Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2016/03/17/jpet.115.231712.DC1

1521-0103/357/2/394–414$25.00 http://dx.doi.org/10.1124/jpet.115.231712 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 357:394–414, May 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Discovery and Characterization of AMPA Receptor Modulators Selective for TARP-g8 s

Michael P. Maher, Nyantsz Wu, Suchitra Ravula, Michael K. Ameriks, Brad M. Savall, Changlu Liu, Brian Lord, Ryan M. Wyatt, Jose A. Matta, Christine Dugovic, Sujin Yun, Luc Ver Donck, Thomas Steckler, Alan D. Wickenden, Nicholas I. Carruthers, and Timothy W. Lovenberg Janssen Research and Development, LLC, Neuroscience Therapeutic Area, San Diego, California (M.P.M., N.W., S.R., M.K.A., B.M.S., C.L., B.L., R.M.W., J.A.M., C.D., S.Y., A.D.W., N.I.C., T.W.L.); and Janssen Research and Development, a Division of Janssen

Pharmaceutica NV, Neuroscience Therapeutic Area, Beerse, Belgium (L.V.D., T.S.) Downloaded from Received December 23, 2015; accepted March 11, 2016

ABSTRACT Members of the a-amino-3-hydroxyl-5-methyl-4-isoxazole- pharmacokinetic properties and achieved high receptor occu- propionic acid (AMPA) subtype of ionotropic glutamate recep- pancy following oral administration. This molecule showed jpet.aspetjournals.org tors mediate the majority of fast synaptic transmission within strong, dose-dependent inhibition of neurotransmission within the mammalian brain and spinal cord, representing attractive the hippocampus, and a strong anticonvulsant effect. At high targets for therapeutic intervention. Here, we describe novel levels of receptor occupancy in rodent in vivo models, JNJ- AMPA receptor modulators that require the presence of the 55511118 showed a strong reduction in certain bands on electro- accessory protein CACNG8, also known as transmembrane encephalogram, transient hyperlocomotion, no motor impairment AMPA receptor regulatory protein g8 (TARP-g8). Using calcium on rotarod, and a mild impairment in learning and memory. JNJ- flux, radioligand binding, and electrophysiological assays of 55511118 is a novel tool for reversible AMPA receptor in- wild-type and mutant forms of TARP-g8, we demonstrate that hibition, particularly within the hippocampus, with potential at ASPET Journals on September 27, 2021 these compounds possess a novel mechanism of action therapeutic utility as an anticonvulsant or neuroprotectant. The consistent with a partial disruption of the interaction between existence of a molecule with this mechanism of action demon- the TARP and the pore-forming subunit of the channel. One strates the possibility of pharmacological targeting of acces- of the molecules, 5-[2-chloro-6-(trifluoromethoxy)phenyl]-1,3- sory proteins, increasing the potential number of druggable dihydrobenzimidazol-2-one (JNJ-55511118), had excellent targets.

Introduction postsynaptic membranes of excitatory synapses in the central nervous system. AMPA receptors (AMPARs) mediate the Glutamate is the primary excitatory neurotransmitter in majority of fast synaptic transmission within the central mammalian brain. The a-amino-3-hydroxyl-5-methyl-4- nervous system (CNS). Thus, inhibition or negative modula- isoxazole-propionic acid (AMPA) subtype of glutamate recep- tion of AMPARs is an attractive strategy for therapeutic tors are ligand-gated ion channels expressed primarily on intervention in CNS disorders characterized by excessive neuronal activity. With the notable exception of pore blockers (which are selective for calcium-permeable AMPA receptors; dx.doi.org/10.1124/jpet.115.231712. s This article has supplemental material available at jpet.aspetjournals.org. see Stromgaard and Mellor, 2004), no AMPAR inhibitors have

ABBREVIATIONS: ACSF, artificial cerebrospinal fluid; AMPA, a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid; AMPAR, AMPA receptor; ANOVA, analysis of variance; CHO, Chinese hamster ovary; CP-465022, 3-(2-Chlorophenyl)-2-[2-[6-[(diethylamino)methyl]-2-pyridinyl]ethenyl]-6- fluoro-4(3H)-quinazolinone hydrochloride; CNS, central nervous system; CT, carboxyl terminus; DMSO, dimethylsulfoxide; DNMTP, delayed non-match to position; EC50, half-maximal effective concentration; ED50, half-maximal effective dose; EEG, electroencephalogram; EMG, electromyogram; EPSC, excitatory postsynaptic current; EX, extracellular domain; FAM, familiar arm; fEPSP, field excitatory postsynaptic potential; f u, unbound fraction; GluA, AMPA subtype of ionotropic glutamate receptor; GYKI-53655, 1-(4-Aminophenyl)-3-methylcarbamyl-4- methyl-3,4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride; HABG, HibernateA supplemented with B27 and Glutamax; HEK-293, human embryonic kidney 293; HPMC, hydroxypropyl methylcellulose; J values, indirect dipole-dipole coupling constants; JNJ-55511118, 5-[2-chloro-6-(trifluoromethoxy)phenyl]-1,3-dihydrobenzimidazol-2-one; JNJ-56022486, 2-(3-chloro-2-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol- 5-yl)phenyl)acetonitrile; LC-MS/MS, liquid chromatography–tandem mass spectrometry; LY-395153, N-[4-[1-(propan-2-ylsulfonylamino)propan- 2-yl]phenyl]benzamide; MES, maximal electroshock; MWM, Morris water maze; NEW, novel arm; NIH, National Institutes of Health; NMDA, N-methyl-D-aspartate;NREM,non-rapideyemovement;NSB,nonspecificbinding;PAM,positiveallostericmodulator;PCR, polymerase chain reaction; Philanthotoxin-74, (S)-N-[7-[(4-Aminobutyl)amino]heptyl]-4-hydroxy-a-[(1-oxobutyl)amino]benzenepropanamide dihydrochloride; p.o., per os; PTZ, pentylenetetrazole; RED, Rapid Equilibrium Dialysis; REM, rapid eye movement; SB, specific binding; TARP, transmembrane AMPA receptor regulatory protein; TB, total binding; TM, transmembrane domain.

394 TARP-g8–Selective AMPAR Modulators 395 been found to have selectivity among the AMPAR subtypes, or Here, we describe the in vitro and in vivo characterization of to exhibit regional specificity. Since AMPAR activity is 5-[2-chloro-6-(trifluoromethoxy)phenyl]-1,3-dihydrobenzimidazol- ubiquitous within the CNS, general antagonism results in 2-one (JNJ-55511118) and 2-(3-chloro-2-(2-oxo-2,3-dihydro-1H- undesired effects, such as ataxia, sedation, and/or dizziness. benzo[d]imidazol-5-yl)phenyl)acetonitrile (JNJ-56022486). These In clinical use, AMPAR antagonists have very narrow thera- compounds are potent negative modulators of AMPA receptors peutic dosing windows: the doses needed to obtain anticon- containing TARP-g8. They show exquisite selectivity, with no vulsant activity are close to or overlap with doses at which measurable effects upon AMPARs containing other TARPs, or undesired effects are observed (Rogawski, 2011). upon TARP-less receptors. Using chimeric proteins comprising Over the past two decades, investigations into the quater- various segments of TARP-g8and-g4 followed by site-directed nary structure of native AMPA receptors have revealed a mutagenesis, we identified the specific amino acids responsible for remarkably large set of interaction partners. Heterologous this remarkable selectivity. We demonstrate in vivo target expression of individual members of the AMPA subtype of occupancy using ex vivo autoradiography, and provide a pre- ionotropic glutamate receptor (GluA) is sufficient to form liminary investigation of the in vivo pharmacological effects of functional AMPA receptors. However, full recapitulation of TARP-g8–selective AMPA receptor inhibition. the trafficking, localization, gating characteristics, and pharmacology of native AMPA receptors requires coassem- bly with a large and diverse set of accessory proteins Materials and Methods Downloaded from (Jackson and Nicoll, 2011; Schwenk et al., 2012; Straub 3-(2-Chlorophenyl)-2-[2-[6-[(diethylamino)methyl]-2-pyridinyl]ethenyl]- and Tomita, 2012). These auxiliary subunits include cyto- 6-fluoro-4(3H)-quinazolinone hydrochloride (CP-465022; Menniti et al., skeletal and anchoring proteins, other signaling proteins, 2000), 1-(4-Aminophenyl)-3-methylcarbamyl-4-methyl-3,4-dihydro- and several intracellular and transmembrane proteins with 7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI-53655; largely unknown functions. The wide variety of proteins Bleakman et al., 1996), and (S)-N-[7-[(4-Aminobutyl)amino]heptyl]- which can participate in AMPA receptor complexes vastly 4-hydroxy-a-[(1-oxobutyl)amino]benzenepropanamide dihydrochlo- increases the ability of a neuron to tune the response ride (Philanthotoxin-74; Kromann et al., 2002) were purchased from jpet.aspetjournals.org characteristics of its synapses. Here, we demonstrate that Tocris (Bristol, UK). N-[4-[1-(propan-2-ylsulfonylamino)propan- these accessory proteins can be used as novel pharmacological 2-yl]phenyl]benzamide (LY-395153; Linden et al., 2001) was pur- chased from Diverchim (Roissy-en-France, France). Perampanel targets. (Hanada et al., 2011) was purchased from Alsachim (Illkirch- Members of the transmembrane AMPA receptor regula- Graffenstaden, France). Unless otherwise noted, all data analyses, tory protein (TARP) family (CACNG2, 3, 4, 5, 7, and 8) are statistics, and data plots were performed using Origin 2015 or associated with most, if not all, AMPARs in the brain. These OriginPro 2015 (OriginLab, Northampton, MA). Grubbs’ test was proteins were originally discovered and named due to their performed prior to statistical analysis; if identified, a single extreme at ASPET Journals on September 27, 2021 homology to the gamma subunit of voltage-gated calcium outlier was excluded from further analysis. Unless otherwise noted, channels (Letts et al., 1998; Burgess et al., 1999; Klugbauer averages are expressed as the mean 6 S.E.M. Significance levels in et al., 2000). TARPs were subsequently found to associate figures are denoted as follows: *P , 0.05, **P , 0.01, and ***P , with and to modulate the activity of AMPA receptors 0.001. Unless otherwise noted, parameters from linear and nonlinear 6 (Hashimoto et al., 1999; Tomita et al., 2003). Several TARPs least-squares fitting procedures are expressed as the value standard error. have distinct region-specific expression in the brain, leading Animal studies described in this article that were performed in the to physiologic differentiation of the AMPA receptor activity. United States were in accordance with the Guide for the Care and Use It has been theorized that targeting individual TARPs may of Laboratory Animals (National Research Council, 2011). Studies enable selective modulation of specific brain circuits without performed in Europe were in accordance with the European Commu- globally affecting synaptic transmission (Gill and Bredt, nities Council Directive 2010/63/EU (European Union, 2010) and local 2011). The expression pattern of TARP-g8 is particularly legislation on animal experimentation. Facilities were accredited by attractive in this respect. Based upon in situ hybridization the Association for the Assessment and Accreditation of Laboratory studies, TARP-g8 is the predominant TARP throughout the Animal Care. Animals were allowed to acclimate for 7 days after hippocampus, and is expressed within essentially all neu- receipt. They were housed in accordance with institutional standards, rons within the stratum pyramidale and stratum granulo- received food and water ad libitum, and were maintained on a 12-hour light/dark cycle. sum. In addition, it is expressed in a substantial proportion of neurons in the amygdala, olfactory bulb, and olfactory nucleus, and in certain layers within the frontal cortex. In Chemical Synthesis contrast, TARP-g8 shows very little expression within the General Synthetic Methods. All reagents were purchased from hindbrain, midbrain, or thalamus (Tomita et al., 2003; Lein Sigma-Aldrich (St. Louis, MO), Strem Chemicals (Newburyport, MA), et al., 2007; http://mouse.brain-map.org/experiment/show/ or Combi-Blocks (San Diego, CA) and used without further purifica- 72108823). tion, except where noted. Solvents were purchased from EMD Negative modulation of AMPA receptors with a molecule Millipore (Cincinnati, OH) and dried by passing through activated selective for TARP-g8 offers the possibility of selectively alumina columns maintained under argon. All reactions were con- reducing excitatory transmission within brain circuits associ- ducted under a nitrogen atmosphere unless otherwise noted. Flash chromatography was performed on Teledyne Isco CombiFlash sys- ated with neuropsychiatric or neurologic disorders. Such an tems using commercially available RediSep silica gel cartridges agent could be a useful therapeutic in pathologic conditions (Teledyne Isco, Lincoln, NE). Reverse-phase high-performance liquid — characterized by hyperactivity within the hippocampus for chromatography purifications were performed on an Agilent 1100 example, temporal lobe epilepsy. This approach should miti- Series system (Agilent Technologies, Santa Clara, CA) with a Waters gate the side-effect profile attributed to nonselective AMPAR XBridge C18 OBD 5 mM preparative column (Waters Corporation, antagonists (Ko et al., 2015). Milford, MA) unless otherwise noted. NMR spectra were recorded on a 396 Maher et al.

Bruker UltraShield-400, Bruker UltraShield-500, or Bruker UltraShield- were generated using overlapping PCRs, except those indicated 600 spectrometer (Bruker AG, Fallanden, Switzerland) and were otherwise. First, two separate PCR reactions (59 end PCR and 39 end referenced to trimethylsilane. Chemical shifts were recorded in parts PCR) that generated overlapping PCR products were performed. Next, per million relative to trimethylsilane, and indirect dipole-dipole the 59 end and 39 end PCR products were mixed to serve as the coupling constants (J values) are reported in Hertz. Combustion template for the PCR reactions that generated the full-length PCR analysis was performed at Intertek Pharmaceutical Services (White- product for molecular clonings. The primers and templates used for house, NJ). Tritium labeling was conducted at Moravek Biochemicals PCR reactions are listed in Supplemental Table 5. DNA coding and (Brea, CA). The reaction scheme for the synthesis of JNJ-55511118 is predicted amino acid sequences for the chimeric constructs are listed shown in Supplemental Fig. 1. The reaction scheme for the synthesis in Supplemental Table 6. and tritiation of JNJ-56022486 is shown in Supplemental Fig. 2. Generation of Point Mutations. All mutant expression con- structs were generated by overlapping PCR using the human wild- type CACNG8 or CACNG4 cDNA as the template. The primers used Molecular Biology for generation of the mutants are listed in Supplemental Table 7. DNA Molecular Cloning of GluA Receptors and Their Accessory coding and predicted amino acid sequences for the chimeric constructs Proteins from Different Species. cDNAs for human GluA1-FLIP; are listed in Supplemental Table 8. GluA1-FLOP; GluA2-FLOP; GluA2-FLIP; GluA3-FLOP; GluA4- FLOP; and and monkey, dog, mouse, and rat GluA1-FLOP, as well Calcium Flux Assay as their accessory proteins, including human CACNG2, CACNG3, A clonal cell line stably expressing the human GluA1o-g8 fusion

CACNG4, CACNG7, CACNG8, CNIH2, monkey CACNG8, mouse Downloaded from construct under geneticin selection in human embryonic kidney 293 CACNG8, and CACNG8, were polymerase chain reaction (PCR) (HEK-293) cells was established for the primary calcium flux assay. amplified from brain cDNAs from respective species. A point mutation All other combinations of GluA subunits and TARPs were performed was introduced into the GluA2 constructs at the Q/R editing site to using cotransfections of the respective plasmids into HEK-293-F cells. allow calcium permeability in the expressed protein (Burnashev et al., AMPA receptors formed by cotransfections are designated with the 1992). Dog CACNG8 was synthesized with codon optimization based plus symbol (e.g., GluA1i cotransfected with TARP-g8 is referred to as on the published sequence (GenBank accession no. KT749896). The GluA1i1g8). sequences for PCR primers are listed in Supplemental Table 1. The jpet.aspetjournals.org For assays with transiently transfected cells, the cells were PCR products were cloned into mammalian expression vectors as generated by bulk transfection. Prior to transfection, 293-F cells were indicated: pCIneo (Promega, Madison, WI), pcDNA3.1(1) (Life Tech- cultured in FreeStyle-293 Expression Medium (Gibco, Grand Island, nologies, Carlsbad, CA), or pcDNA4/TO (Life Technologies). Cloning NY) at 0.5–2 million cells/ml in shaker flasks at 37°C and 8% CO at sites (highlighted in shaded letters) were introduced into primers to 2 120 rpm. At the time of transfection, cells were diluted to 1 million/ml facilitate the cloning process. The insert regions were sequenced to with FreeStyle-293 medium. Cell viability was above 90% for trans- confirm the sequence identities. FLOP and FLIP splice variants are fections to be considered successful. Transfection was performed by designated with o and i suffixes, respectively (e.g., the FLIP variant of

combining equal amounts of pAdvantage vector (Promega) and target at ASPET Journals on September 27, 2021 GluA1 is designated GluA1i). DNA. Total DNA was 50 mg per 40-ml transfection. The DNA ratio of Generation of GluA1o-CACNG8 Fusion Protein Expression AMPA receptor to TARPs was 4:1. The transfection reagent was Constructs. To ensure a 1:1 stoichiometry of GluA1o and g8inthe FreeStyle MAX (Invitrogen, Carlsbad, CA). Cells were seeded into expressed channel, a fusion of the cDNAs for GRIA1o and CACNG8 384-well polylysine-coated plates at 15,000 cells/well at 16–24 hours was used. Following Shi et al. (2009), we fused the cDNA encoding after transfection, and used for assays 24–48 hours after transfection. the C terminus of GluA1o to the cDNA encoding the N terminus of g8. The calcium flux assays were performed as follows. Cell plates were We inserted a linker sequence encoding QQQQQQQQQQEFAT washed with assay buffer (135 mM NaCl, 4 mM KCl, 3 mM CaCl , between the two full-length cDNAs. The channels expressed with 2 1 mM MgCl , 5 mM glucose, and 10 mM HEPES, pH 7.4, 300 mOsm) this construct appear to have identical properties to channels formed 2 using a Biotek EL405 plate washer (Biotek, Winooski, VT). The cells by coexpression of GRIA1o with an excess of CACNG8 (Shi et al., were then loaded with a calcium-sensitive dye according to the 2009). Human, mouse, and rat GluA1o-CACNG8 fusion protein manufacturers’ instructions (Calcium-5 or Calcium-6; Molecular De- expression constructs were generated by overlapping PCR followed vices, Sunnyvale, CA) combined with the test compounds at a range of by cloning into mammalian expression vectors. The human GluA1o- concentrations. Calcium flux following the addition of 15 mM gluta- CACNG8 fusion protein expression DNA was cloned into pCIneo mate was monitored using a FLIPR Tetra (Molecular Devices). between EcoR1 and Not1 sites, whereas the mouse and rat GluA1o- The fluorescent response in each well was normalized to the CACNG8 expression constructs were cloned into pcDNA4/TO be- response of negative and positive control wells. The negative control tween HindIII and Not1 sites. The primers and templates for the wells had no added compounds, and the positive control wells had been overlapping PCRs are listed in Supplemental Table 2. All clones incubated with 50 mM CP-465022 (a non–subtype-selective AMPAR were sequenced, and the identities were confirmed. DNA coding and antagonist; Lazzaro et al., 2002). The responses (R) to glutamate as predicted amino acid sequences for the fusion constructs are listed in functions of the test compound concentrations (x) were fitted to a four- Supplemental Table 3. parameter logistic function (eq.1): Construction of Chimeric Proteins Using TARPs g8, g4, and À Á g2. The sequences for the human variants of each protein were p R 5 A 1 ðA 2 A Þ= 1 1 ðx=x Þ (1) aligned using the UniProt alignment tool, which also predicted the 2 1 2 0 transmembrane segments of the proteins. The protein sequences were The fitted parameter corresponding to the midpoint (ϰ0) was taken divided into nine regions separated near the borders of the predicted to be the potency of inhibition of the compound (IC50; 50% inhibitory transmembrane sections; these nine regions were the N and C termini concentration). Potency is expressed as Equation 2: (CT), the four transmembrane domains (TM1–TM4), the two extra- 52 ð ½ Þ; ð Þ cellular domains (EX1, EX2), and the intracellular domain. The pIC50 log10 IC50 M 2 predicted topology of the TARP is shown in Fig. 3A, and the splice where pIC50 is the negative log of the 50% inhibitory concentration. points between the TARPs are shown in Supplemental Table 4. The chimeras were designated by a nine-digit number; each digit indicates the TARP used for that section of the protein, starting from the Knockout Animals N terminus. Graphical representations of the chimeric TARPs are The TARP-g8 knockout mouse line Cacng8tm1Ran was originally shown in Supplemental Fig. 3. The chimeric expression constructs described by Rouach et al. (2005). This mouse line, generated by TARP-g8–Selective AMPAR Modulators 397 homologous recombination in embryonic stem cells to replace exons 2 potassium fluoride, 30 mM KCl, 10 mM HEPES, and 5 mM EGTA (pH and 3 with a neomycin resistance gene, was rederived by back-crossing 7.4, 290 mOsm). The extracellular buffer was 135 mM NaCl, 4 mM into a C57BL/6J mouse line at Jackson Laboratory (Bar Harbor, ME). KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 10 mM HEPES (pH 7.4, 300 mOsm). The open-tip resistances of the micropipettes using these solutions were 2–4MV. Recordings were performed in voltage- Patch-Clamp Electrophysiology clamp mode using an Axopatch 200B amplifier and Digidata 1440A Heterologous Cells. Studies with GluA1i were performed with digitizer (Axon Instruments, Sunnyvale, CA). Recordings were con- transiently transfected HEK-293 cells. Human GluA1i with or trolled and measured using pClamp 9.2 software (Axon Instruments). without human TARP-g8 was transfected into HEK-293 cells using Current was measured by holding the interior of the cell at 260mV, Lipofectamine 2000 (Life Technologies) following the manufac- using a 5-kHz low-pass filter. The cells were continuously perfused turer’s instructions. Eight to 24 hours after transfection, cells were through 7-mm square glass barrels using a solenoid-controlled solu- plated onto 12-mm glass coverslips in Dulbecco’s modified Eagle’s tion switching device (PF-77B; Warner Instruments, Hamden, CT). medium with high glucose, without glutamine (Sigma-Aldrich), The peak current in response to a 500-ms exposure to 10 mM supplemented with 10% fetal bovine serum, and incubated at 37°C glutamate every 5 seconds was measured before and after exposure in a humidified 5% CO2 incubator. To increase surface expression, to test compound; 10 mM glutamate was chosen as a saturating cells were transferred to a humidified 5% CO2 incubator at 30°C for concentration for the peak responses (Robert and Howe, 2003). 6–24 hours immediately prior to use. Recordings were performed Steady-state currents were measured during the last 50 ms of the 48–72 hours post-transfection. glutamate application. Upon establishing stable glutamate-evoked

