1521-0111/89/5/575–584$25.00 http://dx.doi.org/10.1124/mol.116.103341 MOLECULAR Mol Pharmacol 89:575–584, May 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Photolabeling a Nicotinic Receptor (nAChR) with an (a4)3(b2)2 nAChR-Selective Positive Allosteric Modulator

Ayman K. Hamouda, Farah Deba, Ze-Jun Wang, and Jonathan B. Cohen Department of Pharmaceutical Sciences, Rangel College of Pharmacy, Texas A&M Health Sciences Center, Kingsville, Texas (A.K.H., F.D., Z.-J.W.); and Department of Neurobiology, Harvard Medical School, Boston, Massachusetts (A.K.H., J.B.C.) Received January 11, 2016; accepted March 9, 2016 Downloaded from ABSTRACT Positive allosteric modulators (PAMs) of nicotinic acetylcholine concentration-response curve without altering ACh efficacy. In (ACh) receptors (nAChRs) have potential clinical applications in contrast, CMPI inhibited (∼35% at 10 mM) ACh responses of the treatment of dependence and many neuropsychi- (a4)2(b2)3 nAChRs and fully inhibited human muscle and atric conditions associated with decreased brain Torpedo nAChRs with IC50 values of ∼0.5 mM. Upon irradiation activity, and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)- at 312 nm, [3H]CMPI photoincorporated into each Torpedo

1H-pyrrazol-4-yl)isoxazole (CMPI) has been identified as a PAM [(a1)2b1gd] nAChR subunit. Sequencing of peptide fragments molpharm.aspetjournals.org selective for neuronal nAChRs containing the a4 subunit. In isolated from [3H]CMPI-photolabeled nAChR subunits estab- this report, we compare CMPI interactions with low-sensitivity lished photolabeling of amino acids contributing to the ACh (a4)3(b2)2 and high-sensitivity (a4)2(b2)3 nAChRs, and with binding sites (aTyr190, aTyr198, gTrp55, gTyr111, gTyr117, dTrp57) muscle-type nAChRs. In addition, we use the intrinsic reactivity that was fully inhibitable by and lower-efficiency, state- of [3H]CMPI upon photolysis at 312 nm to identify its binding dependent [3H]CMPI photolabeling within the ion channel. Our sites in Torpedo nAChRs. Recording from Xenopus oocytes, results establish that CMPI is a potent potentiator of nAChRs we found that CMPI potentiated maximally the responses containing an a4:a4 subunit interface, and that its intrinsic of (a4)3(b2)2 nAChR to 10 mMACh(EC10)by400%andwith photoreactivy makes it of potential use to identify its binding an EC50 of ∼1 mM. CMPI produced a left shift of the ACh sites in the (a4)3(b2)2 nAChR. at ASPET Journals on September 26, 2021

Introduction disorders, including Alzheimer’s and Parkinson’s diseases (Jensen et al., 2005). Muscle- and neuronal-type nicotinic acetylcholine (ACh) Drugs that target nAChRs have potential therapeutic uses receptors (nAChRs) are pentameric ligand-gated ion channels in the treatment of nicotine addiction and the behavioral with diverse subunit composition, functions, biophysical prop- symptoms associated with neurodegenerative and neuropsy- erties, and pharmacological characteristics (Jensen et al., chiatric conditions (Taly et al., 2009; Hurst et al., 2013). 2005; Hurst et al., 2013). Muscle-type nAChRs have a subunit nAChR positive allosteric modulators (PAMs) are a novel class stoichiometry of a2bgd or a2b«d, whereas neuronal nAChRs of cholinergic agents that enhance ACh-mediated responses are either homopentamers (e.g., a7 nAChR) or heteropen- by binding at site(s) distinct from the conserved ACh (orthos- tamers (e.g., a4b2 nAChR) and are expressed pre- and/or teric) binding sites (Bertrand and Gopalakrishnan, 2007; postsynaptically at cholinergic and other neurotransmitter Williams et al., 2011). nAChR PAMs are characterized by a synapses (Gotti et al., 2009). Presynaptic neuronal nAChRs wide range of chemical structures, and they potentially bind are involved in regulation of neurotransmitter release and the to different sites in an nAChR or selectively to an nAChR modulation of sleep, attention, learning, and memory (Dani subtype (Changeux and Taly, 2008). PAM binding sites in a and Bertrand, 2007). In addition, neuronal nAChRs play a muscle nAChR have been identified by photoaffinity label- central role in the development of nicotine addiction and are ing at subunit interfaces in the transmembrane domain (TMD; implicated in the underlying pathology of neuropsychiatric Nirthanan et al., 2008) and extracellular domain (ECD; Hamouda et al., 2013). In neuronal nAChRs, mutational analyses identified intersubunit sites in the ECD (Seo et al., This research was supported in part by the Edward and Anne Lefler Center of 2009; Olsen et al., 2013) and an intrasubunit site in the a7 Harvard Medical School (J.B.C.), Faculty Development Fund of Texas A&M nAChR TMD (Young et al., 2008; daCosta et al., 2011). Health Sciences Center (A.K.H.), and the National Institutes of Health National Institute of Neurologic Disorders and Stroke [Grant NS-093590] (A.K.H). PAMs of the a4b2 nAChR, the most abundant and dx.doi.org/10.1124/mol.116.103341. widely distributed neuronal nAChR subtype, include

ABBREVIATIONS: ACh, acetylcholine; BgTx, a-; Carb, carbamylcholine; CMPI, 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H- pyrrazol-4-yl)isoxazole; cpm, count per minute; dFBr, desformylflustrabromine; ECD, extracellular domain; EndoLys-C, Lysobacter enzymogenes endoproteinase Lys-C; nAChR, nicotinic ; NS9283, 3-[3-(pyridin-3-yl)-1,2,4-oxadiazol-5-yl]benzonitrile; PAM, positive allosteric modulator; PCP, ; rpHPLC, reverse-phase high-performance liquid chromatography; TMD, transmembrane domain; TPS, Torpedo physiologic saline; V8 protease, Staphylococcus aureus endoproteinase Glu-C.

