 ␥ such as [ H]phosphatidylinositol 4,5-bisphosphate to mea- optimally to facilitate G q/11-[ S]GTP S binding (Offer- sure phospholipase C activity (Garro et al., 2001). manns et al., 1994; Akam et al., 2001) and sequential, rather We now demonstrate that it is possible to use antibody than simultaneous (Akam et al., 2001; Selkirk et al., 2001), 35 ␥ 35 ␥ capture-[ S]GTP S binding methods to study Gq/11 protein addition of agonist followed by [ S]GTP S, which improved activation by the M1 mAChR, thereby extending the postmor- both the magnitude and reproducibility of agonist-induced tem human brain tissue assay armamentarium to Gq/11 pro- responses. tein-coupled receptors. This is an important development Having validated the assay as a method for measuring Gq/11 35 ␥ because [ S]GTP S-for-GDP exchange on non-Gi/o G protein protein-coupled receptor activation, we deployed it to examine subpopulations is generally masked by the relatively higher whether or not it was feasible to study M1 mAChR pharmacol- rates of exchange occurring on Gi/o proteins (Milligan, 2003), ogy. First, because multiple mAChR subtypes are expressed in an effect that is particularly pronounced in brain tissue cerebral cortex, we used the selective M1 receptor toxin MT-7 where Gi/o (and particularly Go) protein expression is very (Adem and Karlsson, 1997) to establish that the vast majority Ͼ ␣ 35 ␥ high (Sternweis and Robishaw, 1984). It is also noteworthy ( 90%) of oxotremorine-M-stimulated G q/11-[ S]GTP S bind- that these receptor-G protein subtype-specific coupling data ing could be attributed to M1 mAChR activation. In contrast, 3 have been achieved in preparations prepared from donor [ H]NMS binding established that M1 mAChRs constitute brains of elderly individuals (68 and 93 years of age) and Ͻ40% of the total mAChR population. Second, we established after relatively long death-to-snap freezing delays (16 and that this assay is sufficiently sensitive to assess accurately 26 h). the intrinsic activity of orthosteric site partial agonists. In A key aspect of the methodology used here has been the this respect, xanomeline (Shannon et al., 1994), which has pretreatment (at 0°C) of membranes with NEM to uncouple been reported to be an M1 mAChR partial agonist in recom- receptors from Gi/o proteins by irreversible alkylation of the binant systems (Wood et al., 1999) and a functional M1 G protein (Aktories and Jakobs, 1984; Flynn and Potter, mAChR antagonist in rat native tissues, presumably due to 1985). This resulted in a substantial decrease in basal SPA- their low receptor reserve (Watson et al., 1999), behaved as a detected [35S]GTP␥S radioactivity, which created a large partial agonist, producing ϳ40% of the maximal response to 874 Salah-Uddin et al. oxotremorine-M. Third, we established that the assay could Hilf G, Gierschik P, and Jakobs KH (1989) Muscarinic acetylcholine receptor- stimulated binding of guanosine 5Ј-O-(3-thiotriphosphate) to guanine-nucleotide- measure the activity of allosteric site agonists in that AC-42 binding proteins in cardiac membranes. Eur J Biochem 186:725–731. (Spalding et al., 2002; Langmead et al., 2006) was able to Horva´th E, van Rooijen LA, Traber J, and Spencer DG (1986) Effects of ␣ 35 ␥ N-ethylmaleimide on muscarinic acetylcholine receptor subtype autoradiography stimulate G q/11-[ S]GTP S binding to a level that at its and inositide response in rat brain. 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