For patch-clamp electrophysiology on heterologously expressed responses, JNJ-55511118 was applied before and during glutamate Downloaded from GluA1o-g8 and human GluA1o-g2, we established single-cell clones application until a steady-state inhibition was observed (typically stably expressing these constructs in Chinese hamster ovary cells (T-Rex- 50–60 seconds). For analysis, the mean peak current of five traces in CHO; Invitrogen) using a tetracycline-inducible expression vector. the presence of test compound was divided by the mean peak current Cells were cultured in Ham’s F-12 supplemented with 10% fetal of five traces prior to the addition of test compound. bovine serum, 100 mg/ml zeocin, and 5 mg/ml blasticidin. To induce For ultra-fast glutamate perfusion, a piezo-driven perfusion system expression, 1 mg/ml tetracycline was added to the culture medium 1–4 was used (Siskiyou, Grants Pass, OR). Recordings on outside-out days prior to use. Cells were plated onto 12-mm plain glass coverslips patches were performed using an AxoPatch 200B amplifier (Axon jpet.aspetjournals.org and incubated at 37°C in a humidified 5% CO2 incubator. To increase Instruments), and signals were filtered at 10 kHz and digitized at surface expression, cells were transferred to a humidified 5% CO2 50 kHz. Data acquisition and online analysis were performed using incubator at 30°C for 6–24 hours immediately prior to use. pClamp 9 (Axon Instruments). Current decay kinetics were fitted with Hippocampal Neurons. Acute hippocampal neurons were a double exponential function using Origin (OriginLab, Northampton, obtained from 8- to 12-week old C57BL/6J male mice, following the MA) and expressed as a weighted decay time constant. For recovery protocol described by Brewer (1997) with the following modifications. from desensitization, an initial desensitizing pulse of glutamate was Medium was prepared by supplementing HibernateA with 2% B27 followed by a second pulse of glutamate at varying time intervals. The and 0.5 mM Glutamax (HABG medium; all reagents from Life recovery from desensitization was expressed as the current peak at ASPET Journals on September 27, 2021

Technologies). Mice were asphyxiated with CO2 and then decapitated amplitude fraction of the second pulse to the first pulse at a given time in accordance with National Institutes of Health (NIH) animal and use interval, and was fitted using a single exponential function (for TARP- guidelines. The brain was rapidly removed, then placed into ice-cold g8–containing AMPA receptors) or a double exponential function (for HABG medium. Sagittal slices, 300 mm thick, were obtained using a TARP-less AMPA receptors) using Origin (OriginLab). VT1200S microtome (Leica Biosystems, Buffalo Grove, IL). Slices were cut in ice-cold solution composed of 150 mM sucrose, 50 mM Brain Slice Whole-Cell Patch Clamp Electrophysiology NaCl, 25 mM NaHCO3, 10 mM glucose, 7 mM MgSO4, 2.5 mM KCl, 1.25 mM Na3PO4, and 0.5 mM CaCl2 equilibrated with 95% O2 and Male mice (2–3 weeks) were anesthetized with isoflurane and then 5% CO2. The hippocampus was isolated from the rest of the slices and decapitated in accordance with NIH animal care and use guidelines. transferred to a calcium-free HibernateA Minus Calcium solution Transverse hippocampal slices (300 mm thick) were cut in ice-cold high (BrainBits, Springfield, IL) containing 20 mg of papain (Worthington sucrose buffer containing 87 mM NaCl, 2.5 mM KCl, 0.5 mM CaCl2, Biochemical, Lakewood, NJ) and 0.5 mM Glutamax (Life Technolo- 7 mM MgSO4, 1.25 mM NaH2PO4, 25 mM NaHCO3, 25 mM glucose, gies) and digested at 30°C under gentle shaking for 30 minutes. Then, and 75 mM sucrose equilibrated with 95% O2 and 5% CO2. Slices were the papain solution was aspirated and replaced with HABG. Slices then placed in artificial cerebrospinal fluid (ACSF) at 35°C for were gently triturated with fire-polished Pasteur pipettes. The su- 30 minutes, and then allowed to recover for at least 1 hour in ACSF pernatant containing dissociated neurons was collected, and then at room temperature. ACSF for electrophysiological recordings con- centrifuged for 2 minutes at 200g. The cell pellet was collected and tained 119 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1.3 mM MgSO4, then resuspended in HABG. The cell suspension was then plated over 1 mM NaH2PO4, 26.2 mM NaHCO3, and 11 mM glucose equilibrated coverslips, and isolated neurons were picked under visual inspection with 95% O2 and 5% CO2. The intracellular recording solution for whole-cell patch-clamp recordings. The extracellular and intracel- contained 130 mM CsMeSO4, 10 mM HEPES, 8 mM KCl, 4 mM lular solutions were the same as described earlier for transfected MgATP, 0.4 mM NaGTP, 10 mM sodium creatine, and 10 mM 1,2- HEK-293 cells. bis(o-aminophenoxy)ethane-N,N,N9,N9-tetraacetic acid. Cerebellar Granule Cells. Cerebellar granule cell cultures were Excitatory postsynaptic currents (EPSCs) intracellularly recorded prepared following Brewer (1997) with the following modifications. from a neuron in the CA1 pyramidal cell layer were evoked by Cerebella were harvested from newborn Sprague-Dawley rat pups electrical stimulation of the Schaffer collateral/commissural pathway (1–4 days old, males and females were mixed). Tissue was minced using a monopolar glass stimulating electrode (filled with ACSF) manually prior to trypsin digestion. Dissociated cells were plated onto placed in the stratum radiatum of CA1 (0.1-Hz stimulation frequency). glass coverslips coated with poly-D-lysine and fibronectin, and then In cases where trains of five stimulations at 50 Hz were done, the pulse cultured for 2–4 weeks prior to use. train was alternated with one stimulation (0.1 or 0.05 Hz between Electrophysiology. Whole-cell and outside-out patch electrophys- trains). Test compounds were bath-applied. To evoke AMPA EPSCs, iology (Hamill et al., 1981) was performed using 1.5-mm-diameter glass neurons were held at 270 mV. N-methyl-D-aspartate (NMDA) EPSCs capillary tubes (TW150-4; World Precision Instruments, Sarasota, FL) were recorded at 140 mV 50 ms after the AMPA EPSC at 270 mV. pulled to a fine tip with a Sutter P-97 micropipette puller (Sutter NMDA EPSCs were recorded prior to and after establishing the Instruments, Novato, CA). The intracellular buffer was 90 mM steady-state inhibition of peak AMPA EPSCs. Recordings were 398 Maher et al. performed using a Multiclamp 700B patch-clamp amplifier (Axon ligand depletion was ,15% at each concentration. Specific binding Instruments); signals were filtered at 4 kHz, digitized at 10 kHz, and (SB) at each radioligand concentration was calculated as SB 5 displayed and analyzed online using pClamp 9.2 (Axon Instruments). TB – NSB at each radioligand concentration. NSB was fitted to a In some experiments, trains of 50-Hz stimulation were used to linear function using linear least-squares analysis. SB was con- determine the paired-pulse ratio. In this case, the EPSC magnitude for verted to pmol/mg protein, and then fitted to a single-site binding each pulse was measured from the baseline current immediately after model to determine the dissociation coefficient (KD) and total the pulse to the peak current. The paired-pulse ratio was calculated as receptor concentration (Bmax): this EPSC magnitude, divided by the immediately preceding EPSC B ½hot magnitude. SB 5 max ½hot 1 ½cold 1 KD

Brain Slice Field Excitatory Postsynaptic Potentials For competition binding experiments, a serial dilution of the test Electrophysiology compound was prepared in assay buffer and combined with a final concentration of 20 nM radioligand. NSB was determined using eight Male mice (7–9 weeks) were anesthetized with isoflurane and then wells containing a blocking concentration of 50 mM JNJ-55511118 decapitated in accordance with NIH animal care and use guidelines. along with radioligand and tissue membranes, and total binding was Horizontal hippocampal slices (300 mm thick) were cut in ice-cold high- determined using eight wells containing only radioligand and tissue sucrose ACSF (composition described earlier). Slices were then placed membranes. Four replicates for each test compound concentration in ACSF at 35°C for 30 minutes, and then allowed to recover for at were used. After incubation and washing as described earlier, the least 1 hour in ACSF at room temperature. Slices were then placed Downloaded from radioactive counts (SB) in each well were normalized to the total and on a perforated multielectrode array chip (Multichannel Systems, nonspecific binding counts and then fitted to a single-site logistic Reutlingen, Germany) and perfused with ACSF heated to 35°C. function. The equilibrium dissociation constant (K ) was calculated Excitatory postsynaptic potentials were evoked with electrodes i from the midpoint parameter of the fit, adjusted for radioligand placed in the CA1 radiatum once every minute, sampling at 50 kHz. concentration using the Cheng-Prusoff correction (Cheng and Prusoff, Multielectrode array responses across multiple electrodes were chosen 1973). based on stability of the response and amplitude (.0.4 mV), which were then averaged to generate an N 5 1 for each slice. Test compounds were jpet.aspetjournals.org bath-applied. Plasma Protein Binding Plasma protein binding was determined by equilibrium dialysis Radioligand Binding using the Rapid Equilibrium Dialysis (RED) device (Thermo Fisher Scientific), consisting of a Teflon base plate, and RED Device inserts – Male Sprague-Dawley rats (6 14 weeks) were anesthetized with comprising two (sample and buffer) side-by-side chambers separated isoflurane and then decapitated in accordance with NIH animal care by a dialysis membrane (molecular weight cut-off 8000). Compounds and use guidelines. The brain was removed, and hippocampi were were prepared as 100 mM dimethylsulfoxide (DMSO) stocks and 2 at ASPET Journals on September 27, 2021 rapidly dissected and then frozen at 80°C until use. For each assay, spiked into 1 ml of mouse, rat, and human plasma (BioreclamationIVT, two hippocampi per five 96-well plates were used. On the day of the Westbury, NY) to make a final concentration of 1 mM. Plasma (300 ml) experiment, the hippocampal tissue was thawed, and then homoge- was dispensed into the sample well, and dialysis buffer (100 mM nized in assay buffer (50 mM Tris, pH 7.4) for 30 seconds at high speed. potassium phosphate, pH 7.4, 500 ml) was dispensed into the buffer The homogenate was centrifuged at 1500 rpm for 5 minutes followed well. Each compound was tested in triplicate. The RED device was by careful decanting of the supernatant, which was centrifuged at sealed, and equilibrium was permitted for 6 hours in a 37°C incubator 39,000g for 30 minutes. Ice-cold assay buffer was added to the cell with gentle agitation at 100 rpm. After incubation, plasma samples were pellet. The protein concentration within the pellet was determined by prepared by transferring 10 ml from plasma wells to 90 mloffreshdialysis colorimetry using a Pierce bicinchoninic acid protein assay kit buffer, and buffer samples were prepared by transferring 90 mlfrom (Thermo Fisher Scientific, Rockford, IL), then diluted with assay buffer wells to 10 ml of naïve plasma. In addition, a reference sample – buffer to obtain a concentration of 200 400 mg protein per milliliter without equilibration was prepared in triplicate by mixing 10 mlof – (10 20 mg protein per well). plasma containing 1 mM compound with 90 ml of buffer to determine Binding assays were performed in Whatman GF/B 96-well filter compound recovery from the assay. Two volumes of 1:1 acetonitrile: plates (GE Healthcare, Little Chalfont, United Kingdom) presoaked methanol spiked with the internal standard phenytoin (0.2 mg/ml) was with 0.3% polyethylenimine. When manufactured, the stock solution addedtothereferenceandsamples.Precipitation of plasma protein 3 of tracer was 34.5 mM single-labeled [ H]JNJ-56022486 and 10.3 mM binding was allowed for 15 minutes before the reference and samples unlabeled JNJ-56022486 in ethanol. The actual stock concentration were centrifuge clarified. Supernatant (10 ml) was used for liquid was calculated at the time of use based upon the decay rate of tritium. chromatography–tandem mass spectrometry (LC-MS/MS) analyses. Ten microliters of 10 test compound, 40 ml of 2.5 tracer, and 50 mlof membrane homogenate were placed into each well. The reaction was incubated for 2 hours at 4°C on a shaker, then terminated by filtration Brain Tissue Binding followed by washing with ice-cold assay buffer four times. After drying Brain tissue binding was assessed by an equilibrium dialysis for 30 minutes at 50°C, 60 ml/well MicroScint-O (PerkinElmer, technique similar to the procedure described for plasma protein Waltham, MA) was added to the plate. Radioactivity retained on the binding. Rat brain tissue homogenate prepared in phosphate- filters was measured using a TopCount liquid scintillation counter buffered saline buffer [pH 7.4, 1:10 (w/v)] was spiked with compound (PerkinElmer, Waltham, MA). The scintillation counter was cali- DMSO stock solution to yield a final concentration of 5 mM. The brated with a linear least-squares fit to the radioactivity counts from dialysis was carried out in a shaking incubator at 37°C for 5 hours. known quantities of [3H]JNJ-56022486. All analyses were performed After incubation, 25 ml of homogenate or 50 ml of buffer was extracted in Origin 2015 (OriginLab). with 50 ml of DMSO and 300 ml of acetonitrile and analyzed by For saturation binding experiments, a 2 serial dilution of [3H] LC-MS/MS using the calibration curves across an appropriate JNJ-56022486 in quadruplicate wells was used for total binding (TB), concentration range and quality control samples. All determinations with 50 mM JNJ-55511118 in quadruplicate wells for determination of were conducted in triplicate. The apparent unbound fraction (f u,app) nonspecific binding (NSB). Ligand depletion was determined by was determined as the ratio of the concentration measured in the comparing the radioactivity counts of total binding to the counts in a homogenate to the concentration measured in the buffer. The separate plate spiked with an equivalent amount of radioligand; unbound fraction in undiluted brain was calculated as TARP-g8–Selective AMPAR Modulators 399

fu;app intravenous administration of compound. Animals were singly housed, fu;brain 5 ; D 1 fu;app 2 Dfu;app given food and water ad libitum, and maintained on a 12-hour light and dark cycle. where D is a dilution factor of 10. Subsequently, the percentage of Evoked population spikes from the CA1 region of the hippocampus compound bound to brain tissue (%BTB) was determined as were recorded following established procedures (Jeggo et al., 2014) À Á with the following modifications. Animals were anesthetized with %BTB 5 1 2 f ; 100%: u brain isoflurane for the duration of the surgical preparation and recording periods while body temperature was maintained with a homeothermic heating pad. A small piece of skull overlaying the hippocampus was Pharmacokinetic Studies removed using a hand-held drill before a concentric bipolar stimulat- Single-dose pharmacokinetic studies of JNJ-55511118 in male ing electrode (FHC, Bowdoin, ME) and tungsten recording microelec- Sprague-Dawley rats were conducted by BioDuro, LLC (Beijing, trode (World Precision Instruments) were inserted into CA1 using the China) following i.v. (1 mg/kg) and per os (p.o.; 5 mg/kg) administra- following stereotaxic coordinates (from the bregma): tion as a solution in 20% hydroxypropyl-b-cyclodextrin with three Stimulating electrode: anterior-posterior 5 3.4 mm, medial- equivalents of sodium hydroxide. Blood was sampled at predose and at lateral 5 2.75 mm 0.033 (i.v.), 0.083 (i.v.), 0.25, 0.5, 1, 2, 4, 8, and 24 hours postdose. Recording electrode: anterior-posterior 5 4.4 mm, medial- Plasma concentrations were quantitated by LC-MS/MS. Pharmacoki- lateral 5 2.25 mm. netic parameters were derived from noncompartmental analysis of the –

plasma concentration versus time data using WinNonlin software Electrodes were typically inserted to a depth of 2 2.5 mm below the Downloaded from (Pharsight, Palo Alto, CA). pial surface before test stimuli were used to help optimize the evoked Single-dose pharmacokinetic studies of JNJ-55511118 in male C57/BL6 signal and determine the final recording depth. Stimulation intensi- mice were conducted by BioDuro, LLC following p.o. (10 mg/kg) admin- ties evoking a 30–60% maximal response were used. Signals from the istration as a suspension in 0.5% hydroxypropyl methylcellulose recording electrode were amplified and filtered (1 Hz to 10 kHz, (HPMC). Blood was sampled at predose and at 0.5, 1, 2, 4, 8, and DAM80 bio-amplifier; World Precision Instruments), then digitized 24 hours postdose. and collected (40-kHz sampling) using a PowerLab 16/35 data

Blood-Brain Barrier. Adult male animals were dosed by oral acquisition unit controlled by LabChart Pro software (ADInstru- jpet.aspetjournals.org administration of a suspension in HPMC. The animals were eutha- ments, Colorado Springs, CO). Brief stimulus pulses were continu- nized using carbon dioxide and decapitated at specified time points ously delivered to the hippocampus at a rate of 0.33 Hz, and the evoked after drug administration. Brains were rapidly frozen on powdered neural responses were recorded. A stable baseline period of 10 minutes dry ice and stored at 280°C before sectioning for receptor occupancy was obtained before administration of the compound, and evoked studies or for compound concentration determination by LC-MS/MS. responses were obtained for an additional 60 minutes thereafter. JNJ- The blood-brain barrier ratio was calculated as the compound 55511118 was formulated in 5% N-methyl-2-pyrrolidone plus 20% concentration in the brain divided by the concentration in the plasma Cremophor (BASF, Ludwigshafen, Germany) plus 75% water and for each animal. dosed intravenously. At the end of the recording session, the brain was at ASPET Journals on September 27, 2021 LC-MS/MS. JNJ-55511118 was quantified on an API4000 MS/MS electrically lesioned to determine the final positions of the stimulating System (Applied Biosystems, Concord, Ontario, Canada) interfaced and recording electrodes. Additionally, the brain was removed and with an Agilent 1100 Series high-performance liquid chromatogra- plasma samples collected to determine in vivo concentrations of pher. Samples were loaded onto a 2.1 30-mm ACE 5mm C4 100A compound via LC-MS/MS. The population spike amplitude for each column (Advanced Chromatography Technologies Ltd., Aberdeen, stimulus was extracted from the recording following the procedure Scotland) under a flow rate of 0.9 ml/min using 5 mM ammonium outlined by Jeggo et al. (2014), normalized to the mean baseline acetate (0.1% formic acid) as mobile phase A and acetonitrile (0.1% amplitude, then averaged for each dose group according to time formic acid) as mobile phase B. Starting with 87% mobile phase A for relative to the injection of drug. 0.4 minute, mobile phase B was increased from 13 to 90% using a linear gradient for 0.8 minute, held at 90% B for 0.3 minute, and equilibrated at 13% B for 1.0 minute for an overall run time of 2.5 minutes. JNJ- Electroencephalogram Recording and Locomotor Activity 55511118 was quantified by MS/MS in the positive ion mode by Studies in Rats monitoring the transition of 328.95 to 208.10 m/z. Experiments were conducted in male Sprague-Dawley rats (350–450 g; Harlan Laboratories, Livermore, CA). Animals were Ex Vivo Receptor Occupancy chronically implanted with telemetric devices (PhysioTel F40-ETT; Data Sciences International, St. Paul, MN) for the recording of Receptor occupancy was assessed by ex vivo autoradiography using electroencephalogram (EEG) with two epidural electrodes placed in the TARP-g8 receptor antagonist [3H]JNJ-56022486. Coronal and the frontal and parietal cortex, electromyogram (EMG), and locomotor sagittal tissue sections of 20-mm thickness were prepared for autora- activity as described previously (Dugovic et al., 2009). EEG and EMG diography as previously described (Langlois et al., 2001). Tissue signals were digitized at a sampling rate of 100 Hz. High- and low-pass sections were incubated for 10 minutes in 50 mM Tris HCl containing filters were set at 1 and 30 Hz for the EEG signal. Polysomnographic 0.1% bovine serum albumin (pH 7.4) with 5nM [3H]JNJ-56022486 at wave forms were analyzed per 10-second epoch and classified as wake, room temperature. Nonspecific binding was characterized with a non-rapid eye movement (NREM), or rapid eye movement (REM) structurally distinct TARP-g8 receptor antagonist. Sections were sleep using the computer software program SleepSign (Kissei Comtec, rinsed in 50 mM Tris HCl containing 0.1% bovine serum albumin on Nagano, Japan). EEG activity within specific vigilance states was ice four times for 10 minutes per rinse, followed by two dips in ice-cold determined by power spectral analysis (fast Fourier transform) within deionized water, then dried under a stream of cold air. Digitized a frequency range of 1–30 Hz. Values for power spectra were divided images were acquired with b-Imager DFine or TRacer (Biospacelab, into four frequency bands: delta (1–4Hz),theta(4–10 Hz), alpha/sigma Paris, France). (10–15 Hz), and beta (15–30 Hz). Locomotor activity counts were analyzed into 1-minute bins and averaged into 5-minute intervals for In Vivo Electrophysiology each animal. All results were averaged and expressed as the mean 6 Male Sprague-Dawley rats (Charles River Laboratories, San Diego, S.E.M. in defined time intervals for each animal. To determine CA) weighing approximately 300–450 g were used for these experi- whether differences were significant at a given interval, either a ments. The jugular vein was precannulated by the vendor to facilitate one-way analysis of variance (ANOVA) or two-way repeated-measures 400 Maher et al.