575 576 Hamouda et al. desformylflustrabromine (dFBr; Sala et al., 2005), 3-(2- Torpedo nAChR-rich membranes were isolated from freshly dissected chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl) Torpedo californica electric organs as described previously (Middleton isoxazole (CMPI; Albrecht et al., 2008), and 3-[3-(pyridin-3-yl)- and Cohen, 1991). Final membranes in 38% sucrose and 0.02% sodium 2 1,2,4-oxadiazol-5-yl]benzonitrile (NS9283; Timmermann et al., azide were stored at 80°C until the day of use, when they were 2012) (Fig. 1A). dFBr was isolated from the marine bryozoan thawed, pelleted by centrifugation, and resuspended in Torpedo physiologic saline (TPS; 250 mM NaCl, 5 mM KCl, 3 mM CaCl , Flustra foliacea, whereas CMPI and NS9283 were developed 2 2 mM MgCl2, and 5 mM sodium phosphate, pH 7.0). through high-throughput screening and lead optimization of nAChR Expression in Xenopus Oocytes. Plasmids with cDNA chemical libraries. At submicromolar concentrations, dFBr, encoding human a1 (pSPa64T), a4 (pSP64polyA), b1 (pBSII), b2 CMPI, and NS9283 potentiate ACh-induced responses of (pSP64polyA), d [pBS(SK)], and « (pBSII) nAChR subunits were a4-containing nAChRs but not a3b2ora7 nAChRs (Sala provided by Dr. Jon Lindstrom (University of Pennsylvania, Phila- et al., 2005; Albrecht et al., 2008; Timmermann et al., 2012). delphia, PA). Plasmids with cDNA encoding Torpedo a1, g, and d Because a4andb2 subunits are known to assemble in two (pMXT) and Torpedo b1 (pSP6) were gifts from Dr. Michael M. White stoichiometries, 3a4:2b2and2a4:3b2 (Fig. 1B), that have (Drexel University College of Medicine, Philadelphia, PA) and Dr. distinct pharmacological profiles (Zwart and Vijverberg, Henry Lester (California Institute of Technology, Pasadena, CA), 1998; Nelson et al., 2003), the effects of NS9283 and dFBr respectively. Plasmids were linearized with restriction endonucleases [AseI (ha4) and PvuII (hb2), BsaAI (Hd), FspI (ha1 and Tb), ScaI were examined at both (a4)3(b2)2 and (a4)2(b2)3 nAChRs (Hb1), XbaI (Ta, Tg, and Td), and XhoI (H«)], and cRNA transcripts Downloaded from (Timmermann et al., 2012; Weltzin and Schulte, 2015). were prepared using SP6 (Torpedo a, b, g, and d subunits; human a1, Whereas dFBr potentiated both a4b2 nAChRs, NS9283 a4, and b2 subunits) or T3 (human b1, d, and « subunits) mMESSAGE potentiated only (a4)3(b2)2 nAChRs by binding to a site at mMACHINE high yield capped RNA transcription kits (Ambion/ the a4:a4 interface, which is present only in the (a4)3(b2)2 Life Technologies, Grand Island, NY) following the manufacturer’s nAChR and overlaps with a third ACh binding site within protocol. the ECD (Harpsøe et al., 2011; Mazzaferro et al., 2011; Adult female X. laevis were purchased from NASCO (Fort Atkinson, Eaton et al., 2014). WI), and ovarian lobules were surgically harvested following animal- molpharm.aspetjournals.org To further characterize the mode of interaction of CMPI use protocols approved by the Texas A&M Health Sciences Center with neuronal and muscle nAChRs, we used a two-electrode Institutional Animal Care and Use Committee. The lobules were treated with 3 mg/ml collagenase type 2 (Worthington Biomedical, voltage-clamp recording from Xenopus laevis oocytes to com- Lakewood, NJ) with gentle shaking for 3 hours at room temperature in pare the interactions of CMPI with (a4)3(b2)2,(a4)2(b2)3, and Ca12-free OR2 buffer (85 mM NaCl, 2.5 mM KCl, 1 mM MgCl ,5mM 3 2 muscle-type nAChRs. We also prepared [ H]CMPI and estab- HEPES, pH 7.6). Stage V and VI oocytes were selected, injected with lished that the intrinsic photochemical reactivity of CMPI can cRNA, and incubated at 18°C in ND96-gentamicin buffer (96 mM be used to identify its binding sites in the ECD and TMD of the NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, 50 mg/ml muscle-type Torpedo nAChR. gentamicin, pH 7.6) for 24–72 hours to allow receptor expression.

Oocytes were injected with 100 ng of a4 and b2 cRNA combined at at ASPET Journals on September 26, 2021 ratios 9a:1b or 1a:9b to express nAChRs with subunit stoichiometries Materials and Methods of 3a:2b or 2a:3b, respectively. For Torpedo and human muscle nAChR, oocytes were injected with 20–50 ng of total mRNA mixed Materials. CMPI was a generous gift from Drs. Stefan McDonough at ratios of 2a:1b:1g:1d and 2a:1b:1d:1«, respectively. and Alessandro Boezio (Amgen Inc., Cambridge, MA; Albrecht et al., Two-Electrode Voltage-Clamp Recording. A standard two- 2008). [3H]CMPI (16 Ci/mmol) was synthesized by custom tritiation electrode voltage-clamp technique was used to study the effects of (ViTrax, Placentia, CA). dFBr was from Tocris Bioscience R&D CMPI and dFBr on ACh-induced current responses of a4b2 and (Minneapolis, MN). Acetylcholine chloride, carbamylcholine chloride, muscle nAChR. Xenopus oocytes were perfused with ND96 recording and other chemicals were purchased from Sigma-Aldrich (Milwaukee, buffer (100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 0.8 mM MgCl2,1mM WI) unless otherwise indicated in the text. Staphylococcus aureus EGTA, 10 mM Hepes, pH 7.5) and voltage clamped at 250 mV using endoproteinase Glu-C (V8 protease) and Lysobacter enzymogenes Oocyte Clamp OC-725B (Warner Instruments, Hamden, CT). Whole- endoproteinase Lys-C (EndoLys-C) were from MP Biomedical (Santa cell currents were digitized using Digidata 1550A/Clampex 10.4 (Axon Ana, CA) and Roche Diagnostics (Indianapolis, IN), respectively. Instruments/Molecular Devices, LLC. Sunnyvale, CA). An automated