ANOVA followed by Dunnett’s multiple comparison test was Rotarod performed. Immediately prior to seizure testing, all mice were subjected to a rotarod test to assess motor coordination. Animals were placed on a Anticonvulsant Studies 1-inch knurled rod that rotates at a speed of 6 rpm. The animal was considered motor-impaired if it fell off this rotating rod three times Anticonvulsant studies were performed by NeuroAdjuvants, Inc. during a 1-minute period. (Salt Lake City, UT). Unless otherwise noted, male albino CF1 mice (Charles River Laboratories, Portage, MI) were used as experimental Morris Water Maze animals. All animals were allowed free access to both food and water except when they were removed from their cages for the experimental The procedure followed the water maze task described by Atcha procedure. All mice were housed, fed, and handled in a manner et al. (2009), with the following modifications. Video tracking software consistent with the recommendations of the National Research (EthoVision XT 9.0; Noldus, Wageningen, The Netherlands) was used Council (2011). No insecticides capable of altering hepatic drug- to measure the path, time taken, and swim speed for each animal to metabolizing enzymes were used in the animal facilities. All animals reach the platform. Male Long-Evans rats (Janvier, Le Genest-Saint- were euthanized in accordance with the Institute of Laboratory Isle, France; N 5 12 per dose group) were trained for 4 days in three Resources policies on the humane care of laboratory animals. All test daily trials with random starting positions to find the hidden platform substances were administered orally in 0.5% methylcellulose in a (days 1–4). The location of the hidden platform was maintained volume of 10 ml/kg body weight. For the maximal electroshock throughout the study. When an animal failed to find the platform

(MES), 6 Hz, and corneal kindling assays, a drop of anesthetic/ within 60 seconds, it was guided to the platform and allowed to stay Downloaded from electrolyte solution (0.5% tetracaine hydrochloride in 0.9% saline) there for another 5–10 seconds. Directly after the last acquisition trial was applied to the eyes of each animal prior to placement of the on day 4, the animals were subjected to a probe trial for 60 seconds, corneal electrodes. during which the platform was removed. Mouse 6 Hz Psychomotor Seizure and MES Tests. Details of Statistical analyses were performed for the averages per day and the procedures have been described previously (Barton et al., 2001; per trial. For analysis of “latency to platform,” a Cox proportional Rowley and White, 2010). In brief, an acute seizure was induced via hazards model was used. For the other parameters, a repeated- electrical stimulation through electrodes applied to the corneas of test measures ANOVA model was used. For probe trials and percent- jpet.aspetjournals.org animals. For the 6 Hz tests, the stimulus was 32 or 44 mA at 6 Hz for 3 per-quadrant measures in acquisition, one-way ANOVA statistics were seconds, and the induced seizure was characterized by jaw chomping, run using InVivoStat software (Clark et al., 2012; http://invivostat.co. vibrissae twitching, forelimb clonus, and Straub tail. For the MES uk/), with dose used as the treatment factor. test, the stimulus was 50 mA at 50 Hz for 0.2 second, and the induced seizures were characterized by a tonic hindlimb extension. Cohorts of V-maze eight mice for each test concentration and stimulation intensity were Male Long-Evans rats (body weight 250–300 g; Janvier Laborato- treated with a single oral dose 2 hours prior to challenge with the ries) were individually housed for 7 days before testing and habituated electrical stimulation. Mice not displaying the described seizure at ASPET Journals on September 27, 2021 to the experimental procedures. The apparatus consisted of two phenotypes were considered protected. enclosed arms with walls of different visual contexts positioned at a Mouse Corneal Kindling. The corneal kindling assay followed 90° angle to each other, and connected to a center zone (Embrechts and established procedures (Matagne and Klitgaard, 1998; Rowley and Ver Donck, 2014). A top-mounted video camera recorded the move- White, 2010). Kindling was achieved by twice-daily corneal stimu- ments of the animals, and images were analyzed for distance made lation (3 mA, 3 seconds, 60 Hz) until all mice reached an established and time spent in each arm using EthoVision XT 9.0 (Noldus). criteria of five consecutive secondarily generalized seizures (Racine Two hours after dosing with vehicle or test compound, animals were stage 5). For compound testing, a cohort of eight fully kindled mice subjected to a habituation trial (T1), during which one arm was closed were administered a single oral dose of the test compound 2 hours and the other arm was free to be explored for 5 minutes [defined as the prior to challenge with the kindling stimulus. The Racine seizure score familiar arm (FAM)]. Then during the retention trial (T2), the novel (0–5; Racine, 1972) was recorded for each mouse and averaged. arm was opened (NEW arm), and the animal explored both arms of Animals with seizure scores of 3 or lower were considered protected. the maze for 5 minutes. The discrimination index indicating prefer- Timed Intravenous Infusion of Metrazol Test. A single dose of ence for the novel arm was calculated from the time spent in each each test compound or vehicle was administered p.o. to cohorts of arm of the maze during the retention trial: discrimination index 5 10mice2hourspriortothetest.Micewerechallengedwith0.5% (NEW 2 FAM)/(NEW 1 FAM). heparinized Metrazol solution [5 mg/ml; pentylenetetrazol (PTZ), Sigma- Statistical analyses were performed using “InVivoStat” software Aldrich, St. Louis, MO], infused at a constant rate of 0.34 ml/min into a (Clark et al., 2012; http://invivostat.co.uk/). The discrimination index lateral tail vein of an unrestrained mouse (Orloff et al., 1949; White was analyzed using single measures parametric analysis, and the et al., 1997). The time in seconds from the start of the infusion to the overall effects were determined using ANOVA followed by all-to-one appearance of the “first twitch,” and then to the onset of sustained comparisons without adjustment for multiplicity (Fisher’s least- clonus, was recorded. The times to each endpoint were converted to significant differences tests). mg/kg of PTZ for each mouse, taking into account the rate of infusion, concentration of PTZ, and weight of the animal. Amygdala Kindling. Male Sprague-Dawley rats (Charles River Delayed Nonmatch to Position Laboratories) were surgically implanted with stimulation/recording Standard operant chambers (Med Associates, St. Albans, VT) were electrodes unilaterally into the amygdala according to the procedure used. One wall was equipped with two retractable response levers and described by McNamara (1995). Rats received daily subthreshold stimulus lights. The opposite wall contained the reward magazine, stimulation followed by behavioral and electrographic monitoring for equipped with a reward light and an infrared sensor. In addition, each a period of 2–3 weeks, during which time a majority of rats were box was equipped with a small “house” light, a small speaker, and a considered fully kindled (five generalized seizures, Racine scale 4–5, metal grid floor. Forty-five-milligram dustless precision pellets (stan- over a period of 8 days). Fully kindled rats received either vehicle dard chow; Bio-Serv, Flemington, NJ) were used as reward. (0.5% HPMC) or JNJ-55511118 (suspension in 0.5% HPMC, oral Animals (male Lister-Hooded rats, aged approximately 18 months gavage, 10 ml/kg). They were then challenged 2 hours later with the and weighing approximately 350–450 g at the time of testing; Harlan same kindling stimulation, and their behavioral seizure score and Laboratories, Horst, The Netherlands) received treatment within electrographic after-discharge duration were recorded. a counter-balanced design (0, 1, 3, and 10 mg/kg JNJ-55511118, TARP-g8–Selective AMPAR Modulators 401 suspended in 0.5% HPMC, 10 ml/kg p.o., 120 minutes prior to testing). To explore the molecular basis of the selectivity of these During testing, the rats experienced 15 trials at four different delays compounds, we constructed chimeric proteins of TARP-g8 and (1, 10, 20, and 30 seconds) per session. A session ended once animals -g4. Functional interaction with the AMPA receptor was completed all 60 trials or after 45 minutes. A trial started with the established by determining the potency of inhibition of GluA1o illumination of the magazine, followed by a nose poke at the magazine, coexpressed with the chimeric TARPs. Figure 3A shows the turning off the receptacle light, and starting the sample phase. One of topology and nomenclature of the constructs used in these two levers was extended in a pseudorandom fashion, and the rat had 20 seconds to respond. This caused the lever to be retracted and experiments. Figure 3C shows the change in potency of started the delay phase (variable 1–30 seconds). Once the delay had inhibition for each compound as compared with the potency passed, the rat had 10 seconds to respond at the receptacle, causing at GluA1o-g8. In the first group of chimeras, we interchanged the magazine light to go off and the two levers to be extended (choice EX1 and the CT of TARP-g8 and -g4. Neither exchange altered phase). A response at the lever opposite the sample lever within a the potency or efficacy of the compounds: chimeras 448444444 limited hold of 10 seconds was counted as a correct response, and led to and 444444448 (g4 with only EX1 or CT replaced) were the retraction of the levers, magazine illumination, a short tone, and insensitive, whereas 884888888 and 888888884 (g8 with only delivery of a food pellet. Pellet collection turned the magazine light off EX1 or CT replaced) were potently inhibited. and started a 5-second intertrial interval. A response to the same lever In the next set of chimeras (888888884–844444444), we that was presented during the sample phase was counted as an progressively replaced sections of g8 with the corresponding incorrect response and resulted in the retraction of the response “ ” regions of g4, starting from the C terminus. JNJ-55511118

levers, a short time out (10 seconds) in darkness. A lack of response Downloaded from during the sample or choice phases is counted as an omitted trial. and JNJ-56022486 both lost their ability to inhibit the Percentage correct served as a measure of working memory. The response beginning with chimera 888888844, implicating relative number of omitted trials and various response latencies TM4 in the functional activity of the compounds. Indeed, served as measures of responsivity. Delayed nonmatch to position chimera 888888848 was completely insensitive to inhibition. (DNMTP) percentage correct data were fitted to repeated-measures The inverse chimera 444444484 was sensitive to these com- logistic regression mixed-effect models, latencies were analyzed pounds, with a reduced potency. using mixed-effect models, and percentage of errors of omission In chimeras 488888888–444444488, we progressively jpet.aspetjournals.org was determined by a logistic regression model. Main treatment replaced domains of TARP-g8, starting from the N terminus. effects were then analyzed by ANOVA followed by post-hoc The potency of JNJ-55511118 and JNJ-56022486 was un- contrasts. changed in chimeras 488888888–444448888. However, when TM3 was replaced (444444888), the compounds lost approx- Results imately 10-fold in potency. This suggests that TM3 is also involved in the functional activity of the compounds, although

JNJ-55511118 and JNJ-56022486 were discovered through somewhat lower in magnitude than TM4. at ASPET Journals on September 27, 2021 directed medicinal chemistry following a high-throughput We aligned the sequences for the human TARPs to identify screening campaign targeting AMPA receptors containing candidate residues that could account for the pharmacology of TARP-g8. The structures of these molecules are shown in these compounds (Fig. 3B). TM4 contains four amino acids Fig. 1. unique to TARP-g8, and TM3 contains three. We generated Calcium Flux. JNJ-55511118 and JNJ-56022486, along point mutations of g8, in each case mutating individual with the nonselective AMPAR inhibitors CP-465022 and residues to the corresponding one from g4. Figure 3D shows GYKI-53655, were evaluated for their ability to inhibit the change in potency of inhibition for GluA1o coexpressed glutamate-evoked calcium flux in HEK-293 cells heterologously with each of these constructs. Two of these point mutations expressing GluA subunits with and without TARPs. Inhibi- showed altered potency of the TARP-selective compounds: tion as a function of concentration for these compounds in G210A and V177I. G210A completely abolished activity of the assays using various combinations of human GluA and TARP compounds, whereas V177I caused a 10-fold loss of potency. subunits is shown in Fig. 2. The fitted values for the potency of Double mutations of g4 and g2 in the corresponding locations inhibition at each target, averaged over multiple experiments, to the g8 residues conferred sensitivity of those TARPs to are summarized in Table 1. JNJ-55511118 and JNJ-56022486 inhibition by JNJ-55511118 and JNJ-56022486. potently inhibited every tested GluAx subunit, provided To identify additional residues which may be involved with TARP-g8 was present. Both compounds showed little inhibi- the functional activity, we scanned TARP-g8 in the vicinity tory activity up to the highest concentrations tested at TARP- G210 and V177. We made single-point alanine mutations from less AMPARs and at AMPARs coexpressed with other TARPs N173 through G176 in TM3, and G209 through I214 in TM4. or with cornichon family AMPA receptor auxiliary protein Most of these point mutations, when coexpressed with 2 (CNIH2). In contrast, the noncompetitive inhibitors CP- GluA1o, did not alter the potency of the g8-selective inhibitors. 465022 and GYKI-53655 showed no selectivity among AMPA N173A caused a complete loss of inhibitory efficacy, whereas receptor subtypes, or among the TARP-containing AMPARs. the compounds were 10- to 100-fold less potent with G209A and F213A. Selectivity. The selectivity of JNJ-55511118 and JNJ- 56022486 was evaluated at 1 mM against a panel of 52 receptors, ion channels, and transporters using radioligand displacement assays (Cerep S.A., Poitiers, France). Data are summarized in Supplemental Table 9. The compounds showed less than 50% binding at all tested targets, except for activity of Fig. 1. Chemical structures of JNJ-55511118, JNJ-56022486, and [3H] JNJ-55511118 at the serotonin receptor 2B (78% effect) JNJ-56022486. and JNJ-56022486 at the melatonin receptor (57% effect). 402 Maher et al. Downloaded from jpet.aspetjournals.org

Fig. 2. Inhibition of glutamate-evoked calcium flux as functions of test compound concentration. The GluA subunit and the TARP are designated in the captions. A dash between the GluA and the TARP indicates that a fusion construct was used. A plus indicates that the GluA and TARP plasmids were cotransfected. Data points are the means of 2–42 data points from 1–22 individual experiments. (A–D) Concentration-response curves for the human constructs. (E and F) Concentration-response curves for nonhuman constructs. In each case, the GluA and the TARP subunits were both from the designated mammal. at ASPET Journals on September 27, 2021

JNJ-55511118 was further evaluated in a cell-based functional (CHO) cells expressing GluA1o-g8, a saturating concentration assay using the recombinant human serotonin receptor 2B, of JNJ-55511118 produced a partial inhibition. Peak currents and was determined to be an antagonist at this receptor, with with 1 mM JNJ-55511118 were reduced to 57.2 6 2.0% (mean 6 an IC50 of 6 mM (data not shown). Thus, both compounds were S.E.M.; N 5 7) relative to currents in the same patches prior to a minimum of 100-fold selective against all tested targets. addition of the compound. In contrast, peak currents in Electrophysiology. The AMPAR modulation of JNJ- patches of CHO cells expressing GluA1o-g2 were virtually 55511118 was explored in further detail using electrophysio- unaffected by the presence of 1 mM JNJ-55511118 (97.6 6 logical recordings. Figure 4A shows representative traces of 2.0%, N 5 8). the glutamate-evoked responses of cells expressing AMPA We used cultured cerebellar granule cells from neonatal mouse receptors. In outside-out patches of Chinese hamster ovary as our model system for native AMPA receptors expressing

TABLE 1 Potency for inhibition of glutamate-evoked responses in calcium flux assays

Data are represented as the mean pIC50 6 standard deviation of multiple measurements. The numbers in parentheses indicate the number of measurements performed. Unless otherwise stated, all constructs are human.

GYKI-53655 CP-465022 JNJ-55511118 JNJ-56022486 GluA1o-g8 5.65 6 0.46 (14) 6.57 6 0.24 (20) 8.33 6 0.28 (22) 8.02 6 0.35 (10) GluA1o+ g8 5.72 6 0.42 (2) 6.26 6 0.25 (7) 7.95 6 0.21 (7) 7.79 6 0.22 (6) rat(GluA1o- g8) 5.00 6 0.41 (2) 6.18 6 0.37 (7) 7.89 6 0.3 (7) 7.45 6 0.41 (5) mouse(GluA1o- g8) 5.19 6 0.24 (2) 6.03 6 0.23 (8) 7.87 6 0.31 (7) 7.53 6 0.44 (5) monkey(GluA1o+ g8) 5.81 6 0.66 (2) 6.28 6 0.25 (4) 7.88 6 0.15 (7) 7.65 6 0.21 (6) dog(GluA1o+g8) 5.63 6 0.38 (2) 6.16 6 0.16 (4) 7.82 6 0.28 (7) 7.53 6 0.27 (6) GluA1i+g8 5.39 6 0.46 (2) 6.02 6 0.17 (8) 7.91 6 0.39 (7) 7.58 6 0.33 (5) GluA2i+g8 5.25 6–(1) 6.06 6 0.22 (6) 8.13 6 0.35 (2) 7.5 6 0.11 (2) GluA3o+g8 5.24 6 0.09 (2) 6.15 6 0.3 (9) 7.42 6 0.38 (6) 7.03 6 0.1 (4) GluA4o+g8 5.8 6 0 (2) 6.28 6 0.46 (2) 7.8 6 0.26 (4) 7.49 6 0.11 (4) GluA1o+g8+CNIH2 5.54 6 0.5 (2) 6.18 6 0.52 (12) 7.87 6 0.31 (7) 7.66 6 0.28 (4) GluA1i 5.11 6 0.29 (2) 5.94 6 0.15 (4) .5 (6) .5 (4) GluA1o+CNIH2 4.96 6 0.12 (2) 5.93 6 0.27 (7) .5 (6) .5 (4) GluA1o+g2 6.03 6 0.11 (2) 6.55 6 0.38 (5) .5 (5) .5 (4) GluA1o+g3 6.18 6 0.3 (2) 6.72 6 0.23 (8) .5 (6) .5 (4) GluA1o+g4 5.85 6 0.35 (2) 6.45 6 0.46 (8) .5 (6) .5 (4) GluA1o+g7 5.84 6 0.19 (2) 6.55 6 0.34 (5) .5 (6) .5 (3) TARP-g8–Selective AMPAR Modulators 403 Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 3. Determination of location of specificity for TARP-selective compounds. (A) Schematic diagram indicating the sections of the proteins used for representative chimeric TARPs (not drawn to scale). Transmembrane segments are depicted as wider lines. Segments from the different TARPs are color-coded: blue, g8; red, g4; green, g2. The chimeras are labeled with a nine-digit number; each digit indicates the TARP used for that section of the protein, starting from the N terminus (NT). The diagrams for all of the chimeras are shown in Supplemental Fig. 3. (B) Sequence alignment of the human isoforms of the TARPs in the TM3–TM4 regions. Vertical lines mark the predicted positions of the transmembrane domain regions. Highlights indicate positions for which TARP-g8 is different from TARP-g4. (C and D) Potency of inhibition of the glutamate response of GluA1o coexpressed with each of the chimeras and point mutations. Potency is expressed as DpIC50: the difference between pIC50 of GluA1o coexpressed with the construct and pIC50 of GluA1o+g8. (D) g8.DM is g8.G210A.V177I, g4.DM is g4.A189G.I156V, and g2.DM is g2.A184G.I153V. 55511118, JNJ-55511118; 56022486, JNJ- 56022486; CP, CP-465022; GYKI, GYKI-53655; IN1, intracellular domain 1.

TARP-g2, and acutely dissociated hippocampal neurons from variety of cell types; no inhibition was seen in cells expressing adult mouse for native AMPA receptors expressing TARP-g8. GluA1o, GluA1o-g2, GluA1i1g2, cultured cerebellar granule Figure 4A shows representative traces of the glutamate-evoked cells, or hippocampal neurons from TARP-g8 knockout mice. responses of outside-out patches from cultured cerebellar neu- Figure 4B shows the glutamate-evoked peak current as a rons and whole-cell currents from acute hippocampal neurons. function of concentration of JNJ-55511118 in outside-out Analogous to the results with heterologously expressed AMPA patches from CHO cells expressing human GluA1o-g8 and receptors, 1 mM JNJ-55511118 reduced peak glutamate-evoked from acutely dissociated mouse hippocampal neurons. In both currents in hippocampal neurons to 60.7 6 2.6% (N 5 6) relative cell types, the peak currents were partially inhibited at a to control, with virtually no effect on cerebellar currents (98.5 6 saturating concentration. By nonlinear least-squares fitting to 1.7%, N 5 7). Supplemental Fig. 4 shows a summary of the effect a logistic function with slope fixed to unity, the maximal of the glutamate-evoked peak current by JNJ-55511118 in a inhibition was 55.8 6 1.9%, and the midpoint was 3.8 6 1.0 nM for 404 Maher et al. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 4. Electrophysiological evaluation of the effects of JNJ-55511118. (A) Outside-out recordings of glutamate-evoked currents in patches from cells expressing AMPA receptors, in the presence (red) and absence (black) of 1 mM JNJ-55511118. L-Glutamate (10 mM) was applied during the time depicted by the gray bar. (B) Peak current evoked by 10 mM L-glutamate, normalized to control, in patches from cells (heterologous GluA1o-g8or acutely dissociated mouse hippocampal pyramidal cells) as a function of concentration (mean 6 S.E.M.). (C) Bath application of JNJ-55511118 partially inhibited the fEPSPs in CA1 hippocampal slices from wild-type mice, but not from TARP-g8 knockout (KO) mice. (D) Representative currents in outside-out patches from cells expressing GluA1i+g8. The middle panel shows the currents normalized to the peak, to visualize the faster desensitization in the presence of 1 mM JNJ-55511118 (red) or TARP-less GluA1i (blue). The lower panel is a box plot for desensitization time constants across conditions. The box plots show the median (line), mean (square), 25th and 75th percentiles (box), and minimum/maximum (whiskers). (E) Recovery from desensitization. Representative traces for recovery from desensitization in TARP-g8–containing AMPA receptors in the absence (top) or presence of 1 mM JNJ-55511118 (middle). The lower panel shows the recovery from desensitization exponential fits for TARP- g8–containing AMPA receptors in the absence (black) or presence of JNJ-55511118 (red) and GluA1i-only expressing receptors (blue). (F, top panel) JNJ-55511118 (1 mM) inhibited peak currents evoked by a brief (1 ms) pulse of glutamate (10 mM). (F, middle panel) Currents normalized to peak show the differences in current decay kinetics across conditions. (F, bottom panel) Summary bar graph for the deactivation time constants across conditions. (G) Expression of TARP-g8 (top-right panel) enhances the kainate (KA; 1 mM, normalized to 10 mM glutamate) efficacy compared with TARP-less AMPA receptors (top-left panel). JNJ-55511118 (1 mM) did not significantly affect the kainate/glutamate ratio in TARP-g8–containing AMPA receptors (bottom panels).

GluA1o-g8. For hippocampal neurons, the maximal inhibition hippocampal slices to test the activity of JNJ-55511118 was 60.1 6 3.4%, and the midpoint was 15.0 6 4.6 nM. (1 mM) on synaptic AMPAR currents. Electrical stimulation of To assess the activity and selectivity of JNJ-55511118 Schaffer collaterals evoked AMPAR-mediated EPSCs which were during synaptic transmission, we recorded field excitatory inhibited by JNJ-55511118 to 73.0 6 4.0% of control (N 5 10; postsynaptic potentials (fEPSPs) from the CA1 region of P 5 0.001 by paired-sample Student’s t test) (Supplemental hippocampal slices from wild-type and TARP-g8 knockout Fig. 5A). This effect appeared to be specific to postsynaptic animals (Fig. 4C). We did not observe an effect of JNJ- AMPA receptors since we did not observe changes in the 55511118 (1 mM) on the fEPSPs in CA1 of the hippocampus paired-pulse ratio or NMDA EPSCs (123 6 16% of control; N 5 from TARP-g8 knockout animals (105.3 6 6.9% of baseline; 10; P 5 0.59 by paired-sample Student’s t test) (Supplemental N 5 5), whereas the fEPSPs were inhibited in slices from wild- Fig. 5, C and D). We observed reduced synaptic summation type littermates (63.4 6 11.0% of baseline; N 5 6). We used when stimulated at 50 kHz in the presence of JNJ-55511118 whole-cell patch clamp in pyramidal CA1 neurons from (Supplemental Fig. 5B). TARP-g8–Selective AMPAR Modulators 405

To investigate the effect of JNJ-55511118 on desensitization the steady-state currents (P 5 0.58 by paired-sample Stu- of g8-containing AMPA receptors, we used 500-ms applications dent’s t test); thus, the inhibition observed with JNJ-55511118 of 10 mM L-glutamate on outside-out patches of HEK-293 cells does not involve the dissociation of the TARP from the receptor expressing heterologous GluA1i1g8 (Fig. 4D). JNJ-55511118 complex. (1 mM) reduced peak currents to 63.8 6 4.1% of control and Binding Assays. We incorporated tritium into JNJ- steady-state currents to 25.4 6 3.7% of control (N 5 6). In the 55511118 and JNJ-56022486 to explore their utility in radio- presence of cyclothiazide, which removes desensitization, ligand binding assays. [3H]JNJ-56022486 proved to have 1 mM JNJ-55511118 inhibited glutamate-evoked currents to lower nonspecific binding than [3H]JNJ-55511118, likely due 59.5 6 1.7% of control (N 5 6; Supplemental Fig. 6). Inhibition to its lower lipophilicity and higher free fraction in brain tissue in the presence of cyclothiazide was not significantly different (see Supplemental Table 10). Therefore, we focused on [3H] from inhibition of peak currents (P 5 0.52), whereas it was JNJ-56022486 for additional binding studies. significantly reduced compared with inhibition of the steady- Saturation binding in membranes from rat hippocampus state current (P , 0.001; one-way repeat measures ANOVA using [3H]JNJ-56022486 is shown in Fig. 5A. The fitted value followed by Tukey’s test). of the binding affinity was 27 6 3 nM, with Bmax 5 3.8 6 In addition to reducing peak and steady-state currents, the 0.3 pmol/mg protein. In competition binding experiments presence of 1 mM JNJ-55511118 accelerated the rate of (Fig. 5B), JNJ-55511118 and JNJ-56022486 fully displaced desensitization, which can be observed by normalizing to the the radioligand (20 nM) with Ki 5 26 6 7 and 19 6 6 nM, peak currents (Fig. 4D, middle panel). After application of respectively (N 5 3). Neither Philanthotoxin-74 nor gluta- Downloaded from 3 10 mM L-glutamate, GluA1i1g8 desensitized with a time mate showed appreciable displacement of [ H]JNJ-56022486, constant of 7.9 6 0.8 ms (N 5 6). In the presence of 1 mM JNJ- whereas LY-395153 [a positive allosteric modulator (PAM)] 55511118, the time constant decreased to 5.7 6 0.5 ms (P , and perampanel partially displaced the radioligand (Fig. 5B). 0.001 by paired-sample Student’s t test). In the absence of Displacement by LY-395153 required the presence of 500 mM TARP, GluA1i desensitized considerably faster, with a time glutamate; the PAM did not displace the radioligand in the constant of 3.5 6 0.3 ms (N 5 5). We determined the recovery absence of glutamate (data not shown). jpet.aspetjournals.org from desensitization by measuring the peak current in re- Autoradiograms showing total binding of [3H]JNJ-56022486 sponse to paired applications of 10 mM L-glutamate, separated in brain slices from mouse, rat, and monkey are shown in Fig. by a variable delay (Fig. 4E). Recovery time constants were 5, D–H. An image from the Allen Brain Atlas of the expression similar for GluA1i1g8 in the absence or presence of JNJ- of CACNG8 in a corresponding coronal brain slice from an 55511118 (control tr,desens 5 93 6 29 ms, N 5 7; JNJ-55511118 adult mouse (Lein et al., 2007) is shown in Fig. 5C. These tr,desens 102 6 33 ms, N 5 6; P 5 1). Receptors in patches from images indicate a high concentration of specific binding that cells expressing TARP-less GluA1i recovered more slowly corresponds well to the expression pattern of CACNG8. at ASPET Journals on September 27, 2021 than GluA1i1g8 (GluA1i tr,desens 5 163 6 10 ms, N 5 8; P 5 Pharmacokinetics. We determined the pharmacokinetic 0.02 by Student’s t test). and in vivo target occupancy profiles for JNJ-55511118 and We investigated the effects of JNJ-55511118 on the de- JNJ-56022486. JNJ-55511118 achieved high plasma concen- activation of TARP-g8–containing AMPA receptors. Fast trations upon oral (p.o.) and i.v. dosing (Fig. 6A). The compound application (1 ms) of glutamate to excised outside-out patches was orally bioavailable in both species. In vivo clearance and generated rapidly deactivating currents. Figure 4F (top panel) volume of distribution in rats were 4.8 ml/min/kg and 1.8 l/kg, shows that JNJ-55511118 partially inhibited the glutamate- respectively. Target occupancy was determined by ex vivo evoked peak currents to 67 6 2% of control (N 5 9). autoradiography of brain slices of animals dosed with test Normalization of peak currents shows that JNJ-55511118 compound, using [3H]JNJ-56022486 to probe for unoccupied increased the deactivation rate (Fig. 4F, middle panel). After receptors. JNJ-55511118 was highly brain-penetrant, and application of 10 mM L-glutamate, GluA1i1g8 deactivated showed high target occupancy upon oral dosing in both rat with a time constant of 4.6 6 0.9 ms (N 5 9). In the presence of and mouse (Fig. 6, B and C). This compound also displayed 1 mM JNJ-55511118, the time constant decreased to 3.1 6 linear exposure as a function of dose (Fig. 6D). Although JNJ- 0.6 ms (P 5 0.001 by paired-sample Student’s t test). In the 56022486 also showed good oral bioavailability, the brain absence of TARP, GluA1i deactivated considerably faster, penetration and target occupancy at 10 mg/kg were sub- with a time constant of 1.2 6 0.3 ms (N 5 6). stantially lower compared with JNJ-55511118 (Supplemental To test for the possibility that JNJ-55511118 may disrupt the Fig. 7). Therefore, we focused on JNJ-55511118 for addi- interaction between TARP g-8 and AMPA receptors, we looked tional in vivo studies. In vitro measurements of tissue at the effects of JNJ-55511118 on kainate efficacy. TARPs binding of JNJ-55511118 showed 1.48 and 0.88% free enhance kainate efficacy for AMPA receptors (Tomita et al., fraction in rat and mouse plasma, respectively, and 0.24% 2005; Turetsky et al., 2005); the ratio of response to kainate free fraction in rat brain tissue. Supplemental Table 10 versus glutamate is a sensitive assay for TARP/AMPAR shows a summary of the pharmacokinetic parameters de- stoichiometry (Shi et al., 2009). As previously reported, TARP- rived for these two compounds. less AMPA receptors had a considerably lower kainate/glutamate In Vivo Electrophysiology. For a more direct measure- ratio of steady-state currents when compared with g8-containing ment of the functional consequences of target engagement, we AMPARs (Fig. 4G, top panels). In the absence of TARP-g8, the performed in vivo electrophysiological recordings in the kainate/glutamate ratio for GluA1i was 0.21 6 0.04 (N 5 4), pyramidal cell layer of CA1 in the rat hippocampus, while whereas the kainate/glutamate ratio for GluA1i-g8 was 5.1 6 stimulating the Schaffer collateral projections from CA3. 1.1 (N 5 9). In the presence of JNJ-55511118 (Fig. 4G, bottom- When dosed intravenously, JNJ-55511118 showed a rapid, left panel), the ratio was 4.2 6 0.7 (N 5 9). JNJ-55511118 dose-dependent inhibition of the evoked population spike failed to significantly change the kainate/glutamate ratio of amplitude, with a fitted half-maximal effective dose (ED50) 406 Maher et al. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 5. Radioligand binding assays. (A) Saturation binding assay using [3H]JNJ-56022486. Data points are the mean and standard deviation of four individual measurements. Specific binding (blue) was calculated as the difference in the means of total (black) and nonspecific (red) binding. Solid lines are fits to the data. (B) Competition binding assays using 20 nM [3H]JNJ-56022486. Data are the mean of four measurements. Solid lines are fits to the data. (C) Expression pattern of the CACNG8 gene by in situ hybridization in a coronal section of mouse brain, downloaded from the Allen Mouse Brain Atlas (Lein et al., 2007) (ª 2015 Allen Institute for Brain Science, http://mouse.brain-map.org/experiment/show/72108823). (D–H) Receptor density in brain slices using [3H]JNJ-56022486. Scale bars in each image are 5 mm. The color scale on the right shows the image intensity from low (blue) to high (red) radiation counts. Mouse (D) and rat (E) coronal brain slices. Mouse (F), rat (G), and monkey (H) sagittal brain slices. of 0.4 mg/kg (Fig. 7, A and B). One hour after dosing, dose-response experiment. A group of seven animals were recordings were terminated; plasma and brain tissue were orally dosed at 2 hours into the light phase with vehicle (0.5% harvested for bioanalysis to measure the concentration of HPMC) or JNJ-55511118 (1, 3, and 10 mg/kg; 0.5% HPMC JNJ-55511118. Fig. 7C shows the population spike inhibition suspension) in a randomized crossover design. EEG/EMG at 1 hour postdosing as a function of the measured brain signals and locomotor activity were recorded for up to 20 concentration for each animal. Superimposed in this graph is hours after each pharmacological treatment; here, we show the receptor occupancy as a function of brain concentration for the effects during the first 8 hours after administration. rat from the experiments shown in Fig. 6E. The calculated Oral administration of JNJ-55511118 elicited a clear dose- half-maximal effective concentration (EC50) values for these dependent decrease in EEG activity during the wake state in two assays were quite similar (591 ng/ml, in vivo electrophys- all frequency bands above 4 Hz (Fig. 7G). Specifically, a dose- iology; 545 ng/ml, autoradiography). related reduction of the power spectral density in the theta EEG. To investigate the effect of functional g8-selective (4–10 Hz) [F(3, 18) 5 26.73, P , 0.001], alpha (10–15 Hz) [F(3, 18) 5 inhibition on the cortical oscillations in freely behaving rats, 35.80, P , 0.001], and beta (15–30 Hz) [F(3, 18) 5 168.10, P , sleep-wake architecture and EEG power spectral density were 0.001] oscillations was observed from the lowest dose tested. evaluated after oral administration of JNJ-55511118 in a The power spectral density in the delta oscillations was TARP-g8–Selective AMPAR Modulators 407 Downloaded from jpet.aspetjournals.org Fig. 6. Pharmacokinetics and target occupancy for JNJ-55511118 in rat and mouse. (A) Plasma concentration as a function of time in Sprague-Dawley rats following oral (5 mg/kg p.o.) or intravenous (1 mg/kg) administration. (B and C) Brain and plasma concentrations in rat and mouse after a 10-mg/kg p.o. dose. Target occupancy was determined by autoradiography of brain slices. (D) Dose linearity of plasma and brain concentrations after p.o. dosing in rat (t = 4 hours) and mouse (t = 1 hour). (E and F) Target occupancy as determined by autoradiography after p.o. dosing in rat (t = 4 hours) and mouse (t = 1 hour). In all panels except (E), data are presented as the mean and standard deviation. minimally affected, and only a small but significant decrease corneal kindling model, JNJ-55511118 provided near- was revealed at a dose of 1 mg/kg. Consequently, the complete seizure protection at and above 5 mg/kg (p.o., 1 hour at ASPET Journals on September 27, 2021 contribution of the EEG delta activity over the total power postdosing). Twenty-five out of 32 animals had Racine scores (relative delta power) was enhanced, since the absolute values of zero at doses of 5–40 mg/kg, indicating the complete absence for power spectra in all of the higher frequency bands were of observable effects of the stimulus. In comparison, 8/8 decreased. animals in the vehicle control group had Racine scores of 5 The 10-mg/kg dose, which should have achieved approxi- (rearing and falling with forelimb clonus). The curve fits to the mately 90% target occupancy (see Fig. 6F), represents the corneal kindling data in Fig. 7, D and E indicate that ED50 5 near-saturating drug effect. At this dose, power in the wake 3.7 mg/kg, with a brain concentration EC50 of 938 ng/ml. state in the theta, alpha, and beta bands was reduced to 81.2 6 JNJ-55511118 showed partial protection in the 6 Hz 1.0, 70.6 6 1.3, and 63.8 6 1.2%, respectively, relative to models. At doses of 10 mg/kg and above, projected to give control. This substantial reduction in EEG power was accom- target occupancies of 80% or greater, 12/32 (37.5%) animals panied by a significant increase in locomotor activity for were protected in the 6 Hz 32 mA model, and 11/40 (27.5%) approximately 1 hour postdose (Fig. 7H). In addition, there were protected in the 6 Hz 44 mA model. This compound was was a dose-dependent increase in the latency to REM and also tested in the MES model; at 40 mg/kg p.o., 50% of the NREM sleep (Supplemental Fig. 8). animals showed seizure protection (N 5 8; data not shown). JNJ-55511118 also produced dose-dependent decreases in The curve fits to the 6 Hz 32 mA data indicate that ED50 5 absolute EEG power during REM and NREM sleep states 18.3 mg/kg, with a plasma concentration EC50 of 4644 ng/ml (Supplemental Fig. 9). During NREM sleep, EEG oscillations and 76% maximum protection. For the 6 Hz 44 mA test, ED50 5 were significantly reduced from the 1-mg/kg dose onward in 6.5 mg/kg, with a plasma concentration EC50 of 2533 ng/ml all measured frequency bands (1–30 Hz). During REM sleep, and 22% maximum protection. delta (1–4 Hz), theta (4–10 Hz), and beta (15–30 Hz) EEG The Metrazol (PTZ) test was performed at t 5 0.5 hour after oscillations were significantly reduced from the dose of 1 mg/kg dosing with 40 mg/kg JNJ-55511118. The compound showed onward. In contrast, EEG oscillations in the sigma frequency strong protection (Fig. 7F); the mean threshold for clonus range (10–15 Hz) were minimally affected. increased from 33.3 6 4.9 to 49.5 6 10.0 mg/kg PTZ (P , 0.001, Anticonvulsant Assays. We explored the anticonvul- two-sample t test), and for twitch, from 30.7 6 4.6 to 42.4 6 sant profile of JNJ-55511118 using several in vivo models. 7.5 mg/kg (P , 0.001, two-sample t test). Figure 7, D and E shows the dose-response relationship of the JNJ-55511118 was also tested in the amygdala kindling activity in the corneal kindling and 6 Hz models, as well as model in rats. This compound protected 11/12 rats from the rotarod test. Based on the target occupancy in mouse seizure (2 hours postdose, 10 mg/kg p.o., 0.5% HPMC suspen- shown in Fig. 6F, target occupancy should be well above 90% sion), compared with 1/12 rats protected with vehicle alone. at doses above 40 mg/kg. Any additional pharmacology above The after-discharge duration, shown in Supplemental Fig. 10, that dose would likely be due to off-target effects. In the was significantly reduced by JNJ-55511118 to 35 6 7 seconds, 408 Maher et al. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 7. Effects of JNJ-55511118 on in vivo measures. (A–C) Evoked neurotransmission in hippocampus using in vivo electrophysiology. Population spikes (PS) were recorded in the pyramidal cell layer of rat hippocampal CA1, evoked by stimulation of Schaffer collaterals in CA3. (C) Response amplitudes, normalized to predose baseline amplitudes, plotted as a function of time following i.v. administration of drug. Each data point represents the average response from two to four animals per dosing group. Drug was administered at time t = 0. (B) Dose-response profile of population spike amplitude, averaged over the last 5 minutes of each 60-minute recording period in (A). (C) Population spike amplitude (black squares) for each animal as a function of brain concentration, as measured immediately after the recording. Superimposed is the target occupancy (blue circles), measured in rats with oral dosing, from the autoradiography experiments shown in Fig. 6. Curves in (B) and (C) indicate nonlinear least-squares fits to a Hill function. (D–F) Anticonvulsant assays in mouse. (D and E) Dose response in corneal kindling (black) and 6 Hz models (red and green). Seizure protection was calculated as the fraction of animals with Racine seizure scores of 3 or lower (N = 8 mice per cohort). Also shown is the fraction of animals from the 6 Hz tests with motor impairment as measured on the rotarod (blue). Lines represent fits to a Hill function. (D) Data plotted as a function of dose. (E) Same data as in (D), plotted using the average measured brain concentration in a parallel cohort of animals. (F) Protection in Metrazol (PTZ) test. The thresholds for the amount of PTZ delivered to achieve twitch and clonus in mice (N = 10 per cohort) were significantly increased after dosing with JNJ- 55511118 (40 mg/kg suspension, p.o.). The box plot shows the median (line), 25th and 75th percentiles (box), minimum/maximum (whiskers), and individual data for each animal (circles). (G) EEG power activity during the wake state in rats. Power spectral density in the delta (1–4Hz),theta(4–10 Hz), alpha (10–15 Hz), and beta (15–30 Hz) bands were determined for the 8-hour period after compound or vehicle administration. Data are represented as means 6 S.E.M. of the same seven animals per dose. *P , 0.05, **P , 0.01, and ***P , 0.001 versus vehicle, based on one-way ANOVA followed by Dunnett’s multiple-comparison post-hoc test. (H) Locomotor activity in rats. Activity counts were determined per 5-minute interval for the 120-minute period after compound or vehicle administration. Data are presented as means 6 S.E.M. of the same seven animals per dose. compared with 92 6 12 seconds with vehicle alone (mean 6 hippocampal plasticity and learning/memory mechanisms S.E.M., N 5 12; P 5 4 1024,two-samplet test). important for performance on spatial working memory tasks. Effects in Models of Learning/Memory/Cognition. In- The Morris water maze (MWM) provides a key method to hibition of AMPA receptors, particularly in the hippocampus investigate spatial learning and memory in rodents (D’Hooge and prefrontal cortical areas, might be expected to impact and De Deyn, 2001). Figure 8, A and B shows that vehicle-treated TARP-g8–Selective AMPAR Modulators 409 Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 8. Effects of JNJ-55511118 in assays of learning and memory in rodents. (A and B) Behavioral measures in the Morris water maze test. (A) Acquisition performance for each training day, measured as the time to locate the submerged platform. Animals were dosed 2 hours before the start of the procedure, and each animal had three training periods over 4 consecutive days. Data points are the mean and S.E.M. for N = 12 animals. (B) Performance in the probe trial. Immediately after the acquisition trials were completed on day 4, the platform was removed. Data shown are the fraction of time that the animals spent in each quadrant of the maze, for the first 30 seconds after entering the water maze. The submerged platform had originally been in quadrant A (QA). (C and D) Primary measures in the DNMTP assay (N = 10 animals per dose group). (C) Overall fraction of correct responses. (D) Fraction of correct responses separated by delay time. (E and F) Behavioral measures for the V-maze (N = 12 animals per dose group). (E) Discrimination index for the preference of rats for the new versus the familiar arm of the maze. Statistics are from comparisons to the vehicle control group. (F) Total distance moved during the habituation and test phases for each dose group. The statistics are from comparisons to the vehicle cohort of each phase.