Fig. 1. (A) The chemical structures of nAChR PAMs: dFBr, NS9283, and CMPI. (B) Top view of a homology model of an a4b2 nAChR containing two a4 and two b2 subunits and a fifth subunit that is either an a4orb2 subunit. Both (a4)3(b2)2 and (a4)2(b2)3 nAChRs have two ACh binding sites within the ECD at the a4:b2 subunit interfaces, and the (a4)3(b2)2 nAChR has a low-affinity ACh binding site within the ECD at the a4:a4 subunit interface. A Photoreactive PAM of (a4)3(b2)2 nAChR 577 perfusion system (Warner Instruments) was used to control recording 1 mM[3H]CMPI (80 mCi per condition) in the absence and presence of chamber perfusion and drug application. Each recording run included 1 mM Carb, and the nAChR subunits were resolved on an 8% SDS-PAGE three to six drug applications (10 seconds each) separated by 1- to gel. The nAChR b, g,andd subunits were recovered by passive elution 4-minute wash intervals. Oocytes were washed with ND96 recording from the gel bands excised from the stained gel and resuspended in buffer for 3–5 minutes between runs. ACh currents in the absence or digestionbuffer(15mMTris,0.5mMEDTA,0.1%SDS,pH8.1).Thegel presence of CMPI and/or dFBr were quantified using pCLAMP 10.4 bands containing the [3H]CMPI-photolabeled nAChR a subunit were (Axon Instruments). For ACh concentration-response curves in the excised and transferred to 15% polyacrylamide mapping gels and subject- absence or presence of CMPI and/or dFBr, ACh currents were ed to in-gel digestion with 100 mgofV8proteasetogeneratenAChRa normalized to current elicited by 1000 mM ACh. For calculating the subunit fragments (aV8-20, aV8-18, aV8-10, and aV8-4; Blanton and concentration-dependent effects of CMPI and dFBr, ACh currents in Cohen 1994) that were recovered from the gel and resuspended in di- the presence of increasing concentrations of CMPI and/or dFBr were gestion buffer. Greater than 95% of the Carb-inhibitable 3H cpm was normalized to the current elicited by ACh alone. Data (average 6 S.E.) recovered in aV8-20, which begins at aSer173 and contains the core of multiple replicas were plotted and fit using SigmaPlot (Systat aromatics aTyr190 and aTyr198 of ACh binding site segment C and Software, San Jose, CA). transmembrane helices M1–M3. aV8-20 was digested for 2 weeks with Radioligand Binding Assays. The effects of CMPI on the equi- 0.5 units of EndoLys-C, and the digest was then fractionated using librium binding of agonist ([3H]ACh) and ion-channel blocker reverse-phase high-performance liquid chromatography (rpHPLC). ([3H]phencyclidine [PCP]) to Torpedo nAChR–rich membranes in TPS All radioactivity in that rpHPLC fractionation eluted as a single,

were determined using a centrifugation assay (Hamouda et al., 2011). Carb-inhibitable peak centered at ∼80% organic in three fractions Downloaded from For [3H]ACh, membranes (40 nM ACh binding sites) were treated for which were pooled and loaded onto glass fiber filters for sequencing 30 minutes with 0.5 mM diisopropylphosphofluoridate to inhibit on a PROCISE 492 protein sequencer (Applied Biosystems, Foster acetylcholinesterase and then incubated for 60 minutes with 15 nM City, CA). At each cycle of Edman degradation, 1/6 was used for [3H]ACh (1.9 Ci/mmol) in the absence of any competitor or in the amino acid identification/quantification and 5/6 for 3Hcounting. presence of increasing concentrations of CMPI or 1 mM carbamylcho- Photolabeling efficiency (in cpm/pmol) was calculated using the 3 x line (Carb) (to determine nonspecific binding). For [ H]PCP, mem- equation [cpmx 2 cpm(x-1)]/5IoR , where x is the cycle number, I0 is branes (0.7 mM ACh binding sites) in TPS were equilibrated with 5 mM the initial mass of the peptide sequenced, and R is the repetitive yield molpharm.aspetjournals.org a-bungarotoxin (BgTx) or 1 mM Carb to stabilize the nAChR in the of Edman degradation. resting or desensitized state, respectively, then incubated with 10 nM [3H]CMPI-photolabeled g nAChR subunits were digested for 18 [3H]PCP (27 Ci/mmol) in the absence or presence CMPI or 100 mM hours at room temperature with 200 mg of V8 protease or for 2 weeks (to determine nonspecific binding). After incubation at room with 0.5 units of EndoLys-C. When the V8 digest was fractionated by temperature for 1 hour, the bound and free 3H were separated by rpHPLC, the peak of 3H was recovered in fractions centered at ∼40% centrifugation (18,000g for 1 hour) and quantified in count per minute organic known to contain the g subunit fragment beginning at gVal102 (cpm) by liquid scintillation counting. For each CMPI concentration, that contains ACh binding site segment E (g109 – g119; Chiara and 3 specific H binding was calculated (cpmtotal 2 cpmnonspecific) and Cohen, 1997). When the EndoLys-C digest was fractionated by small- normalized to the specifically bound 3H cpm in the absence of competitor. pore Tricine-SDS PAGE (Schagger and von Jagow, 1987; Hamouda 3 The average 6 S.D. of parallel duplicate aliquots was plotted and fit using et al., 2013), the peak of H was recovered in a gel band with an at ASPET Journals on September 26, 2021 SigmaPlot (Systat Software). [3H]CMPI Photolysis and Analytical Photolabeling. The CMPI UV absorption spectrum was characterized by a peak at 21 21 270 nm (extinction coefficient, «270 5 17,700 M cm ), whereas 21 21 «312 was 600 M cm . Using a hand-held lamp (model EB-280C; Spectronics Corp., Westbury, NJ), conditions for photolabeling were determined based upon the observed changes of UV absorp- tion following irradiation of CMPI (20 mM) in TPS at 312 nm, which resulted in a decrease in the 270-nm peak and the formation of a peak at 315 nm with a half-life (t1/2) of 3 minutes. Based upon this result, 3HincorporationintoTorpedo nAChR–rich membranes was examined by photolabeling at 312 nm on an analytical scale. Membrane suspensions (2 mg of protein/ml in TPS supplemented with 1 mM oxidized glutathione; 130 mg of protein and 170 pmol ACh binding sites per condition) were equilibrated with 1 mM[3H]CMPI (1 mCi per condition) for 40 minutes at 4°C and then irradiated on ice for 3 or 6 minutes at a distance of less than 1 cm. The membrane polypeptides were then resolved by SDS-PAGE as previously de- scribed (Hamouda et al., 2011) on two parallel 8% polyacrylamide gels. Polypeptides were visualized by Coomassie Blue R-250 stain, and one gel was prepared for fluorography using Amplify (GE Healthcare, Pittsburgh, PA). Polypeptide gel bands were excised from both gels, and 3H incorporation was determined by liquid scintillation counting. Since irradiation for 3 and 6 minutes resulted in the same level of 3H Fig. 2. ACh concentration-response curves of (a4)3(b2)2 and (a4)2(b2)3 nAChRs. Xenopus oocytes injected with nAChR subunit mRNAs mixed at a incorporation into nAChR subunits, subsequent analytical and pre- ratio of 9a4:1b2[m,(a4)3(b2)2 nAChR] or 1a4:9b2[d,(a4)2(b2)3 nAChR] parative photolabelings were irradiated at 312 nm for 3 minutes. were voltage clamped at 250 mV, and currents elicited by 10-second [3H]CMPI Preparative Photolabeling. Preparative-scale pho- applications of increasing concentrations of ACh were recorded. (A) tolabeling, isolation of photolabeled Torpedo nAChR fragments, and Representative traces of ACh-induced current responses of (a4)3(b2)2 identification of photolabeled amino acids by automated Edman and (a4)2(b2)3 nAChRs. For each concentration, the peak currents were 6 protein microsequencing were performed as detailed previously normalized to the peak current elicited by 1 mM ACh, and averages ( S.E.) of replicas from multiple oocytes (m,4;d, 6) were plotted (B) and fit to a (Chiara et al., 2009; Hamouda et al., 2013). In brief, Torpedo three-parameter Hill equation (Materials and Methods, eq. 1). ACh EC – 50 nAChR rich membranes (2 mg of protein/ml; 10 mg of protein and values/Hill coefficients for (a4)3(b2)2 and (a4)2(b2)3 nAChRs were 160 6 ∼13 nmol ACh binding sites per condition) were photolabeled with 24 mM/0.9 6 0.1 and 1.1 6 0.04 mM/1.260.04, respectively. 578 Hamouda et al.