animals obtained a typical learning curve during the 4 first (P 5 0.05, Cox proportional hazards model) and second (P 5 training days evidenced by shortening of latency times (Fig. 0.04) training days; however, on the third and fourth training 8A) and path length (Supplemental Fig. 11) to reach the days, their performance was indistinguishable from vehicle- platform. Based upon the occupancy as a function of dose treated animals. shown in Fig. 6E, the dose range of 0.63, 2.5, and 10 mg/kg p.o. In the DNMTP test, there was an overall effect of dose (P , should have given target occupancies of 50–90%. Surprisingly, 0.01), with JNJ-55511118 decreasing the percentage of correct the performance was not severely impacted; at the 0.63- and responses at the two highest doses tested (3 and 10 mg/kg; P , 2.5-mg/kg doses during the training days, there was no 0.05 and P , 0.01, respectively), although the effect was rather statistically significant deviation from performance of moderate (approximately 5% reduction; Fig. 8C). This was vehicle-treated animals, apart from a small transient but accompanied by a small, but significant, reduction in respon- statistically significant improved latency time to platform on sivity, as indicated by an increase in errors of omission and day 2 (which was not confirmed in an independent repeat response latencies (Supplemental Fig. 12). Although there study; data not shown). The animals in the 10-mg/kg cohort was no significant drug delay interaction (P . 0.05), post-hoc showed a modest deficit in learning the platform location contrasts were computed against the vehicle group for the (increased time and path length to locate the platform) on the percentage of correct responses, showing significant effects of 410 Maher et al. the two higher doses at the shortest delay (1 second), but not at alter the pharmacology of the backbone TARP, again suggest- longer delays (Fig. 8D). All tested doses increased the percent- ing that domain EX1 is not involved with the pharmacological age of trials omitted, and the two highest doses tested induced a activity. Instead, domain scanning and direct substitutions significant increase on latency measures (see Supplemental indicated that TM3 and TM4 govern the selectivity. Fig. 12). The magnitudes of these effects were generally modest The point-mutation studies allowed us to determine that (less than 0.5 second for most measures). selectivity is entirely determined by two amino acids predicted The V-maze was used to study the effects of JNJ-55511118 to lie within TM3 and TM4. These two amino acids (G210 and on working memory in rodents. This paradigm exploits the V177) are unique to TARP-g8. Altering these two amino acids natural tendency of rodents to explore a novel, nonthreatening to their corresponding components of TARP-g4 completely environment. The V-maze avoids the use of aversive test abolished the potency of the compounds, whereas the TARP- conditions, such as electric shocks or deprivation, that may g8 versions of these same two amino acids added to both have nonspecific influences on the responses, and does not TARP-g2 and TARP-g4 confer identical sensitivity of these require prior learning of a rule. TARPs to the compounds. According to the predicted topology Vehicle-treated animals show a clear preference for the new of the TARP (Fig. 3A), G210 and V177 are both 2–3 residues arm over the familiar arm they explored just before (discrim- deep within the outer membrane surface. ination index 5 0.40 6 0.06; Fig. 8E). A partial impairment of Data and alignments were retrieved from the UniProt this parameter is found in the 10-mg/kg treatment group (P 5 database (UniProt Consortium, 2015) for genes identified as 0.0055, ANOVA). Concomitantly, a small increase in distance CACNG8, from a selection of species. Sequence alignments of Downloaded from moved is seen in a dose-dependent manner in the habituation TM3 and TM4 for the TARP-g8 in selected species are shown phase (P , 0.0001) and at a dose of 10 mg/kg only in the test in Supplemental Fig. 13; these regions are highly conserved phase (P 5 0.0075; Fig. 8F). across species. Curiously, the two key amino acids that determine selectivity against TARP-g2and-g4 (G210A and V177I) are also conserved across species (Supplemental Fig.

Discussion jpet.aspetjournals.org 14). Indeed, we found no species differences in potency or AMPA receptor signaling is a critical component of normal selectivityinrat,mouse,dog,monkey,orhumanisoforms and pathophysiological excitatory neuronal function, and has (Fig. 2, E and F; Table 1). These two key amino acid changes been an attractive pharmacological target for both positive are relatively small; each involves adding a single methyl and negative modulation. To date, all negative modulators of group in making the wild-type TARP-g8 protein completely AMPA receptors target the pore-forming GluA subunits. insensitive to JNJ-55511118. In addition to these two key Although the presence of TARPs can modify potency and residues, alanine scanning indicated that N173 in TM4 and efficacy of modulators (Cokic and Stein, 2008), no published G209 and F213 in TM3 influence the potency of the com- at ASPET Journals on September 27, 2021 reports have shown any selectivity of inhibitors among the pounds. These amino acids are adjacent to, or one alpha helix TARPs. Here, we report a novel pharmacological approach to turn away from, the key selectivity locations. These five negatively modulate AMPA receptor signaling, with mole- amino acids may form or contribute to the binding site for cules selective for TARP-g8. these TARP modulators. JNJ-55511118 and JNJ-56022486 are highly potent, and Mechanism of Action. The radioligand binding assays are exquisitely selective for AMPA receptors containing showed that JNJ-55511118 and JNJ-56022486 fully displaced TARP-g8, with no detectable functional effect on TARP-less [3H]JNJ-56022486, with affinities comparable to the potencies AMPA receptors or those containing TARP-g2, -g3, -g4, or -g7. as measured in the calcium flux assays. In contrast, ligands The data presented here suggest that these compounds exert that bind to the agonist site (glutamate) and the pore their effects through a novel mechanism of action, via partial (Philanthotoxin-74) showed no displacement. Ligands that disruption of a protein-protein interaction. JNJ-55511118 bind to the GluA PAM site (LY-395153) and the noncompet- exhibits excellent pharmacokinetics and brain penetration, itive antagonist site (perampanel), which couple allosterically and achieves high-target occupancy upon oral and intra- when TARPs are associated with the AMPA receptor (Schober venous dosing. Tritiated JNJ-56022486 is useful as a radio- et al., 2011), partially displaced [3H]JNJ-56022486. Partial ligand for competitive binding studies, and for ex vivo displacement implies allosteric coupling between these sites autoradiography to determine target occupancy. and the binding site for the TARP-g8 modulators; further TARP Selectivity. The g8–g4 chimeras were designed to studies are required to investigate this phenomenon. locate the site of selectivity. Previous studies have demon- The electrophysiology experiments allowed detailed char- strated that the TARP C terminus and EX1 domains have acterization of the effects of JNJ-55511118 upon the AMPA strong effects on trafficking and functional interaction with receptor currents. The results are consistent with a partial the GluA subunits (Tomita et al., 2005; Turetsky et al., 2005; disruption of the interaction between TARP-g8 and the pore- Cais et al., 2014). Thus, a drug interacting at one of these two forming GluA subunits. Type I TARPs provide a positive regions could differentially impact the function of the AMPA modulatory influence upon the AMPA receptor (Howe, 2015). receptor complex. Chimera pairs (448444444, 884888888) and The electrophysiological studies showed that JNJ-55511118 (444444448, 888888884) directly probed this question. As reverses some, but not all, of the effects of the TARP on the shown in Fig. 3C, JNJ-55511118 and JNJ-56022486 inhibited AMPA receptor complex. A saturating concentration of JNJ- 888888884 (g8 with the C terminus from g4) and failed to 55511118 reduces peak responses by 36–44%, and steady- inhibit 444444448 (g4 with the C terminus from g8). This pair state currents by ∼75%. This reduction is consistent with of results suggests that the C terminus is not involved with the masking the increases in single-channel conductance ob- pharmacological activity of these two compounds. The chi- served in studies of single-channel kinetics of TARPed versus mera pair (448444444, 884888888) showed a similar failure to TARP-less AMPA receptors (Shelley et al., 2012; Zhang et al., TARP-g8–Selective AMPAR Modulators 411

2014). This inhibition is accompanied by an increase in the (Chen et al., 2000). Whether JNJ-55511118 impacts these desensitization and deactivation kinetic rates. additional interactions between TARPs and AMPARs remains Other aspects of the inhibition by JNJ-55511118 indicate an open question. Indeed, this molecule may prove useful in that TARP-g8 remains associated with the receptor complex. determining the structure-function relationships of these First, the kainate/glutamate ratio does not change as would be diverse phenomena. expected with changes in TARP stoichiometry (Shi et al., 2009). Synaptic Transmission. We tested the activity of JNJ- TARPs enhance kainate efficacy for AMPA receptors (Tomita 55511118 in hippocampal slices, a preparation in which the et al., 2005; Turetsky et al., 2005), and the kainate-to-glutamate native synaptic receptors are found in association with compo- response ratio is a sensitive assay for TARP/AMPAR stoichio- nents of the postsynaptic densities and associated auxiliary metry (Shi et al., 2009). As previously reported, TARP-less subunits. Here, we found that JNJ-55511118 inhibited the AMPA receptors had a considerably lower kainate/glutamate synaptic responses in the hippocampal CA1 region from wild- ratio of steady-state currents when compared with AMPA type mice but not from TARP-g8 knockout littermates. Consis- receptors containing TARP-g8. JNJ-55511118 failed to signif- tent with specificity of JNJ-55511118 on postsynaptic AMPA icantly change the kainate/glutamate ratio of the steady-state receptors, the compound only inhibited the AMPA EPSCs from currents, strongly suggesting that the inhibition observed CA1 pyramidal neurons, but not the NMDA EPSCs, and did not with JNJ-55511118 does not involve the dissociation of the affect the paired-pulse ratio. Taken together, these data suggest TARP from the receptor complex. Second, the deactivation and that JNJ-55511118 does not affect the presynaptic glutamate desensitization kinetics observed are considerably faster in release probability, but instead acts directly on postsynaptic Downloaded from the TARP-less receptors when compared with the TARP- AMPA receptors containing TARP-g8. We also observed re- g8–containing receptors in the presence of JNJ-55511118. duced synaptic summation when stimulated at 50 kHz in the Third, JNJ-55511118 does not affect the recovery from de- presence of JNJ-55511118 (Supplemental Fig. 5), suggesting a sensitization, whereas the TARP-containing AMPA receptors potential indication of this compound in states of hyperactive recover from desensitization faster than TARP-less AMPA hippocampal activity, such as seizures. These results confirm receptors (Priel et al., 2005). JNJ-55511118 may partially the activity of JNJ-55511118 on native postsynaptic AMPA jpet.aspetjournals.org affect the GluA-TARP interaction required for reduced de- receptors under basal and high-frequency synaptic stimulation, sensitization and deactivation, leaving intact those interac- and set the stage for a physiologic role of this compound in tions required for kainate efficacy. Alternatively, TARP-g8 hippocampal activity. may induce a unique conformational state on AMPARs In anesthetized rats, JNJ-55511118 caused near-complete not observed with other TARPs, enabling a binding site on inhibition of population spikes in CA1 driven by Shaffer AMPARs for the inhibitors to reduce open-channel collateral stimulation. The concentration dependence of probability. population spike inhibition closely matched the target occu- at ASPET Journals on September 27, 2021 These features suggest that JNJ-55511118 does not cause pancy. The magnitude of inhibition of the population spike the TARP to dissociate from the AMPA receptor complex, but amplitude is somewhat surprising, given the partial inhibi- instead modifies the protein-protein interaction between the tion of AMPA receptor current in in vitro electrophysiology TARP and GluA subunits. In a simplified gating model (Fig. 4, A and B) and of the fEPSP slope in ex vivo slices (Fig. containing single open, resting, and desensitized states (Sun 4C). Partial inhibition of AMPA receptors to the extent et al., 2002), these effects are consistent with destabilization of observed with JNJ-55511118 is apparently sufficient to the open state by JNJ-55511118. Additional kinetic studies reduce the synaptic drive below the spiking threshold of the comparing TARP-less AMPA receptors to those containing postsynaptic neurons. TARP-g8 with and without JNJ-55511118 should reveal more details of the mechanism of action. In addition, mutations of TARP-g8 at the key amino acids described earlier may provide TABLE 2 additional mechanistic insight and structure-activity relation- Summary of results of behavioral experiments with JNJ-55511118 ships regarding the interaction between TARPs and the GluA Experiment Result Notes subunits. Anticonvulsant Electrophysiological measurements in acutely dissociated PTZ 2 Increased threshold to seizure hippocampal neurons indicate that the modulation by JNJ- Corneal kindling 22 Complete seizure protection 22 55511118 closely recapitulates the behavior in heterologous Amygdala kindling Complete seizure protection 6Hz32mA 2 Incomplete protection systems. This suggests that, consistent with previous findings, 6Hz44mA 2 Incomplete protection native hippocampal AMPA receptors are highly TARPed MES 0 (i.e., contain a high level of TARP proteins), primarily with General behavior Rotarod 0 TARP-g8. It also indicates that the presence of the additional Basal motor activity + Transient hyperlocomotion accessory proteins in the native system does not substantially Sleep/wake cycle 2 Decreased sleep duration alter the functional impact of the compound or the binding Sedation 0 Learning/memory pocket. Water maze 2 Minor decrease in learning rate In addition to their modulatory effects on gating and V-maze 2 Minor decrease in performance channel conductance, TARPs have multiple additional effects DNMTP 2 Minor decrease in performance upon AMPARs: they modify surface expression (Rouach et al., EEG oscillations Wake 2 Decrease in theta, alpha, beta 2005; Tomita et al., 2005); alter the pharmacology of the PAM, NREM 2 Decrease in delta, theta, sigma, beta agonist, and noncompetitive antagonist binding sites (Tomita REM 2 Decrease in delta, theta, beta et al., 2005; Turetsky et al., 2005; Schober et al., 2011); and 0, no effect; + increased response; –, decreased response; —, strong decreased participate in anchoring AMPARs to the synaptic scaffolding response. 412 Maher et al.

Effects In Vivo. Table 2 summarizes the results of behav- et al., 1998b; Smith et al., 2011), although effects of AMPA ioral tests performed using a saturating dose of JNJ- receptor blockade in the delayed match to position test, a 55511118. Quantitative EEG analysis in freely behaving rats closely related paradigm to DNMTP, has been reported to during the wake state revealed a dose-dependent decrease in be more subtle than effects of NMDA receptor blockade the power bands of theta, alpha, and beta activity. These data (Stephens and Cole, 1996). indicate that JNJ-55511118 produced some degree of EEG In the MWM, animals in the highest-dose cohort showed slowing, starting at a dose corresponding to 60% receptor attenuated learning within training days 1 and 2 (as evi- occupancy. This effect is consistent with the anticonvulsant denced by nearly flat learning curves), but it is remarkable properties of the compound. JNJ-55511118 gave strong pro- that the latency time during the first trial on the second and tection in the corneal kindling and PTZ models in mice, and in third training day is close to that of the vehicle group on the the amygdala kindling model in rats. Efficacy in the corneal last trial of the day before, suggesting that the animals kindling model was dose-dependent, and roughly approxi- consolidate memories during the nights between the training mated the target occupancy as a function of plasma concen- sessions. This is consistent with absence of treatment effects tration. The compound also showed partial protection in the on performance during the probe trial: all treatment groups 6 Hz and MES tests in mice. showed indistinguishable preference for the area in the pool Modulation of AMPA receptor signaling has long been where the platform was located during the training days. considered an attractive strategy for the treatment of epilepsy JNJ-55511118 showed impairment in V-maze, MWM, and (Rogawski, 2011). AMPAR antagonists show strong anticon- DNMTP. However, compared with effects following systemic Downloaded from vulsant activity in preclinical models, and the noncompetitive administration of the NMDA receptor antagonists dizocilpine AMPAR inhibitor perampanel was recently approved as an or phencyclidine (Willmore et al., 2001), or the muscarinic adjunctive treatment of partial-onset seizures (Ko et al., antagonist scopolamine (Chudasama and Muir, 1997), the 2015). The efficacy of topiramate may be mediated in part by impairment induced by JNJ-55511118 was relatively small. modulation of phosphorylation of neuronal AMPA receptors. Our data are in line with those reported for GluA1 knockout As with all known anticonvulsant medications, both of these mice, which also show relatively mild learning and memory jpet.aspetjournals.org drugs exhibit dose-limiting side-effect profiles that limit their impairments compared with animals with hippocampal le- clinical utility (Perucca and Gilliam, 2012). Modulation of sions (Sanderson and Bannerman, 2012). AMPAR signaling using a TARP-g8–selective mechanism has Even at saturating doses, JNJ-55511118 showed a benign two distinct advantages that may result in an improved side-effect profile, with no loss of motor coordination or therapeutic margin. First, the expression of TARP-g8 within sedation (Fig. 7; Supplemental Fig. 8). Overall behavior of the brain indicates that the drug will have its largest effect animals dosed with JNJ-55511118 appeared largely normal, within the hippocampus, while avoiding direct inhibitory with only transient hyperlocomotion immediately after dosing at ASPET Journals on September 27, 2021 effects on brain regions involved with motor coordination and upon transfer into a novel environment. Considering the and wakefulness. Second, the compounds negatively modulate robust anticonvulsant profile, the strong inhibition of EEG but do not completely inhibit AMPAR signaling. This in- signals, and the expression of the target within the hippo- herently limits the maximal effect of the drug. campus and cortex, the relatively mild impact upon learning AMPA receptors have been associated with regulation of and memory in the Morris water maze, DNMTP, and V-maze hippocampal plasticity and short-term memory mechanisms assays is quite surprising. Thus, TARP-g8 inhibition with important for performance on spatial and working memory molecules such as JNJ-55511118 shows strong potential tasks. GluA1 knockout mice show impaired short-term habit- clinical utility as an anticonvulsant, particularly for those uation to recently experienced stimuli, which has been forms of epilepsy with a strong hippocampal component, such suggested to affect performance in hippocampus-dependent as temporal lobe epilepsy. spatial working memory tasks (Sanderson et al., 2009; In summary, TARP-g8 modulators represent a novel phar- Sanderson and Bannerman, 2012). Hippocampal lesions affect macological class of molecules which possess an unprecedented learning and memory in both human (Manns et al., 2003) and mechanism of action: partial disruption of a protein-protein nonhuman species (Morris et al., 1982). Similarly, knockout of interaction between the pore-forming GluA subunit of AMPA TARP-g8 reduces hippocampal AMPA receptor function and receptors and the TARP-g8 accessory protein. These com- synaptic plasticity (Rouach et al., 2005; Fukaya et al., 2006), pounds provide important tools at several levels: 1) the although the impact of learning, memory, and cognition in molecular pharmacology of this interaction,2)dissectionofthe these transgenic animals has not yet been reported. structure-function relationship of the GluA-TARP interaction, We addressed this issue by testing JNJ-55511118 in rats and 3) the in vivo behavioral and therapeutic potential of using the delayed nonmatch to position, V-maze, and Morris partial inhibition of hippocampal excitability. Neuropsychiatric water maze tests. Performance in MWM is severely attenu- disorders can be viewed as pathologic disruption of the ated by manipulations that negatively impact hippocampal excitation/inhibition balance of specific structures, circuits, or signaling (Morris et al., 1982; Riedel et al., 1999). DNMTP is sets of neurons. TARP-g8 modulators have the potential to be generally thought to be dependent upon hippocampal function transformational anticonvulsants, particularly for medial (Steckler et al., 1998a), with degradation in performance temporal lobe epilepsy. By avoiding the midbrain and hind- following hippocampal lesions (Chudasama and Muir, 1997; brain, AMPA receptor modulators selective for TARP-g8 may Winters and Dunnett, 2004) and direct intrahippocampal attenuate seizures without side effects such as ataxia and infusion of drugs affecting cholinergic, GABAergic, or gluta- sedation that are often seen with less-selective anticonvul- matergic function (Robinson and Mao, 1997; Mao and Robinson, sants. Other clinical applications for this mechanism include 1998). Moreover, numerous studies have found that DNMTP schizophrenia, particularly in the early stages where exces- is sensitive to systemic glutamatergic manipulations (Steckler sive limbic activity has been observed (Schobel et al., 2013), TARP-g8–Selective AMPAR Modulators 413 and anxiety disorders, given the reciprocal relationship be- Burnashev N, Monyer H, Seeburg PH, and Sakmann B (1992) Divalent ion perme- ability of AMPA receptor channels is dominated by the edited form of a single tween hippocampal function and the effects of anxiolytic drugs subunit. Neuron 8:189–198. (Gray and McNaughton, 2003). Cais O, Herguedas B, Krol K, Cull-Candy SG, Farrant M, and Greger IH (2014) Mapping the interaction sites between AMPA receptors and TARPs reveals a role The existence of this mechanism of action has the potential for the receptor N-terminal domain in channel gating. Cell Reports 9:728–740. to greatly expand the number of druggable targets. There are Chen L, Chetkovich DM, Petralia RS, Sweeney NT, Kawasaki Y, Wenthold RJ, Bredt DS, and Nicoll RA (2000) Stargazin regulates synaptic targeting of AMPA recep- at least 30 additional proteins in the AMPAR proteome tors by two distinct mechanisms. Nature 408:936–943. (Schwenk et al., 2012), each with unique expression profiles Cheng Y and Prusoff WH (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an and functional impact upon the AMPA receptor complex. enzymatic reaction. Biochem Pharmacol 22:3099–3108. Thus, just as TARP-g8 shows tissue specificity for the Chudasama Y and Muir JL (1997) A behavioural analysis of the delayed non-matching hippocampus, it may be possible to tune the effects of a drug to position task: the effects of scopolamine, lesions of the fornix and of the prelimbic region on mediating behaviours by rats. Psychopharmacology (Berl) 134:73–82. targeting other subunits for specific brain regions or neuronal Clark RA, Shoaib M, Hewitt KN, Stanford SC, and Bate ST (2012) A comparison of subtypes. Beyond AMPA receptors, most ion channels, and InVivoStat with other statistical software packages for analysis of data generated from animal experiments. J Psychopharmacol 26:1136–1142. indeed drug targets from other protein classes, associate with Cokic B and Stein V (2008) Stargazin modulates AMPA receptor antagonism. Neu- accessory proteins, some of which show expression patterns ropharmacology 54:1062–1070. D’Hooge R and De Deyn PP (2001) Applications of the Morris water maze in the study more specific than the pore-forming subunit. of learning and memory. Brain Res Brain Res Rev 36:60–90. Dugovic C, Shelton JE, Aluisio LE, Fraser IC, Jiang X, Sutton SW, Bonaventure P, Yun S, Li X, and Lord B, et al. (2009) Blockade of orexin-1 receptors attenuates Acknowledgments orexin-2 receptor antagonism-induced sleep promotion in the rat. J Pharmacol Exp – Downloaded from The authors acknowledge the contributions of the following indi- Ther 330:142 151. Embrechts S and Ver Donck L(2014) NMDA receptor antagonism improves visuo- viduals associated with Janssen Research and Development for their spatial recognition memory in a two trial discrimination task, in FENS Forum of contributions to the previously described experiments. Ning Qin Neuroscience; 2014 July 5-9; Milan, Italy. Vol 9, pp287, Meeting Expert, Bucharest, cloned several GluA and TARP constructs. Raymond Rynberg de- Romania. European Union (2010) Directive 2010/63/EU of the European parliament and of the veloped formulations for the in vivo studies. Nancy Aerts and John council of 22 September 2010 on the protection of animals used for scientific pur- Talpos performed the DNMTP experiments. Steven Sutton managed poses. Official Journal of the European Union OJ L 276:33–79. Fukaya M, Tsujita M, Yamazaki M, Kushiya E, Abe M, Akashi K, Natsume R, Kano the back-crossing of the transgenic mice. Sofie Embrechts performed jpet.aspetjournals.org M, Kamiya H, and Watanabe M, et al. (2006) Abundant distribution of TARP the water maze and V-maze experiments. Tom Van de Casteele gamma-8 in synaptic and extrasynaptic surface of hippocampal neurons and its performed the statistical analysis of the behavioral data from major role in AMPA receptor expression on spines and dendrites. Eur J Neurosci – learning/memory assays. Caroline Lanigan advised on statistical 24:2177 2190. Gill MB and Bredt DS (2011) An emerging role for TARPs in neuropsychiatric dis- analyses. Leslie Nguyen, Minerva Batugo, and Brian Scott performed orders. Neuropsychopharmacology 36:362–363. the bioanalytical studies. The authors also thank the following Gray JA and McNaughton N (2003) The Neuropsychology of Anxiety: An enquiry into individuals associated with NeuroAdjuvants, Inc. for performing the the function of the septo-hippocampal system, Oxford University Press, Oxford, UK. anticonvulsant experiments: H. Steve White, University of Utah Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ (1981) Improved patch-

(study oversight and consultation, study reporting); Cameron S. clamp techniques for high-resolution current recording from cells and cell-free at ASPET Journals on September 27, 2021 – Metcalf, NeuroAdjuvants, Inc. (6 Hz, MES, corneal kindling, amyg- membrane patches. Pflugers Arch 391:85 100. Hanada T, Hashizume Y, Tokuhara N, Takenaka O, Kohmura N, Ogasawara A, dala kindling, study design and management, data analysis and Hatakeyama S, Ohgoh M, Ueno M, and Nishizawa Y (2011) Perampanel: a novel, reporting); Misty D. Smith (study review/quality control, tissue orally active, noncompetitive AMPA-receptor antagonist that reduces seizure ac- – collection); Timothy Pruess, University of Utah (i.v. Metrazol test); tivity in rodent models of epilepsy. Epilepsia 52:1331 1340. Hashimoto K, Fukaya M, Qiao X, Sakimura K, Watanabe M, and Kano M (1999) Fabiola Vanegas, University of Utah (novel object and open field Impairment of AMPA receptor function in cerebellar granule cells of ataxic mutant assays); Jenny Huff, University of Utah (amygdala kindling); Carlos mouse stargazer. J Neurosci 19:6027–6036. H. Rueda, University of Utah (surgical amygdala electrode implanta- Howe JR (2015) Modulation of non-NMDA receptor gating by auxiliary subunits. J Physiol 593:61–72. tion). Staff members at BioDuro, LLC (Beijing, China) performed the Jackson AC and Nicoll RA (2011) The expanding social network of ionotropic gluta- pharmacokinetic studies in rats and mice. mate receptors: TARPs and other transmembrane auxiliary subunits. Neuron 70: 178–199. Jeggo R, Zhao FY, and Spanswick D (2014) Electrophysiological techniques for Authorship Contributions studying synaptic activity in vivo. Curr Protocols Pharmacol 11:11.1–11.11, 17. Klugbauer N, Dai S, Specht V, Lacinová L, Marais E, Bohn G, and Hofmann F (2000) Participated in research design: Maher, Ameriks, Savall, Liu, A family of gamma-like calcium channel subunits. FEBS Lett 470:189–197. Dugovic, Wickenden, Carruthers, Lovenberg. Ko D, Yang H, Williams B, Xing D, and Laurenza A (2015) Perampanel in the Conducted experiments: Maher, Wu, Ravula, Liu, Lord, Wyatt, treatment of partial seizures: Time to onset and duration of most common adverse events from pooled Phase III and extension studies. Epilepsy Behav 48:45–52. Matta, Dugovic, Yun. Kromann H, Krikstolaityte S, Andersen AJ, Andersen K, Krogsgaard-Larsen P, Contributed new reagents or analytic tools: Ravula. Jaroszewski JW, Egebjerg J, and Strømgaard K (2002) Solid-phase synthesis of Performed data analysis: Maher, Wu, Liu, Lord, Wyatt, Matta, polyamine toxin analogues: potent and selective antagonists of Ca21-permeable AMPA receptors. J Med Chem 45:5745–5754. Dugovic, Ver Donck, Steckler. Langlois X, te Riele P, Wintmolders C, Leysen JE, and Jurzak M (2001) Use of the Wrote or contributed to the writing of the manuscript: Maher, beta-imager for rapid ex vivo autoradiography exemplified with central nervous Ameriks, Liu, Lord, Wyatt, Matta, Dugovic, Ver Donck, Steckler. system penetrating neurokinin 3 antagonists. J Pharmacol Exp Ther 299:712–717. Lazzaro JT, Paternain AV, Lerma J, Chenard BL, Ewing FE, Huang J, Welch WM, Ganong AH, and Menniti FS (2002) Functional characterization of CP-465,022, a References selective, noncompetitive AMPA receptor antagonist. Neuropharmacology 42: – Atcha Z, Chen WS, Ong AB, Wong FK, Neo A, Browne ER, Witherington J, 143 153. and Pemberton DJ (2009) Cognitive enhancing effects of ghrelin receptor agonists. Lein ES, Hawrylycz MJ, Ao N, Ayres M, Bensinger A, Bernard A, Boe AF, Boguski Psychopharmacology (Berl) 206:415–427. MS, Brockway KS, and Byrnes EJ, et al. (2007) Genome-wide atlas of gene ex- – Barton ME, Klein BD, Wolf HH, and White HS (2001) Pharmacological character- pression in the adult mouse brain. Nature 445:168 176. ization of the 6 Hz psychomotor seizure model of partial epilepsy. Epilepsy Res 47: Letts VA, Felix R, Biddlecome GH, Arikkath J, Mahaffey CL, Valenzuela A, Bartlett 217–227. FS, 2nd, Mori Y, Campbell KP, and Frankel WN (1998) The mouse stargazer gene Bleakman D, Ballyk BA, Schoepp DD, Palmer AJ, Bath CP, Sharpe EF, Woolley ML, encodes a neuronal Ca21-channel gamma subunit. Nat Genet 19:340–347. Bufton HR, Kamboj RK, and Tarnawa I, et al. (1996) Activity of 2,3-benzodiazepines Lindén AM, Yu H, Zarrinmayeh H, Wheeler WJ, and Skolnick P (2001) Binding of an at native rat and recombinant human glutamate receptors in vitro: stereospecificity AMPA receptor potentiator ([3H]LY395153) to native and recombinant AMPA and selectivity profiles. Neuropharmacology 35:1689–1702. receptors. Neuropharmacology 40:1010–1018. Brewer GJ (1997) Isolation and culture of adult rat hippocampal neurons. J Neurosci Manns JR, Hopkins RO, Reed JM, Kitchener EG, and Squire LR (2003) Recognition Methods 71:143–155. memory and the human hippocampus. Neuron 37:171–180. Burgess DL, Davis CF, Gefrides LA, and Noebels JL (1999) Identification of three Mao JB and Robinson JK (1998) Microinjection of GABA-A agonist muscimol into the novel Ca(21) channel gamma subunit genes reveals molecular diversification by dorsal but not the ventral hippocampus impairs non-mnemonic measures of tandem and chromosome duplication. Genome Res 9:1204–1213. delayed non-matching-to-position performance in rats. Brain Res 784:139–147. 414 Maher et al.

Matagne A and Klitgaard H (1998) Validation of corneally kindled mice: a sensitive Schwenk J, Harmel N, Brechet A, Zolles G, Berkefeld H, Müller CS, Bildl W, screening model for partial epilepsy in man. Epilepsy Res 31:59–71. Baehrens D, Hüber B, and Kulik A, et al. (2012) High-resolution proteomics un- McNamara JO (1995) Analyses of the molecular basis of kindling development. ravel architecture and molecular diversity of native AMPA receptor complexes. Psychiatry Clin Neurosci 49:S175–S178. Neuron 74:621–633. Menniti FS, Chenard BL, Collins MB, Ducat MF, Elliott ML, Ewing FE, Huang JI, Shelley C, Farrant M, and Cull-Candy SG (2012) TARP-associated AMPA receptors Kelly KA, Lazzaro JT, and Pagnozzi MJ, et al. (2000) Characterization of the display an increased maximum channel conductance and multiple kinetically binding site for a novel class of noncompetitive alpha-amino-3-hydroxy-5-methyl-4- distinct open states. J Physiol 590:5723–5738. isoxazolepropionic acid receptor antagonists. Mol Pharmacol 58:1310–1317. Shi Y, Lu W, Milstein AD, and Nicoll RA (2009) The stoichiometry of AMPA receptors Morris RG, Garrud P, Rawlins JN, and O’Keefe J (1982) Place navigation impaired in and TARPs varies by neuronal cell type. Neuron 62:633–640. rats with hippocampal lesions. Nature 297:681–683. Smith JW, Gastambide F, Gilmour G, Dix S, Foss J, Lloyd K, Malik N, National Research Council (2011) Guide for the Care and Use of Laboratory Animals, and Tricklebank M (2011) A comparison of the effects of ketamine and phencycli- 8th ed, The National Academies Press, Washington, DC. dine with other antagonists of the NMDA receptor in rodent assays of attention Orloff MJ, Williams HL, and Pfeiffer CC (1949) Timed intravenous infusion of and working memory. Psychopharmacology (Berl) 217:255–269. metrazol and strychnine for testing anticonvulsant drugs. Proc Soc Exp Biol Med Steckler T, Drinkenburg WH, Sahgal A, and Aggleton JP (1998a) Recognition 70:254–257. memory in rats–II. Neuroanatomical substrates. Prog Neurobiol 54:313–332. Perucca P and Gilliam FG (2012) Adverse effects of antiepileptic drugs. Lancet Steckler T, Sahgal A, Aggleton JP, and Drinkenburg WH (1998b) Recognition Neurol 11:792–802. memory in rats–III. Neurochemical substrates. Prog Neurobiol 54:333–348. Priel A, Kolleker A, Ayalon G, Gillor M, Osten P, and Stern-Bach Y (2005) Stargazin Stephens DN and Cole BJ (1996) AMPA antagonists differ from NMDA antagonists reduces desensitization and slows deactivation of the AMPA-type glutamate re- in their effects on operant DRL and delayed matching to position tasks. Psycho- ceptors. J Neurosci 25:2682–2686. pharmacology (Berl) 126:249–259. Racine RJ (1972) Modification of seizure activity by electrical stimulation. II. Motor Straub C and Tomita S (2012) The regulation of glutamate receptor trafficking and seizure. Electroencephalogr Clin Neurophysiol 32:281–294. function by TARPs and other transmembrane auxiliary subunits. Curr Opin Riedel G, Micheau J, Lam AG, Roloff EL, Martin SJ, Bridge H, de Hoz L, Poeschel Neurobiol 22:488–495. B, McCulloch J, and Morris RG (1999) Reversible neural inactivation reveals Strømgaard K and Mellor I (2004) AMPA receptor ligands: synthetic and pharma- hippocampal participation in several memory processes. Nat Neurosci 2: cological studies of polyamines and polyamine toxins. Med Res Rev 24:589–620. 898–905. Sun Y, Olson R, Horning M, Armstrong N, Mayer M, and Gouaux E (2002) Mecha- Downloaded from Robert A and Howe JR (2003) How AMPA receptor desensitization depends on re- nism of glutamate receptor desensitization. Nature 417:245–253. ceptor occupancy. J Neurosci 23:847–858. Tomita S, Adesnik H, Sekiguchi M, Zhang W, Wada K, Howe JR, Nicoll RA, Robinson JK and Mao JB (1997) Differential effects on delayed non-matching-to- and Bredt DS (2005) Stargazin modulates AMPA receptor gating and trafficking by position in rats of microinjections of muscarinic receptor antagonist scopolamine or distinct domains. Nature 435:1052–1058. NMDA receptor antagonist MK-801 into the dorsal or ventral extent of the hip- Tomita S, Chen L, Kawasaki Y, Petralia RS, Wenthold RJ, Nicoll RA, and Bredt DS pocampus. Brain Res 765:51–60. (2003) Functional studies and distribution define a family of transmembrane Rogawski MA (2011) Revisiting AMPA receptors as an antiepileptic drug target. AMPA receptor regulatory proteins. J Cell Biol 161:805–816. Epilepsy Curr 11:56–63. Turetsky D, Garringer E, and Patneau DK (2005) Stargazin modulates native AMPA Rouach N, Byrd K, Petralia RS, Elias GM, Adesnik H, Tomita S, Karimzadegan S, receptor functional properties by two distinct mechanisms. J Neurosci 25: jpet.aspetjournals.org Kealey C, Bredt DS, and Nicoll RA (2005) TARP gamma-8 controls hippocampal 7438–7448. AMPA receptor number, distribution and synaptic plasticity. Nat Neurosci 8: UniProt Consortium (2015) UniProt: a hub for protein information. Nucleic Acids Res 1525–1533. 43:D204–D212. Rowley NM and White HS (2010) Comparative anticonvulsant efficacy in the corneal White HS, Brown SD, Woodhead JH, Skeen GA, and Wolf HH (1997) Topiramate kindled mouse model of partial epilepsy: Correlation with other seizure and epi- enhances GABA-mediated chloride flux and GABA-evoked chloride currents in lepsy models. Epilepsy Res 92:163–169. murine brain neurons and increases seizure threshold. Epilepsy Res 28:167–179. Sanderson DJ and Bannerman DM (2012) The role of habituation in hippocampus- Willmore CB, LaVecchia KL, and Wiley JL (2001) NMDA antagonists produce site- dependent spatial working memory tasks: evidence from GluA1 AMPA receptor selective impairment of accuracy in a delayed nonmatch-to-sample task in rats. subunit knockout mice. Hippocampus 22:981–994. Neuropharmacology 41:916–927.