Fig. 3. CMPI potentiates low-sensitivity (a4)3(b2)2 but not high-sensitivity (a4)2(b2)3 nAChRs. Xenopus oocytes expressing human (a4)3(b2)2 (m)or(a4)2(b2)3 (d, s) nAChRs were voltage clamped at 250 mV, and currents elicited by 10-second applications of 10 mM ACh (d, m)or 3 mM ACh (s) were recorded in the absence or presence of CMPI (A–D) or dFBr (E and F). Shown in (A and B) are representative traces of the effects of CMPI on ACh-induced current responses of (a4)3(b2)2 and (a4)2(b2)3 nAChRs, respectively. (C–F) At each PAM concentration, the current response was normalized to the current elicited by ACh alone, and the average 6 S.E. of responses from multiple oocytes [11 (C); 3 (d), 4 (s) (D); 14 (E); 5 (F)] is plotted. Potentiation data (C, E, and F) were fit to a three-parameter equation (eq. 2). Inhibition data (D) were fit to a single site model of inhibition (eq. 3). (C) The concentration depen- dence of CMPI potentiation of the (a4)3(b2)2 nAChR was characterized by EC50 = 0.26 6 0.05 mM, n = 1.2 6 0.2, and Imax = 314 6 17%. CMPI also potentiated responses of

(a4)3(b2)2 nAChR to 1 mM ACh (three oocytes; data not Downloaded from shown) with EC50 = 0.4 6 0.03 mM, n = 1.4 6 0.2, and Imax = 238 6 8%. (D) CMPI at 10 mM inhibited (a4)2(b2)3 nAChR by 39 6 3% with IC50 = 0.6 6 0.2 mM. (E) dFBr potentiation of (a4)3(b2)2 nAChRs was characterized by EC50 = 0.36 6 0.02 mM, n =1.66 0.2, and Imax =4176 7%. (F) dFBr potentiation of (a4)2(b2)3 nAChRs was characterized by EC50 = 1.6 6 0.04 mM, n = 1.2 6 0.03, and Imax =3046 4%. molpharm.aspetjournals.org apparent molecular mass of ∼24 kDa. When that material was further (Garcia et al., 2007). For the 14-kDa band, the major peak of 3H eluted purified by rpHPLC, all 3H eluted as a broad peak (∼60% organic), at ∼70% organic, the region known to contain the fragment beginning which was identified by N-terminal sequencing as the fragment begin- at dMet257, the N terminus of the dM2 transmembrane helix (Arevalo ning at gGlu47 that contains ACh binding site segment D (gTrp55; et al., 2005). For the 21-kDa band, the major peak of 3H eluted at ∼50% Chiara and Cohen, 1997) . organic and was identified by sequence analysis as the fragment [3H]CMPI-photolabeled nAChR d subunits were digested for 2 beginning at Glu47 that contains ACh binding site segment D (dTrp57) weeks with 0.5 units of EndoLys-C. The digest was fractionated by (Wang et al., 2000). Tricine SDS-PAGE, and material eluted from gel bands with apparent Data Analyses. Data analyses and parameter calculations molecular masses of ∼21 and 14 kDa was fractionated by rpHPLC were performed using SigmaPlot version 11 (Systat Software). ACh at ASPET Journals on September 26, 2021

Fig. 4. CMPI increases ACh potency at low sensitivity (a4)3(b2)2 nAChRs. ACh current responses of Xenopus oocytes expressing (a4)3(b2)2 nAChR were recorded in the absence of any other drug (n) and in the presence of 1 mM CMPI or 1 mM dFBr. (A) A representative current trace showing the effect of 1 mM CMPI on currents elicited by 10, 100, and 1000 mM ACh. (B) ACh concentration-response curves for (a4)3(b2)2 nAChRs in the absence (n) and presence of 1 mM CMPI (m). (C) A representative current trace showing the effect of increasing concentrations of dFBr on currents elicited by 1000 mM ACh. (D) ACh concentration-response curves for (a4)3(b2)2 nAChRs in the absence (n) and presence of 1mM dFBr (m). For each ACh concentration (2/+ drug), the current was normalized to the current elicited by 1000 mM ACh in the same oocyte. Averages (6S.E.) for responses from multiple oocytes [14 (n), 12 (m)(B);6(n), 4 (m)] were plotted and fit to eq. 1. Values of ACh (EC50/n/Imax) were as follows: ACh (control), 92 6 17 mM/0.8 6 0.07/110 6 6%; ACh (+CMPI), 0.7 6 0.2 mM/0.6 6 0.1/97 6 4% (B); ACh (control), 190 6 30 mM/0.73 6 0.04/130 6 6%; ACh (+1 dFBr), 139 6 112 mM/0.43 6 0.06/339 6 52% (D). For high-sensitivity (a4)2(b2)3 nAChRs (not shown), the effect of dFBr on the ACh concentration response was characterized by ACh EC50/n/Imax values of 1.4 6 0.08 mM/1.2 6 0.08/111 6 1.4% (2dFBr) and 2.3 6 0.05 mM/0.8 6 0.1/464 6 23% (+dFBr). A Photoreactive PAM of (a4)3(b2)2 nAChR 579 concentration-response curves in the absence or presence of CMPI membranes were normalized to their control values in the absence of and/or dFBr were fit to a three-parameter Hill equation: inhibitor and fit to a single site inhibition model: À Á n 5 =ð 1 ð = Þ Ix 5 Imax= 1 1 ðEC50=xÞ (1) f x 100 1 x IC50 (3) where f is ACh-induced current or specific [3H]PCP binding in the where Ix is the current at ACh concentration x, normalized to the x presence of CMPI at concentration x, and IC is the CMPI concen- current at 1 mM ACh; Imax is the maximum current response; n is the 50 tration producing 50% inhibition. Hill coefficient; and EC50 is the ACh concentration producing 50% of the maximum response. The concentration-dependent potentiation of ACh-induced currents by CMPI or dFBr was fit to a three-parameter Hill equation: Results À Á a b n CMPI Potentiates Low-Sensitivity ( 4)3( )2 but Not Ix 5 100 1 Imax= 1 1 ðEC50=xÞ (2) High-Sensitivity (a4)2(b)3 nAChRs. Initial characteriza- tion of CMPI as an nAChR PAM established that it poten- where Ix is the ACh current in the presence of PAM at concentration x, normalized to the current in the absence (as percentage), Imax is the tiated responses of a4b2 nAChRs expressed in human maximum potentiation of current (as percent control); n is the embryonic kidney cells to submaximal doses of nicotine or