Sanderson DJ, Good MA, Skelton K, Sprengel R, Seeburg PH, Rawlins JN, Winters BD and Dunnett SB (2004) Selective lesioning of the cholinergic septo- at ASPET Journals on September 27, 2021 and Bannerman DM (2009) Enhanced long-term and impaired short-term spatial hippocampal pathway does not disrupt spatial short-term memory: a comparison memory in GluA1 AMPA receptor subunit knockout mice: evidence for a dual- with the effects of fimbria-fornix lesions. Behav Neurosci 118:546–562. process memory model. Learn Mem 16:379–386. Zhang W, Devi SP, Tomita S, and Howe JR (2014) Auxiliary proteins promote modal Schobel SA, Chaudhury NH, Khan UA, Paniagua B, Styner MA, Asllani I, Inbar BP, gating of AMPA- and kainate-type glutamate receptors. Eur J Neurosci 39: Corcoran CM, Lieberman JA, and Moore H, et al. (2013) Imaging patients with 1138–1147. psychosis and a mouse model establishes a spreading pattern of hippocampal dysfunction and implicates glutamate as a driver. Neuron 78:81–93. Schober DA, Gill MB, Yu H, Gernert DL, Jeffries MW, Ornstein PL, Kato AS, Felder Address correspondence to: Dr. Michael P. Maher, Janssen Research and CC, and Bredt DS (2011) Transmembrane AMPA receptor regulatory proteins and Development, LLC, Neuroscience Therapeutic Area, 3210 Merryfield Row, San cornichon-2 allosterically regulate AMPA receptor antagonists and potentiators. J Diego, CA 92121. E-mail: [email protected] Biol Chem 286:13134–13142. JPET #231712

Discovery and characterization of AMPA receptor modulators

selective for TARP-8

Michael P. Maher, Nyantsz Wu, Suchitra Ravula, Michael K. Ameriks, Brad M. Savall, Changlu Liu,

Brian Lord, Ryan M. Wyatt, Jose Matta, Christine Dugovic, Sujin Yun, Luc Ver Donck, Thomas Steckler,

Alan D. Wickenden, Nicholas I. Carruthers, Timothy W. Lovenberg

Journal of Pharmacology and Experimental Therapeutics

Supplemental Materials

Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

Supplemental Tables

Supplemental Table 1. Primers, cloning sites, and vectors used in generating mammalian expression vectors for the wild-type genes used in this study. Cloning sites (highlighted in shaded letters) were introduced into primers to facilitate the cloning process. Genes Forward primer (5’ to 3’) Reverse primer (5’ to 3’) Cloning sites Vector Human ATGTCAGAATTCATGCAGCACATTTTT TCGTCAGCGGCCGCGCAGCTTCGGGGG EcoR1/Not1 pCIneo GCCTTCTTCTGCAC CTCTGTGAGTTGCGACA GluA1o Monkey AGTCTAGGATCCGCCACCATGCAGCAC CTGTCAGCGGCCGCTTACAATCCCGTG BamH1/Not1 pcDNA4/TO ATTTTTGCCTTCTTCTGCACC GCTCCCAAGGGCATCCCTGAAC GluA1o Dog AGTCATGGATCCGCCACCATGCAGCAC ATGTCTGCGGCCGCTTACAATCCCGTG BamH1/Not1 pcDNA4/TO ATTTTTGCCTTCTTCTGCACC GCTCCCAAGGGCATCC GluA1o Mouse ATCTCAAAGCTTGCCACCATGCCGTAC GTCTCAGCGGCCGCTTACAATCCTGTG HindIII/Not1 pcDNA4/TO ATCTTTGCCTTTTTCTGCAC GCTCCCAAGGGCATCC GluA1o Rat GluA1o TCGTCAAAGCTTGCCACCATGCCGTAC ACGTCTGCGGCCGCTTACAATCCTGTG HindIII/Not1 pcDNA4/TO ATCTTTGCCTTTTTCTGCACCGGTT GCTCCCAAGGGCATC Human ATGTCAGAATTCATGCAGCACATTTTT TCGTCAGCGGCCGCGCAGCTTCGGGGG EcoR1/Not1 pcDNA4/TO GCCTTCTTCTGCAC CTCTGTGAGTTGCGACA GluA1i Human AGTCATAAGCTTGCCACCATGCAAAAG ATGTCAGGCCGCCTAAATTTTAACACT HindIII/Not1 pcDNA4/TO ATTATGCATATTTCTGTCCT TTCGATGCCATATAC GluA2o Human ATGTCAGGTACCGCCACCATGGCCAGG ATGTCTGCGGCCGCACTAGATCTTAAC BamH1/Not1 pcDNA4/TO CAGAAGAAAATGGGGCAAA ACTCTCTGTTCCATAC GluA3o Human ATGTCTAAGCTTGCCACCATGAGGATT AGTCTCGCGGCCGCTCTAAATTTTAAT HindIII/Not1 pcDNA4/TO ATTTCCAGACAGATTGTCTTGTTAT ACTTTCGGTTCCATATACGT GluA4o Human AGTCGTGGATCCGCCACCATGGGGCTG TCTACTGCGGCCGCTTATACGGGGGTG BamH1/Not1 pcDNA3.1(+) TTTGATCGAGGTGTTC GTCCGGCGGTTGGCTGT CACNG2 Human ATGTCAGAATTCATGAGGATGTGTGAC ATGTCAGCGGCCGCTCAGACGGGCGTG EcoR1/Not1 pcDNA3.1/ AGAGGTATC GTGCGCCTGTTGGCCGGATT CACNG3 hygro(+) Human ATGTCTGGATCCGCCACCATGGTGCGA CTGTCAGCGGCCGCTCACACAGGGGTC BamH1/Not1 pcDNA3.1(+) TGCGACCGCGGGCTGCAG GTCCGTCGGTTCAGCATGC CACNG4 Human GTCATCGGATCCGCCACCATGAGTCAC CTGTACGCGGCCGCTCAGCAGGGCGAG BamH1/Not1 pcDNA3.1(+) TGCAGCAGCCGCGCCCTGA GTGGAGATGTGCAGGTG CACNG7 Human ATGTCAGAATTCCATGGAGTCGCTGAA GTCATCGCGGCCGCCTACACAGGCGTG EcoR1/Not1 pcDNA3.1(+) GCGCTGGAACGAAGA GTTTTCCTGTTGAGCGTGTT CACNG8 Mouse AGTCTAGAATTCCAACAGCAGCAACAA AGTCTAGCGGCCGCCTACACGGGCGTG EcoR1/Not1 pcDNA4/TO CAGCAACAGCAACAAGCCACCATGGAG GTTTTCCTGTTGAGC CACNG8 TCATTGAAACGCTGGAATGA Rat AGTCTAGAATTCCAACAGCAGCAACAA CTGTCAGCGGCCGCCTACACGGGCGTG EcoR1/Not1 pcDNA3.1 CAGCAACAGCAACAAGCCACCATGGAA GTTTTCCTGTTGAGCGTG CACNG8 TCATTGAAACGCTGGAATGAAG Monkey CTGTACGAATTCGCCACCATGGAGTCG GTCTACGCGGCCGCCTACACAGGCGTG EcoR1/Not1 pcDNA4/TO CTGAAGCGCTGGAACGA GTTTTCCTGTTGAGCGTGTTG CACNG8 Dog CTGTCAGAATTCGCCACCATGGAGTCG CTGTCAGCGGCCGCCTACACTGGAGTA EcoR1/Not1 pcDNA3.1 CTGAAGCGCTGGAACGA GTTTTTCTGTTGAGA CACNG8 Human GTCTACGGATCCGCCACCATGGCGTTC CTGTCAGCGGCCGCTTAGAAACTCACC BamH1/Not1 pcDNA3.1(+) ACCTTCGCCGCGTTCTGCT AACGTATAAACCATACT CNIH2

2 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

Supplemental Table 2. Primers, cloning sites, and vectors used to generate expression vectors for fusions of GluA1o and TARP-8 used in this study. Shaded sequences in primers are restriction site introduced to facilitate the cloning process. Primers and templates for PCR to generate GluA1-FLOP fusion protein expression constructs 5’ end PCR 3’ end PCR Full length PCR Forward Reverse Template Forward Reverse Template Forward Reverse Template primer primer primer primer primer primer Human P1 P2 Human P3 P4 Human P1 P4 5’end + 3’ GluA1o- GluA1o CACNG8 end CACNG (codon 8 optimized)* Mouse P5 P6 Mouse P7 P8 Mouse P5 P8 5’end + 3’ GluA1o- GluA1o CACNG8 end CACNG 8 Rat P9 P10 Rat GluA1o P11 P12 Rat P9 P12 5’end + 3’ GluA1o- CACNG8 end CACNG 8

Primer sequences (5’ to 3’)

P1 ATGTCAGAATTCATGCAGCACATTTTTGCCTTCTTCTGCAC P2 CTGCTGCTGCTGCTGCAGTCCTGTTGCTCCCAGAGGCATCCCGCTTGAATGGCTCATACATGGAATCGATTGCATGGACTTGGGGAA GTC P3 AGCAACAGGACTGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGGAGTTCGCAACTATGGAGAGCCTGAAGCGGTGGAACGAGGAAC P4 ACGTCAGCGGCCGCTCAGACGGGTGTGGTTTTTCGGTTCAGTGTA P5 ATCTCAAAGCTTGCCACCATGCCGTACATCTTTGCCTTTTTCTGCAC P6 TTGTTGCTGTTGCTGTTGTTGCTGCTGTTGGAATTCCAATCCTGTGGCTCCCAAGGGCATCCCT P7 CAGCAGCAACAACAGCAACAGCAACAAGCCACCATGGAGTCATTGAAACGCTGGAATGAAG P8 AGTCTAGCGGCCGCCTACACGGGCGTGGTTTTCCTGTTGAGC P9 TCGTCAAAGCTTGCCACCATGCCGTACATCTTTGCCTTTTTCTGCACCGGTT P10 GGCTTGTTGCTGTTGCTGTTGTTGCTGCTGTTGGAATTCCAATCCTGTGGCTCCCAAGGGCATCC P11 AGCAGCAACAACAGCAACAGCAACAAGCCACCATGGAATCATTGAAACGCTGGAATGAAGAG P12 CTGTCAGCGGCCGCCTACACGGGCGTGGTTTTCCTGTTGAGCGTG

3 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

Supplemental Table 3. Coding sequences and predicted amino acid sequences for fusion proteins comprising human, mouse, and rat gluA1o and CACNG8. Construct DNA Sequence Amino acid sequence ATGCAGCACATTTTTGCCTTCTTCTGCACCGGTTTCCTAGGCGCGGTAGTAGGTGCCAATTTCCCCAACAATATCCAGATCGGGGGATTATTTCCAAAC MQHIFAFFCTGFLGAVVGANFPNNIQIGGLFPN Human CAGCAGTCACAGGAACATGCTGCTTTTAGATTTGCTTTGTCGCAACTCACAGAGCCCCCGAAGCTGCTCCCCCAGATTGATATTGTGAACATCAGCGAC QQSQEHAAFRFALSQLTEPPKLLPQIDIVNISD AGCTTTGAGATGACCTATAGATTCTGTTCCCAGTTCTCCAAAGGAGTCTATGCCATCTTTGGGTTTTATGAACGTAGGACTGTCAACATGCTGACCTCC SFEMTYRFCSQFSKGVYAIFGFYERRTVNMLTS GluA1o- TTTTGTGGGGCCCTCCACGTCTGCTTCATTACGCCGAGCTTTCCCGTTGATACATCCAATCAGTTTGTCCTTCAGCTGCGCCCTGAACTGCAGGATGCC FCGALHVCFITPSFPVDTSNQFVLQLRPELQDA CACNG8 CTCATCAGCATCATTGACCATTACAAGTGGCAGAAATTTGTCTACATTTATGATGCCGACCGGGGCTTATCCGTCCTGCAGAAAGTCCTGGATACAGCT LISIIDHYKWQKFVYIYDADRGLSVLQKVLDTA GCTGAGAAGAACTGGCAGGTGACAGCAGTCAACATTTTGACAACCACAGAGGAGGGATACCGGATGCTCTTTCAGGACCTGGAGAAGAAAAAGGAGCGG AEKNWQVTAVNILTTTEEGYRMLFQDLEKKKER CTGGTGGTGGTGGACTGTGAATCAGAACGCCTCAATGCTATCTTGGGCCAGATTATAAAGCTAGAGAAGAATGGCATCGGCTACCACTACATTCTTGCA LVVVDCESERLNAILGQIIKLEKNGIGYHYILA AATCTGGGCTTCATGGACATTGACTTAAACAAATTCAAGGAGAGTGGCGCCAATGTGACAGGTTTCCAGCTGGTGAACTACACAGACACTATTCCGGCC NLGFMDIDLNKFKESGANVTGFQLVNYTDTIPA AAGATCATGCAGCAGTGGAAGAATAGTGATGCTCGAGACCACACACGGGTGGACTGGAAGAGACCCAAGTACACCTCTGCGCTCACCTACGATGGGGTG KIMQQWKNSDARDHTRVDWKRPKYTSALTYDGV AAGGTGATGGCTGAGGCTTTCCAGAGCCTGCGGAGGCAGAGAATTGATATATCTCGCCGGGGGAATGCTGGGGATTGTCTGGCTAACCCAGCTGTTCCC KVMAEAFQSLRRQRIDISRRGNAGDCLANPAVP TGGGGCCAAGGGATCGACATCCAGAGAGCTCTGCAGCAGGTGCGATTTGAAGGTTTAACAGGAAACGTGCAGTTTAATGAGAAAGGACGCCGGACCAAC WGQGIDIQRALQQVRFEGLTGNVQFNEKGRRTN TACACGCTCCACGTGATTGAAATGAAACATGACGGCATCCGAAAGATTGGTTACTGGAATGAAGATGATAAGTTTGTCCCTGCAGCCACCGATGCCCAA YTLHVIEMKHDGIRKIGYWNEDDKFVPAATDAQ GCTGGGGGCGATAATTCAAGTGTTCAGAACAGAACATACATCGTCACAACAATCCTAGAAGATCCTTATGTGATGCTCAAGAAGAACGCCAATCAGTTT AGGDNSSVQNRTYIVTTILEDPYVMLKKNANQF GAGGGCAATGACCGTTACGAGGGCTACTGTGTAGAGCTGGCGGCAGAGATTGCCAAGCACGTGGGCTACTCCTACCGTCTGGAGATTGTCAGTGATGGA EGNDRYEGYCVELAAEIAKHVGYSYRLEIVSDG AAATACGGAGCCCGAGACCCTGACACGAAGGCCTGGAATGGCATGGTGGGAGAGCTGGTCTATGGAAGAGCAGATGTGGCTGTGGCTCCCTTAACTATC KYGARDPDTKAWNGMVGELVYGRADVAVAPLTI ACTTTGGTCCGGGAAGAAGTTATAGATTTCTCCAAACCATTTATGAGTTTGGGGATCTCCATCATGATTAAAAAACCACAGAAATCCAAGCCGGGTGTC TLVREEVIDFSKPFMSLGISIMIKKPQKSKPGV TTCTCCTTCCTTGATCCTTTGGCTTATGAGATTTGGATGTGCATTGTTTTTGCCTACATTGGAGTGAGTGTTGTCCTCTTCCTGGTCAGCCGCTTCAGT FSFLDPLAYEIWMCIVFAYIGVSVVLFLVSRFS CCCTATGAATGGCACAGTGAAGAGTTTGAGGAAGGACGGGACCAGACAACCAGTGACCAGTCCAATGAGTTTGGGATATTCAACAGTTTGTGGTTCTCC PYEWHSEEFEEGRDQTTSDQSNEFGIFNSLWFS CTGGGAGCCTTCATGCAGCAAGGATGTGACATTTCTCCCAGGTCCCTGTCTGGTCGCATCGTTGGTGGCGTCTGGTGGTTCTTCACCTTAATCATCATC LGAFMQQGCDISPRSLSGRIVGGVWWFFTLIII TCCTCATATACAGCCAATCTGGCCGCCTTCCTGACCGTGGAGAGGATGGTGTCTCCCATTGAGAGTGCAGAGGACCTAGCGAAGCAGACAGAAATTGCC SSYTANLAAFLTVERMVSPIESAEDLAKQTEIA TACGGGACGCTGGAAGCAGGATCCACTAAGGAGTTCTTCAGGAGGTCTAAAATTGCTGTGTTTGAGAAGATGTGGACATACATGAAGTCAGCAGAGCCA YGTLEAGSTKEFFRRSKIAVFEKMWTYMKSAEP TCAGTTTTTGTGCGGACCACAGAGGAGGGGATGATTCGAGTGAGGAAATCCAAAGGCAAATATGCCTACCTCCTGGAGTCCACCATGAATGAGTACATT SVFVRTTEEGMIRVRKSKGKYAYLLESTMNEYI GAGCAGCGGAAACCCTGTGACACCATGAAGGTGGGAGGTAACTTGGATTCCAAAGGCTATGGCATTGCAACACCCAAGGGGTCTGCCCTGAGAAATCCA EQRKPCDTMKVGGNLDSKGYGIATPKGSALRNP GTAAACCTGGCAGTGTTAAAACTGAACGAGCAGGGGCTTTTGGACAAATTGAAAAACAAATGGTGGTACGACAAGGGCGAGTGCGGCAGCGGGGGAGGT VNLAVLKLNEQGLLDKLKNKWWYDKGECGSGGG GATTCCAAGGACAAGACAAGCGCTCTGAGCCTCAGCAATGTGGCAGGCGTGTTCTACATCCTGATCGGAGGACTTGGACTAGCCATGCTGGTTGCCTTA DSKDKTSALSLSNVAGVFYILIGGLGLAMLVAL ATCGAGTTCTGCTACAAATCCCGTAGTGAATCCAAGCGGATGAAGGGTTTTTGTTTGATCCCACAGCAATCCATCAACGAAGCCATACGGACATCGACC IEFCYKSRSESKRMKGFCLIPQQSINEAIRTST CTCCCCCGCAACAGCGGGGCAGGAGCCAGCAGCGGCGGCAGTGGAGAGAATGGTCGGGTGGTCAGCCATGACTTCCCCAAGTCCATGCAatcgatTCCA LPRNSGAGASSGGSGENGRVVSHDFPKSMQSIP TGTATGAGCCATTCAAGCGGGATGCCTCTGGGAGCAACAGGACTGcagcagcagcagcagcagcagcagcagcaggagttcgcaactATGGAGAGCCTG CMSHSSGMPLGATGLQQQQQQQQQQEFATMESL AAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATC KRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTI GCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGA AISTDYWLYTRALICNTTNLTAGGDDGTPHRGG GGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAA GGASEKKDPGGLTHSGLWRICCLEGLKRGVCVK ATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCA INHFPEDTDYDHDSAEYLLRVVRASSIFPILSA ATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCA ILLLLGGVCVAASRVYKSKRNIILGAGILFVAA GGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTAT GLSNIIGVIVYISANAGEPGPKRDEEKKNHYSY GGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCAT GWSFYFGGLSFILAEVIGVLAVNIYIERSREAH TGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTC CQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRF AGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGT RYRRRSRSSSRSSEPSPSRDASPGGPGGPGFAS ACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGC TDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVG GCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACC AFGGAAGGAGGGGGGGGGAGAERDRGGASGFLT CTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCC LHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSA CCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA PAPGTLAKEAAASNTNTLNRKTTPV ATGCCGTACATCTTTGCCTTTTTCTGCACCGGTTTTCTAGGTGCGGTTGTGGGTGCCAATTTCCCCAACAATATCCAGATAGGGGGATTATTTCCAAAC MPYIFAFFCTGFLGAVVGANFPNNIQIGGLFPN Mouse CAACAATCACAGGAACATGCGGCTTTTAGGTTTGCTTTGTCACAACTCACGGAGCCCCCCAAGCTGCTTCCCCAGATCGATATTGTGAACATCAGCGAC QQSQEHAAFRFALSQLTEPPKLLPQIDIVNISD AGCTTTGAGATGACTTACCGATTCTGTTCCCAGTTCTCCAAAGGAGTGTACGCCATCTTTGGATTTTATGAACGAAGGACTGTCAACATGCTGACCTCC SFEMTYRFCSQFSKGVYAIFGFYERRTVNMLTS GluA1o- TTCTGTGGGGCCCTCCATGTGTGCTTCATCACTCCAAGTTTTCCCGTTGACACATCCAATCAGTTTGTCCTTCAGCTGCGCCCGGAACTACAGGAAGCT FCGALHVCFITPSFPVDTSNQFVLQLRPELQEA CACNG8 CTCATTAGCATTATCGACCATTACAAGTGGCAGACTTTTGTCTACATTTATGATGCTGACCGGGGCCTGTCAGTCCTGCAGAGAGTCTTGGATACAGCC LISIIDHYKWQTFVYIYDADRGLSVLQRVLDTA GCCGAGAAGAACTGGCAGGTGACGGCTGTCAACATTCTAACAACCACGGAGGAAGGATACCGGATGCTCTTTCAGGACCTGGAGAAGAAAAAGGAGAGG AEKNWQVTAVNILTTTEEGYRMLFQDLEKKKER CTGGTGGTGGTGGACTGTGAATCAGAACGCCTCAACGCCATCCTGGGCCAGATTGTGAAGCTAGAAAAGAACGGCATCGGGTACCACTACATCCTCGCC LVVVDCESERLNAILGQIVKLEKNGIGYHYILA AACCTGGGCTTCATGGACATTGACTTAAATAAGTTCAAGGAGAGTGGAGCCAATGTGACAGGTTTCCAACTGGTGAACTACACAGACACGATCCCAGCC NLGFMDIDLNKFKESGANVTGFQLVNYTDTIPA AGAATCATGCAGCAGTGGAGGACAAGTGACGCTCGAGACCACACACGGGTGGACTGGAAGAGACCCAAGTACACCTCTGCGCTCACCTACGATGGGGTG RIMQQWRTSDARDHTRVDWKRPKYTSALTYDGV AAGGTGATGGCTGAGGCTTTCCAGAGCCTGCGGAGGCAGAGAATTGATATATCTCGCCGGGGGAATGCTGGGGATTGTCTGGCTAACCCAGCTGTTCCC KVMAEAFQSLRRQRIDISRRGNAGDCLANPAVP TGGGGCCAAGGGATCGACATCCAGAGAGCTCTGCAGCAGGTGCGATTTGAAGGTTTAACAGGAAACGTGCAGTTTAATGAGAAAGGACGCCGGACCAAC WGQGIDIQRALQQVRFEGLTGNVQFNEKGRRTN TACACGCTCCACGTGATTGAAATGAAACATGACGGCATCCGAAAGATTGGTTACTGGAATGAAGATGATAAGTTTGTCCCTGCAGCCACCGATGCCCAA YTLHVIEMKHDGIRKIGYWNEDDKFVPAATDAQ GCTGGGGGCGATAATTCAAGTGTTCAGAACAGAACATACATCGTCACAACAATCCTAGAAGATCCTTATGTGATGCTCAAGAAGAACGCCAATCAGTTT AGGDNSSVQNRTYIVTTILEDPYVMLKKNANQF GAGGGCAATGACCGTTACGAGGGCTACTGTGTAGAGCTGGCGGCAGAGATTGCCAAGCACGTGGGCTACTCCTACCGTCTGGAGATTGTCAGTGATGGA EGNDRYEGYCVELAAEIAKHVGYSYRLEIVSDG AAATACGGAGCCCGAGACCCTGACACGAAGGCCTGGAATGGCATGGTGGGAGAGCTGGTCTATGGAAGAGCAGATGTGGCTGTGGCTCCCTTAACTATC KYGARDPDTKAWNGMVGELVYGRADVAVAPLTI ACTTTGGTCCGGGAAGAAGTTATAGATTTCTCCAAACCATTTATGAGTTTGGGGATCTCCATCATGATTAAAAAACCACAGAAATCCAAGCCGGGTGTC TLVREEVIDFSKPFMSLGISIMIKKPQKSKPGV TTCTCCTTCCTTGATCCTTTGGCTTATGAGATTTGGATGTGCATTGTTTTTGCCTACATTGGAGTGAGTGTTGTCCTCTTCCTGGTCAGCCGCTTCAGT FSFLDPLAYEIWMCIVFAYIGVSVVLFLVSRFS CCCTATGAATGGCACAGTGAAGAGTTTGAGGAAGGACGGGACCAGACAACCAGTGACCAGTCCAATGAGTTTGGGATATTCAACAGTTTGTGGTTCTCC PYEWHSEEFEEGRDQTTSDQSNEFGIFNSLWFS CTGGGAGCCTTCATGCAGCAAGGATGTGACATTTCTCCCAGGTCCCTGTCTGGTCGCATCGTTGGTGGCGTCTGGTGGTTCTTCACCTTAATCATCATC LGAFMQQGCDISPRSLSGRIVGGVWWFFTLIII TCCTCATATACAGCCAATCTGGCCGCCTTCCTGACCGTGGAGAGGATGGTGTCTCCCATTGAGAGTGCAGAGGACCTAGCGAAGCAGACAGAAATTGCC SSYTANLAAFLTVERMVSPIESAEDLAKQTEIA TACGGGACGCTGGAAGCAGGATCCACTAAGGAGTTCTTCAGGAGGTCTAAAATTGCTGTGTTTGAGAAGATGTGGACATACATGAAGTCAGCAGAGCCA YGTLEAGSTKEFFRRSKIAVFEKMWTYMKSAEP TCAGTTTTTGTGCGGACCACAGAGGAGGGGATGATTCGAGTGAGGAAATCCAAAGGCAAATATGCCTACCTCCTGGAGTCCACCATGAATGAGTACATT SVFVRTTEEGMIRVRKSKGKYAYLLESTMNEYI GAGCAGCGGAAACCCTGTGACACCATGAAGGTGGGAGGTAACTTGGATTCCAAAGGCTATGGCATTGCAACACCCAAGGGGTCTGCCCTGAGAAATCCA EQRKPCDTMKVGGNLDSKGYGIATPKGSALRNP GTAAACCTGGCAGTGTTAAAACTGAACGAGCAGGGGCTTTTGGACAAATTGAAAAACAAATGGTGGTACGACAAGGGCGAGTGCGGCAGCGGGGGAGGT VNLAVLKLNEQGLLDKLKNKWWYDKGECGSGGG GATTCCAAGGACAAGACAAGCGCTCTGAGCCTCAGCAATGTGGCAGGCGTGTTCTACATCCTGATCGGAGGACTTGGACTAGCCATGCTGGTTGCCTTA DSKDKTSALSLSNVAGVFYILIGGLGLAMLVAL ATCGAGTTCTGCTACAAATCCCGTAGTGAATCCAAGCGGATGAAGGGTTTTTGTTTGATCCCACAGCAATCCATCAACGAAGCCATACGGACATCGACC IEFCYKSRSESKRMKGFCLIPQQSINEAIRTST CTCCCCCGCAACAGCGGGGCAGGAGCCAGCGGAGGAAGTGGCAGTGGAGAGAATGGCAGAGTGGTCAGCCAGGACTTCCCCAAGTCCATGCAATCCATT LPRNSGAGASGGSGSGENGRVVSQDFPKSMQSI CCCTGCATGAGCCACAGTTCAGGGATGCCCTTGGGAGCCACAGGATTGGAATTCCAACAGCAGCAACAACAGCAACAGCAACAAGCCACCATGGAGTCA PCMSHSSGMPLGATGLEFQQQQQQQQQQATMES TTGAAACGCTGGAATGAAGAGAGGGGTTTGTGGTGTGAAAAGGGCGTTCAGGTACTACTGACCACCATCGGCGCCTTCTCGGCTTTTGGCCTCATGACC LKRWNEERGLWCEKGVQVLLTTIGAFSAFGLMT ATCGCCATCAGCACTGACTACTGGCTCTACACAAGAGCTCTCATCTGCAACACCACCAACCTCACAGCAGGTGATGACGGACCACCCCATCGTGGGGGC IAISTDYWLYTRALICNTTNLTAGDDGPPHRGG AGTGGCTCCTCCGAGAAGAAGGACCCTGGGGGCCTCACACATTCAGGCCTCTGGCGGATATGCTGCCTGGAAGGGTTGAAAAGAGGTGTCTGCGTGAAG SGSSEKKDPGGLTHSGLWRICCLEGLKRGVCVK ATCAACCACTTCCCGGAGGACACGGACTACGACCACGACAGCGCGGAGTACCTGCTCCGAGTAGTCCGGGCTTCCAGCATCTTTCCTATCCTGAGCGCC INHFPEDTDYDHDSAEYLLRVVRASSIFPILSA ATCCTGCTGCTGCTCGGGGGCGTGTGCGTAGCTGCCTCCCGCGTCTACAAGTCCAAAAGGAACATCATCCTGGGCGCAGGGATCCTGTTCGTGGCAGCA ILLLLGGVCVAASRVYKSKRNIILGAGILFVAA GGCTTGAGCAACATCATCGGGGTGATTGTGTACATATCGGCCAACGCCGGCGAGCCTGGCCCCAAGAGGGACGAGGAGAAGAAAAACCACTACTCGTAC GLSNIIGVIVYISANAGEPGPKRDEEKKNHYSY GGCTGGTCCTTCTACTTCGGCGGGCTGTCCTTCATCCTGGCTGAGGTGATCGGAGTACTGGCCGTCAACATCTACATCGAGCGCAGCCGCGAGGCACAC GWSFYFGGLSFILAEVIGVLAVNIYIERSREAH TGCCAATCACGCTCGGACCTGCTCAAGGCCGGCGGCGGCGCGGGCGGCAGTGGCGGGAGCGGCCCCTCGGCCATCCTCCGTCTGCCCAGTTACCGCTTC CQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRF CGCTACCGCCGCCGCTCCCGCTCCAGCTCCCGAGGCTCCAGCGAGGCGTCGCCATCCCGGGATGCGTCTCCCGGCGGCCCCGGGGGCCCGGGCTTCGCC RYRRRSRSSSRGSSEASPSRDASPGGPGGPGFA TCCACGGACATCTCCATGTACACGCTCAGCCGCGACCCGTCCAAGGGCAGCGTGGCTGCGGGGCTGGCGAGCGCCGGTGGCGGCGGCAGCGGTGCCGGC STDISMYTLSRDPSKGSVAAGLASAGGGGSGAG GTGGGTGCCTACGGCGGGGCGGCCGGGGCGGCGGGGGGCGGCGGGGCGGGCTCGGAGCGGGACCGCGGGAGCTCGGCGGGTTTTCTCACGCTGCACAAC VGAYGGAAGAAGGGGAGSERDRGSSAGFLTLHN GCCTTCCCCAAGGAAGCGGCGTCCGGCGTCACGGTCACAGTCACCGGACCGCCCGCTGCACCCGCGCCCGCGCCCGCGCCGCCCGCTCCTGCAGCGCCC AFPKEAASGVTVTVTGPPAAPAPAPAPPAPAAP GCGCCCGGGACCCTGTCCAAAGAGGCCGCGGCGTCCAACACCAACACGCTCAACAGGAAAACCACGCCCGTGTAG APGTLSKEAAASNTNTLNRKTTPV