Hill coefficient; and EC50 is the PAM concentration producing 50% ACh (Albrecht et al., 2008). To further characterize the maximal potentiation. pharmacological profile of CMPI at a4b2nAChRs,weused Downloaded from CMPI inhibition of ACh-induced currents in muscle-type nAChRs two-electrode voltage-clamp recording from Xenopus laevis 3 – and CMPI inhibition of [ H]PCP binding to Torpedo nAChR rich oocytes to characterize the effects of CMPI on the ACh responses of (a4)3(b2)2 (low ACh sensitivity) and (a4)2(b2)3 (high ACh sensitivity) nAChR isoforms. Expression of (a4)3(b2)2 (ACh EC50 5 160 6 24 mM) and (a4)2(b2)3 (ACh molpharm.aspetjournals.org EC50 5 1.14 6 0.04 mM) nAChRs was achieved by injecting oocytes with a mixture of a4 and b2 mRNAs at ratios of 9a4: 1b2or1a4:9b2, respectively (Fig. 2). For (a4)3(b2)2 nAChRs, at ASPET Journals on September 26, 2021

Fig. 6. CMPI inhibits muscle-type nAChRs. Xenopus oocytes injected with Fig. 5. CMPI and dFBr act independently to potentiate low-sensitivity mRNA for human muscle (2a:1b:1d:1«; ,)orTorpedo (2a:1b:1g:1d; m) (a4)3(b2)2 nAChRs. (A and B) Representative current responses elicited by nAChR were voltage clamped at 250 mV, and currents elicited by 10- 10 mM ACh (A) or 1000 mM ACh (B) in the absence and presence of CMPI second applications of 10 mM ACh were recorded in the absence or presence and/or dFBr. (C) ACh concentration response in the absence (n) and of increasing concentrations of CMPI. Shown in (A) and (B) are represen- presence of 1 mM CMPI and 1 mM dFBr (j). For each ACh concentration tative traces for human a2b1«d and Torpedo a2b1gd nAChRs. (C) Currents (2/+ drug), the current was normalized to the current elicited by 1000 mM were normalized to the peak current elicited by 10 mM ACh alone, and ACh in the same oocyte. Average (6S.E.) from multiple oocytes [n (9), j averages (6S.E.) of multiple replicas from six oocytes were plotted and fit (8)] were plotted and fit to eq. 1. Values of ACh (EC50/n/Imax) were as to a single site inhibition model (eq. 3). CMPI inhibited human muscle and follows: ACh (control), 106 6 29 mM/0.62 6 0.06/124 6 7%; ACh (+CMPI Torpedo nAChR with IC50 values of 0.7 6 0.05 and 0.2 6 0.03 mM, +dFBr), 0.20 6 0.04 mM/1.2 6 0.3/223 6 8%. respectively. 580 Hamouda et al.

potentiated responses for 1 mM ACh (Fig. 4C) with an EC50 of ∼0.3 mM, i.e., a value similar to that seen for 10 mM ACh (Fig. 3E). dFBr increased the ACh maximal response (Imax 5 339 6 52%) with little effect on ACh potency (Fig. 4D). The differences between CMPI and dFBr in terms of a4b2 nAChR isoform selectivity and their effects on the ACh responses led us to examine the effect of CMPI on (a4)3(b2)2 nAChR potentiation by dFBr and vice versa. In the presence of Fig. 7. CMPI binds in the Torpedo nAChR ion channel. Equilibrium 1 mM CMPI, a concentration that alone elicited maximum binding of [3H]ACh and [3H]PCP to Torpedo nAChR–rich membranes was potentiation of the ACh response at submaximal concentra- determined in the absence and presence of increasing concentrations of tions, dFBr still produced a further potentiation of ACh at CMPI. Specific binding was calculated as the difference between the total 10 mM (EC ; Fig. 5A) or at 1 mM (Fig. 5B). Simultaneous and nonspecific binding, which was determined in the presence of 1 mM 10 Carb ([3H]ACh) or 100 mM proadifen ([3H]PCP), and then normalized to application of 1 mM CMPI and 1 mM dFBr produced a left shift the specific binding in the absence of CMPI. Data were fit to a single site in the ACh concentration-response curve, increasing ACh model of inhibition (eq. 3). Binding (total/nonspecific) was as follows: for potency by ∼100-fold and increasing efficacy (Imax 5 223 6 [3H]ACh (2/+1 mM Carb, 10,680 6 65/130 6 3 cpm); for [3H]PCP (+Carb, 2 6 6 2 8%) (Fig. 5C). These results indicate that CMPI and dFBr /+100 mM proadifen, 7640 75/1580 15 cpm; +BgTx, /+100 mM Downloaded from proadifen, 4240 6 47/1933 6 22 cpm). CMPI inhibited [3H]PCP binding in bind at different sites and act independently to enhance the 6 6 the presence of Carb or BgTx with IC50 values of 20 1 mM and 35 3 mM, potency or efficacy of ACh at (a4)3(b2)2 nAChRs. respectively. CMPI Is an Inhibitor of Muscle-Type nAChRs. Be- cause the interaction of CMPI with muscle-type nAChRs has CMPI, when coapplied, enhanced the response to 10 mM not been characterized, we examined the effect of CMPI on (EC10) ACh, producing a maximal potentiation (Imax)of ACh-induced currents in Xenopus oocytes expressing human molpharm.aspetjournals.org 314 6 17% with an EC50 of 0.26 6 0.05 mM (Fig. 3, A and C). In muscle (2a:1b:1d:1«)orTorpedo (2a:1b:1g:1d) nAChRs (Fig. oocytes expressing (a4)2(b2)3 nAChRs, CMPI (0.01–10 mM) 6). CMPI inhibited human muscle and Torpedo nAChRs with did not enhance current responses for 1, 3, or 10 mM ACh. IC50 values of 0.7 6 0.05 and 0.2 6 0.03 mM, respectively. Instead, CMPI reduced the ACh response, maximally by ∼35% Additionally, we characterized the effect of CMPI on the at 10 mM CMPI (Fig. 3, B and D). In contrast to the selective equilibrium binding of [3H]ACh and an ion-channel blocker 3 potentiation of (a4)3(b2)2 nAChRs by CMPI, in the same ([ H]PCP) to Torpedo nAChR (Fig. 7). CMPI up to 100 mM did 3 oocytes, dFBr enhanced ACh responses of (a4)2(b2)3 nAChRs not displace [ H]ACh binding. However, CMPI competitively 3 (Fig. 3, B and F; EC50 5 1.6 6 0.05 mM; Imax 5 304 6 4%) as inhibited [ H]PCP binding to Torpedo nAChR stabilized well as (a4)3(b2)2 nAChRs (Fig. 3E; EC50 5 0.36 6 0.02 mM; either in the resting state, closed-channel state (1BgTx, IC50 5

Imax 5 417 6 7%). 20 mM), or desensitized state (1Carb, IC50 5 35 mM). These at ASPET Journals on September 26, 2021 When coapplied with ACh to oocytes expressing (a4)3(b2)2 results indicate that CMPI, at concentrations that produced nAChRs, CMPI at 1 mM produced a left shift of the ACh functional inhibition of muscle-type nAChRs, binds to a concentration response, decreasing EC50 from 92 617 mMto site within the ion channel but not to the orthosteric ACh 0.760.2 mM without any change in the maximum response binding sites. 3 (Imax 5 97 6 4%) (Fig. 4, A and B). This effect of CMPI on the Photolabeling of Torpedo nAChR with [ H]CMPI. UV ACh concentration response is similar to that of NS9283 photolysis conditions for optimal [3H]CMPI photoincorpora- (Olsen et al., 2013) but differs from that of dFBr, which tion into Torpedo nAChR subunits were established as

Fig. 8. Photoincorporation of [3H]CMPI into Torpedo nAChR–rich membranes. (A) Coomassie Blue stain (lane 1) and fluorograph (lanes 2–3) of an SDS- PAGE gel for membranes (130 mg, 170 pmol ACh sites per condition) photolabeled with 1 mM[3H]CMPI in the absence of other ligand (lane 2) or in the presence of 1 mM Carb (lane 3). The electrophoretic mobilities of the nAChR a, b, g,andd subunits; rapsyn (Rn); the Na+/K+-ATPase a subunit (90K); and calelectrin (37K) are indicated. (B) Quantification of the effects of Carb, BgTx, and CMPI (100 mM) on [3H]CMPI incorporation into nAChR subunits. Data in (B) are from the same analytical [3H]CMPI photolabeling experiment, with photolabeling in the control condition (no drug added) done in duplicate. Aliquots of nAChRs from each photolabeling condition were resolved on two parallel SDS-PAGE gels, and polypeptide bands were excised from both gels for 3H quantification by liquid scintillation counting. Data for Carb, BgTx, and CMPI conditions are average 6 1/2 range of gel bands excised from the two parallel gels. Data for the control condition are average 6 S.D. of four gel bands from the two parallel gels. A Photoreactive PAM of (a4)3(b2)2 nAChR 581 described in Materials and Methods. To examine the pharma- cological specificity of [3H]CMPI photoincorporation into the nAChR, Torpedo nAChR–rich membranes were photolabeled with [3H]CMPI (1 mM) in the absence (no drug added) and presence of 1 mM Carb, 2 mM BgTx, or 100 mM CMPI, and covalent incorporation within the subunits was determined by SDS-PAGE followed by fluorography or liquid scintillation counting (Fig. 8). In the absence of any other drug, [3H]CMPI photoincorporated most prominently into the nAChR a, b, and g subunits and into non-nAChR proteins that bind to the nAChR (Rn, rapsyn) or are present in contaminating mem- brane fragments [calelectrin (37K), a member of the annexin family of Ca21-binding proteins, and the a subunit of Na1/K1- ATPase (90K)] (Blanton et al., 2001). Photolabeling of a and g nAChR subunits was reduced by the same extent (∼50%) in the presence of Carb, BgTx, or CMPI, indicating that a major component of subunit photolabeling was likely to be within Downloaded from the amino acids contributing to the ACh binding site at the a:g subunit interface. Photolabeling in the nAChR d sub- unit, which contributes to the ACh binding site at the a:d subunit interface, was ∼30% that of the g subunit and was decreased by ∼10 and 20% in the presence of BgTx and CMPI, respectively. molpharm.aspetjournals.org [3H]CMPI Photolabeling of Agonist Binding Sites. The ACh binding sites of the muscle-type nAChR are located within the ECD at the two a subunit interfaces with adjacent g and d subunits. Core aromatic residues contributing to the ACh binding sites are located in three distinct regions within the primary sequence of the a subunit (segments A, B, and C) 3 3 that contribute to the principal surface of the binding pocket, Fig. 9. [ H]CMPI photolabeling of ACh binding sites. H(s, d) and phenylthiohydantoin amino acids (⬜, j) released during sequence and amino acids that contribute to the complementary sur- analyses of nAChR subunit fragments beginning at aHis186 containing face are located in three regions within the g or d subunits segment C of the ACh binding sites (ABS) (A), gVal102 containing ACh at ASPET Journals on September 26, 2021 47 (segments D, E, and F) (Brejc at al. 2001; Changeux and Taly, binding site segment E (B), and gGlu and dGlu47 containing ACh binding site segment D (C and D). The fragments were isolated from Torpedo 2008). To identify amino acids photolabeled within the a nAChR photolabeled with [3H]CMPI in the absence (⬜, s) and presence of 3 subunit, the [ H]CMPI-photolabeled a subunit was subjected Carb (j,d), as described under Materials and Methods. (A) The fragment to in-gel digestion with V8 protease (data not shown). The beginning at aHis186 (⬜,2Carb/j, +Carb, 5.2/7.5 pmol) was present at a 3 10-fold higher level than any other fragment, and the Carb-inhibitable majority (.95%) of Carb-inhibitable H was contained 3 173 peaks of H release at cycles 5, 7, and 13 established photolabeling of with aV8-20, which begins at aSer and contains the core aTyr190 and aTyr198, the core aromatics in a subunit ACh binding site aromatics in ACh binding site segment C (aTyr190/aTyr198) segment C, at 16 and 29 cpm/pmol, respectively, and of aCys192 at 102 and transmembrane helices M1–M3. When the subunit frag- 8 cpm/pmol. (B) The fragment beginning at gVal was present (⬜, 186 2Carb/j, +Carb, 9/2.5 pmol) as a secondary sequence along with an ment beginning at aHis was isolated from an EndoLys-C N-terminal fragment of V8 protease (Val1; 2Carb/+Carb, 200/140 pmol). The 3 digest of aV8-20 and sequenced (Fig. 9A), the peaks of H release major peaks of 3H release at cycles 10 and 16 established Carb-inhibitable 3 photolabeling of gTyr111 and gTyr117 at 56 and 82 cpm/pmol, respectively. (C) in cycles 5, 7, and 13 indicated [ H]CMPI photolabeling of 47 190 192 198 With the fragment beginning at gGlu (⬜,2Carb/j,+Carb,3/6pmol) aTyr , aCys ,andaTyr (16, 8, and 29 cpm/pmol, 3 present at a 10-fold higher level than any other fragment, the peak of H respectively), which was inhibited by .90% in the presence release at cycle 9 indicates photolabeling of the core aromatic in g subunit of Carb. To identify amino acids photolabeled within g ACh binding sites segment D, gTrp55 (s, 2Carb/d, +Carb, 30/0.7 cpm/pmol). 55 (D) Fragments beginning at dGlu47 and dHis26 were present (⬜, 2Carb/j, subunit ACh binding site segments D (core aromatic gTrp ) 3 109 119 47 102 +Carb, 11/10 and 3/2.6 pmol, respectively). The peak of Hreleaseat and E (g -g ), peptides beginning at gGlu and gVal cycle 11 was consistent with Carb-inhibitable photolabeling of the core were isolated from EndoLys-C and V8 protease digests of g aromatic in d subunit ACh binding site segment D, dTrp57 (s, 2Carb/d, subunit, respectively. Sequencing of the peptide beginning +Carb, 2/0.1 cpm/pmol). at gVal102 established [3H]CMPI photolabeling of gTyr111 (56 cpm/pmol) and gTyr117 (84 cpm/pmol) that was com- pletely inhibitable by Carb (Fig. 9B). Amino acid sequencing CMPI Photolabeling of the Ion Channel. While the of a peptide beginning at gGlu47 (Fig. 9C) identified Carb- majority of [3H]CMPI photoincorporation was associated with inhibitable photolabeling of gTrp55 (2/1Carb, 30/0.7 cpm/pmol). the ACh binding sites, CMPI inhibition of [3H]PCP and [3H] Sequencing of a peptide beginning at dGlu47 that contains d ACh binding to the Torpedo nAChR established that CMPI subunit ACh binding site segment D (core aromatic dTrp57) binds with higher affinity in the ion channel than in the ACh (Fig. 9D) established that there was also Carb-inhibitable binding sites. To identify the CMPI binding site within the ion photolabeling of dTrp57 (2/1Carb, 2/0.1 cpm/pmol), but it was channel, we examined [3H]CMPI photolabeling of amino acid at less than 10% the efficiency of photolabeling of gTrp55, the residues within the M2 helices that line the ion channel corresponding amino acid in g subunit ACh binding site (Unwin, 2005). As shown in Fig. 10, N-terminal sequencing segment D. of a subunit fragment beginning at the N terminus of dM2 582 Hamouda et al.

that CMPI photoincorporated into valine and serine side chains in the ion channel and into tyrosines and trypto- phans in the ACh binding sites demonstrates that CMPI is a photoreactive PAM possessing broad amino acid side-chain reactivity, which makes it well suited to identify PAM binding sites in neuronal nAChRs. Pharmacology of CMPI at a4b2 nAChRs. The selective potentiation of (a4)3(b2)2 nAChRs indicates that the a4:a4 interface is required for CMPI binding and/or modulation of channel gating. In addition to CMPI, physostigmine (a non- 3 Fig. 10. [ H]CMPI photolabeling in the Torpedo nAChR (dM2) ion selective nAChR PAM) and NS9283 [a PAM selective for channel. 3H(s, d) and phenylthiohydantoin amino acids (⬜, j) released during sequence analyses of the d subunit fragment beginning at dMet257 nAChRs containing a2 and a4 subunits (Timmermann et al., (2/+Carb, 19/11 pmol) isolated from nAChRs photolabled by [3H]CMPI in 2012)] also require the presence of an a4:a4 subunit interface the absence (2Carb; s, ⬜) or presence (+Carb; d, j) of 1 mM Carb. For the for a4b2 nAChR potentiation (Olsen et al., 2013, 2014; Jin –Carb condition, the peak of 3H release in cycle 13 indicates photolabeling of dVal271 (dM2-13; 0.6 cpm/pmol) that was inhibited by 76% in the et al., 2014). Although there is no established relationship presence of Carb. For the +Carb condition, the peak of 3H release in cycle 6 between the location of a PAM binding site and its effect on indicates [3H]CMPI photolabeling at dSer262 (dM2-6; 0.3 cpm/pmol). ACh responses, these nAChR PAMs with a4:a4 subunit Downloaded from interface selectivity each increase ACh potency without altering the ACh maximal response (Pandya and Yakel, (dMet257) established state-dependent photolabeling of resi- 2011; Olsen et al., 2013), similar to the effect of benzodiaz- dues known to line the ion channel lumen. For nAChRs epines on GABA responses in abg GABA type A receptors photolabeled with [3H]CMPI in the absence of Carb (resting 3 (Sigel and Steinmann, 2012). state), there was a peak of H release in cycle 13 that indicated molpharm.aspetjournals.org In contrast, consistent with previous results (Kim et al., 2007; labeling of dVal271 (dM2-13; 0.6 cpm/pmol). In the presence of Weltzin and Schulte, 2015), we found that dFBr potentiates both Carb (desensitized state), photolabeling of dVal271 was re- (a4) (b2) and (a4) (b2) nAChRs, increasing ACh maximal duced by 75%. The major peak of 3H release was in cycle 6, 3 2 2 3 responses with little effect on ACh EC .Wealsofoundthat indicating photolabeling of dSer262 (dM2-6; 0.3 cpm/pmol). 50 CMPI at 1 mM, a concentration that produces maximal poten- tiation, did not prevent further potentiation of (a4)3(b2)2 nAChR Discussion by dFBr and vice versa (Fig. 5, A and B). The effects of both CMPI (decreased ACh EC50) and dFBr (increased ACh maximal In this report, we demonstrate that CMPI, a potent PAM response) were maintained when CMPI and dFBr were coap- selective for nAChRs containing the a4 subunit (Albrecht et al., plied with ACh (Fig. 5C). These results indicate that CMPI and at ASPET Journals on September 26, 2021 2008), potentiates responses of (a4)3(b2)2 (low-sensitivity) dFBr potentiate (a4)3(b2)2 nAChR by independent mechanisms. nAChRs, but not (a4)2(b2)3 (high-sensitivity) nAChRs. By re- They also establish the presence of at least two classes of PAM cording from Xenopus oocytes expressing (a4)3(b2)2,(a4)2(b2)3,or binding sites within a4b2 nAChRs: one at the a4:a4 subunit muscle-type (a2b«d or a2bgd)nAChRs,wefoundthatCMPI interface that mediates potentiation by CMPI, and one or more at enhances ACh potency of (a4)3(b2)2 nAChRs without changing the a4:b2and/orb2:a4 subunit interface that mediate potenti- ACh efficacy (maximal response). At concentrations producing ation by dFBr. In a4b2 nAChRs, there can be a single binding a maximal enhancement of (a4)3(b2)2 nAChR responses, CMPI site per receptor for PAMs such as CMPI and NS9283, which are acts as an allosteric inhibitor of (a4)2(b2)3 nAChRs and of selective for (a4)3(b2)2 nAChRs and bind at the a4-a4 subunit human (a2b«d)andTorpedo (a2bgd) muscle-type nAChRs. In interface. For NS9283, mutational analyses predicted a binding the Torpedo nAChR, CMPI binds with .30-fold higher affinity site in the ECD at the a4:a4 subunit interface that overlaps with in the ion channel than in the agonist binding sites. Identifica- the third ACh binding site in that receptor (Olsen et al., 2013). tion of the Torpedo nAChR amino acids photolabeled by However, further studies will be required to determine whether, 3 [ H]CMPI confirmed binding in the ion channel and the ACh in the (a4)3(b2)2 nAChR, CMPI binds to the same site as NS9283. binding sites, and no evidence was found of photolabeling of ForPAMssuchasdFBrthatpotentiate(a4)2(b2)3 and other sites in the nAChR ECD or TMD. However, the fact (a4)3(b2)2 nAChRs, there will be at least two binding sites

Fig. 11. Location of [3H]CMPI-photolabeled amino acids within the Torpedo nAChR ECD and TMD. Side views of the Torpedo nAChR (Unwin, 2005; a, gold; b, cyan; g, green; and d, brown) from the outside showing the ECD of a-g subunits (A) and from the inside of the ion channel showing the TMD of the b-d-a subunits (B). [3H]CMPI-photolabeled amino acids within the ACh binding site at the extracellular interface of a:g subunits and within the dM2 helix that contribute to the ion channel are shown in stick format and colored by elements (carbone, gray; oxygen, red; nitrogen, blue). A Photoreactive PAM of (a4)3(b2)2 nAChR 583 within a receptor, either at subunit interfaces (intersubunit side chains, but we can only speculate on the photoreactive sites) or within a subunit’s helix bundle (intrasubunit sites). intermediates formed upon irradiation at 312 nm. CMPI In agreement with this, substitutions of an amino acid within has a complex ring structure (Fig. 1) containing piperidine, the b subunit ECD that projects into the b-a interface (two chlorobenzene, and pyrazole isoxazole rings. CMPI photo- per receptor) reduced a4b2 nAChR potentiation by dFBr incorporation into valine in the ion channel indicates the (Weltzin and Schulte, 2015). likely involvement of a radical intermediate, whereas reac- [3H]CMPI Is an nAChR Photoreactive Probe. Photo- tions with serines or aromatic amino acid side chains can affinity labeling and protein microsequencing techniques have occur via radical or electrophilic intermediates. The 3,5- been used extensively for identification of amino acids con- disubstituted isoxazole ring forms a reactive azirine inter- tributing to drug binding sites in many targets (Vodovozova, mediate upon irradiation at 313 nm or shorter wavelengths 2007; Das, 2011), including muscle-type nAChR binding sites (Price and Ratajczak, 1984), and UV irradiation of pyrazoles for orthosteric ligands, channel blockers, and allosteric mod- atwavelengthsbelow250nmresultsinH-atomlossand ulators (Hamouda et al., 2014; Forman et al., 2015). Photo- formation of radical intermediates (King et al., 2010). affinity labeling identifies amino acid residues within the Similarly, photodehalogenation (C-Cl cleavage) of the chlo- protein structure that are in direct contact with a bound drug robenzene ring would form a radical intermediate with and differentiates them from amino acids not contributing to broad amino acid side-chain reactivity. Even in the absence the binding site but important for drug action, which can be of further definition of CMPI’s photoreactive intermediates, Downloaded from identified by mutational analyses. Even though CMPI bound the fact that it photoincorporates into a broad range of with .30-fold higher affinity in the Torpedo nAChR ion amino acid side chains makes it well suited for use in the 3 channel than in the transmitter binding sites, [ H]CMPI identification of its binding sites in (a4)3(b2)2 nAChRs, 55 photoincorporated with .3-fold higher efficiency into gTrp , where it acts as a PAM, and in (a4)2(b2)3 nAChRs, where it gTyr111, and gTyr117 in the ACh binding sites than into acts as a negative allosteric modulator. dVal271, the amino acid labeled most efficiently in the ion molpharm.aspetjournals.org channel (Fig. 11). Since reaction with aromatic amino acid side Acknowledgments chains is likely to occur by a different reactive intermediate The authors thank Dr. Stefan I. McDonough and Dr. Alessandro than incorporation into aliphatic amino acid side chains, this A. Boezio from Amgen Inc. for generously providing CMPI. result is not unexpected. However, the fact that [3H]CMPI 55 incorporates into gTrp with 15-fold higher efficiency than Authorship Contributions 57 into dTrp , the amino acid in d subunit ACh binding site Participated in research design: Hamouda, Cohen. segment D equivalent to gTrp55 in the g subunit, suggests that Conducted experiments: Hamouda, Deba, Wang. CMPI binds preferentially to the ACh binding site at the a-g Performed data analysis: Hamouda, Deba, Cohen. interface, as is seen for d-tubocurarine, the classic muscle-type Wrote or contributed to the writing of the manuscript: Hamouda, at ASPET Journals on September 26, 2021 nAChR competitive antagonist, which photolabels the same Cohen. amino acids in the ACh binding site as CMPI (Chiara and References Cohen, 1997; Chiara et al., 1999). 3 Albrecht BK, Berry V, Boezio AA, Cao L, Clarkin K, Guo W, Harmange JC, Hierl M, As seen for ACh binding site photolabeling by [ H]d- Huang L, and Janosky B et al. (2008) Discovery and optimization of substituted piperidines as potent, selective, CNS-penetrant alpha4beta2 nicotinic acetylcholine tubocurarine and other photoaffinity and competi- – 3 receptor potentiators. Bioorg Med Chem Lett 18:5209 5212. tive antagonists, [ H]CMPI photolabeling of the ACh binding Arevalo E, Chiara DC, Forman SA, Cohen JB, and Miller KW (2005) Gating- site core aromatics (aTyr190, aTyr198, gTrp55,anddTrp57) enhanced accessibility of hydrophobic sites within the transmembrane region 111 117 of the nicotinic acetylcholine receptor’s d-subunit. A time-resolved photolabeling and of gTyr and gTyr was fully inhibited in the presence study. J Biol Chem 280:13631–13640. of agonist (Chiara and Cohen, 1997; Nirthanan et al., 2005; Bertrand D and Gopalakrishnan M (2007) Allosteric modulation of nicotinic acetyl- receptors. Biochem Pharmacol 74:1155–1163. Srivastava et al., 2009). This full inhibition of photolabeling Blanton MP and Cohen JB (1994) Identifying the lipid-protein interface of the Tor- by agonist contrasts with what has been seen for [3H]dFBr pedo nicotinic acetylcholine receptor: secondary structure implications. Bio- 3 chemistry 33:2859–2872. and the nonselective nAChR PAMs [ H]physostigmine and Blanton MP, Lala AK, and Cohen JB (2001) Identification and characterization of 3 [ H]galanthamine, which also photolabeled amino acids at membrane-associated polypeptides in Torpedo nicotinic acetylcholine receptor-rich 190 198 membranes by hydrophobic photolabeling. Biochim Biophys Acta 1512:215–224. the a/g extracellular subunit interface (aTyr , aTyr , Brejc K, van Dijk WJ, Klaassen RV, Schuurmans M, van Der Oost J, Smit AB, 111 117 gTyr ,andgTyr ) but with different Carb sensitivity and Sixma TK (2001) Crystal structure of an ACh-binding protein reveals the (Hamouda et al., 2013, 2015). [3H]dFBr, [3H]physostigmine, ligand-binding domain of nicotinic receptors. 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