4 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

ATGCCGTACATCTTTGCCTTTTTCTGCACCGGTTTTCTAGGTGCGGTTGTGGGTGCCAATTTCCCCAACAATATCCAGATAGGGGGATTATTTCCAAAC MPYIFAFFCTGFLGAVVGANFPNNIQIGGLFPN Rat CAACAATCACAGGAACATGCGGCTTTTAGGTTTGCTTTGTCACAACTCACGGAGCCCCCCAAGCTGCTTCCCCAGATCGATATTGTGAACATCAGCGAC QQSQEHAAFRFALSQLTEPPKLLPQIDIVNISD AGCTTTGAGATGACTTACCGATTCTGTTCCCAGTTCTCCAAAGGAGTGTACGCCATCTTTGGATTTTATGAACGAAGGACTGTCAACATGCTGACCTCC SFEMTYRFCSQFSKGVYAIFGFYERRTVNMLTS GluA1o- TTCTGTGGGGCCCTCCATGTGTGCTTCATCACTCCAAGTTTTCCCGTTGACACATCCAATCAGTTTGTCCTTCAGCTGCGCCCGGAACTACAGGAAGCT FCGALHVCFITPSFPVDTSNQFVLQLRPELQEA CACNG8 CTCATTAGCATTATCGACCATTACAAGTGGCAGACTTTTGTCTACATTTATGATGCTGACCGGGGCCTGTCAGTCCTGCAGAGAGTCTTGGATACAGCC LISIIDHYKWQTFVYIYDADRGLSVLQRVLDTA GCCGAGAAGAACTGGCAGGTGACGGCTGTCAACATTCTAACAACCACGGAGGAAGGATACCGGATGCTCTTTCAGGACCTGGAGAAGAAAAAGGAGAGG AEKNWQVTAVNILTTTEEGYRMLFQDLEKKKER CTGGTGGTGGTGGACTGTGAATCAGAACGCCTCAACGCCATCCTGGGCCAGATTGTGAAGCTAGAAAAGAACGGCATCGGGTACCACTACATCCTCGCC LVVVDCESERLNAILGQIVKLEKNGIGYHYILA AACCTGGGCTTCATGGACATTGACTTAAATAAGTTCAAGGAGAGTGGAGCCAATGTGACAGGTTTCCAACTGGTGAACTACACAGACACGATCCCAGCC NLGFMDIDLNKFKESGANVTGFQLVNYTDTIPA AGAATCATGCAGCAGTGGAGGACAAGTGACTCTCGAGACCACACACGGGTGGACTGGAAGAGACCCAAGTACACCTCTGCGCTCACCTACGATGGGGTG RIMQQWRTSDSRDHTRVDWKRPKYTSALTYDGV AAGGTGATGGCTGAGGCTTTCCAGAGCCTGCGGAGGCAGAGAATTGATATATCTCGCCGGGGGAATGCTGGGGATTGTCTGGCTAACCCAGCTGTTCCC KVMAEAFQSLRRQRIDISRRGNAGDCLANPAVP TGGGGCCAAGGGATCGACATCCAGAGAGCTCTGCAGCAGGTGCGATTTGAAGGTTTAACAGGAAACGTGCAGTTTAATGAGAAAGGACGCCGGACCAAC WGQGIDIQRALQQVRFEGLTGNVQFNEKGRRTN TACACGCTCCACGTGATTGAAATGAAACATGACGGCATCCGAAAGATTGGTTACTGGAATGAAGATGATAAGTTTGTCCCTGCAGCCACCGATGCCCAA YTLHVIEMKHDGIRKIGYWNEDDKFVPAATDAQ GCTGGGGGCGATAATTCAAGTGTTCAGAACAGAACATACATCGTCACAACAATCCTAGAAGATCCTTATGTGATGCTCAAGAAGAACGCCAATCAGTTT AGGDNSSVQNRTYIVTTILEDPYVMLKKNANQF GAGGGCAATGACCGTTACGAGGGCTACTGTGTAGAGCTGGCGGCAGAGATTGCCAAGCACGTGGGCTACTCCTACCGTCTGGAGATTGTCAGTGATGGA EGNDRYEGYCVELAAEIAKHVGYSYRLEIVSDG AAATACGGAGCCCGAGACCCTGACACGAAGGCCTGGAATGGCATGGTGGGAGAGCTGGTCTATGGAAGAGCAGATGTGGCTGTGGCTCCCTTAACTATC KYGARDPDTKAWNGMVGELVYGRADVAVAPLTI ACTTTGGTCCGGGAAGAAGTTATAGATTTCTCCAAACCATTTATGAGTTTGGGGATCTCCATCATGATTAAAAAACCACAGAAATCCAAGCCGGGTGTC TLVREEVIDFSKPFMSLGISIMIKKPQKSKPGV TTCTCCTTCCTTGATCCTTTGGCTTATGAGATTTGGATGTGCATTGTTTTTGCCTACATTGGAGTGAGTGTTGTCCTCTTCCTGGTCAGCCGCTTCAGT FSFLDPLAYEIWMCIVFAYIGVSVVLFLVSRFS CCCTATGAATGGCACAGTGAAGAGTTTGAGGAAGGACGGGACCAGACAACCAGTGACCAGTCCAATGAGTTTGGGATATTCAACAGTTTGTGGTTCTCC PYEWHSEEFEEGRDQTTSDQSNEFGIFNSLWFS CTGGGAGCCTTCATGCAGCAAGGATGTGACATTTCTCCCAGGTCCCTGTCTGGTCGCATCGTTGGTGGCGTCTGGTGGTTCTTCACCTTAATCATCATC LGAFMQQGCDISPRSLSGRIVGGVWWFFTLIII TCCTCATATACAGCCAATCTGGCCGCCTTCCTGACCGTGGAGAGGATGGTGTCTCCCATTGAGAGTGCAGAGGACCTAGCGAAGCAGACAGAAATTGCC SSYTANLAAFLTVERMVSPIESAEDLAKQTEIA TACGGGACGCTGGAAGCAGGATCCACTAAGGAGTTCTTCAGGAGGTCTAAAATTGCTGTGTTTGAGAAGATGTGGACATACATGAAGTCAGCAGAGCCA YGTLEAGSTKEFFRRSKIAVFEKMWTYMKSAEP TCAGTTTTTGTGCGGACCACAGAGGAGGGGATGATTCGAGTGAGGAAATCCAAAGGCAAATATGCCTACCTCCTGGAGTCCACCATGAATGAGTACATT SVFVRTTEEGMIRVRKSKGKYAYLLESTMNEYI GAGCAGCGGAAACCCTGTGACACCATGAAGGTGGGAGGTAACTTGGATTCCAAAGGCTATGGCATTGCAACACCCAAGGGGTCTGCCCTGAGAAATCCA EQRKPCDTMKVGGNLDSKGYGIATPKGSALRNP GTAAACCTGGCAGTGTTAAAACTGAACGAGCAGGGGCTTTTGGACAAATTGAAAAACAAATGGTGGTACGACAAGGGCGAGTGCGGCAGCGGGGGAGGT VNLAVLKLNEQGLLDKLKNKWWYDKGECGSGGG GATTCCAAGGACAAGACAAGCGCTCTGAGCCTCAGCAATGTGGCAGGCGTGTTCTACATCCTGATCGGAGGACTTGGACTAGCCATGCTGGTTGCCTTA DSKDKTSALSLSNVAGVFYILIGGLGLAMLVAL ATCGAGTTCTGCTACAAATCCCGTAGTGAATCCAAGCGGATGAAGGGTTTTTGTTTGATCCCACAGCAATCCATCAACGAAGCCATACGGACATCGACC IEFCYKSRSESKRMKGFCLIPQQSINEAIRTST CTCCCCCGCAACAGCGGGGCAGGAGCCAGCGGAGGAGGTGGCAGTGGAGAGAATGGCAGAGTGGTCAGCCAGGACTTCCCCAAGTCCATGCAATCCATT LPRNSGAGASGGGGSGENGRVVSQDFPKSMQSI CCCTGCATGAGCCACAGTTCAGGGATGCCCTTGGGAGCCACAGGATTGGAATTCCAACAGCAGCAACAACAGCAACAGCAACAAGCCACCATGGAATCA PCMSHSSGMPLGATGLEFQQQQQQQQQQATMES TTGAAACGCTGGAATGAAGAGAGGGGTTTGTGGTGCGAAAAGGGCGTTCAGGTACTACTGACCACCATAGGCGCCTTTGCAGCTTTTGGCCTCATGACC LKRWNEERGLWCEKGVQVLLTTIGAFAAFGLMT ATCGCCATCAGCACTGACTACTGGCTCTACACAAGAGCTCTCATCTGCAACACCACCAACCTCACAGCAGGTGATGATGGACCACCCCATCGTGGGGGC IAISTDYWLYTRALICNTTNLTAGDDGPPHRGG AGTGGCTCCTCAGAGAAGAAGGACCCTGGGGGCCTCACACATTCAGGCCTCTGGCGGATATGCTGCCTGGAAGGGTTGAAAAGAGGTGTCTGCGTGAAG SGSSEKKDPGGLTHSGLWRICCLEGLKRGVCVK ATCAACCACTTCCCGGAGGACACGGACTACGACCACGACAGCGCGGAGTACCTGCTCCGAGTAGTCCGGGCCTCCAGCATCTTTCCTATCCTGAGCGCC INHFPEDTDYDHDSAEYLLRVVRASSIFPILSA ATCCTGCTGCTGCTCGGGGGCGTGTGCGTAGCTGCCTCTCGCGTCTACAAATCCAAAAGGAACATCATCCTGGGCGCAGGGATCCTGTTCGTGGCAGCA ILLLLGGVCVAASRVYKSKRNIILGAGILFVAA GGCCTGAGCAACATCATCGGGGTGATCGTGTACATATCGGCAAACGCGGGCGAGCCAGGCCCCAAGAGGGACGAGGAGAAGAAAAACCACTATTCGTAT GLSNIIGVIVYISANAGEPGPKRDEEKKNHYSY GGCTGGTCCTTCTACTTCGGCGGGCTGTCATTCATCCTGGCCGAGGTGATCGGCGTGCTAGCCGTCAACATCTACATCGAGCGCAGCCGCGAGGCACAC GWSFYFGGLSFILAEVIGVLAVNIYIERSREAH TGCCAATCACGCTCGGACCTACTCAAGGCCGGCGGCGGCGCGGGCGGCAGTGGCGGGAGCGGCCCCTCGGCCATCCTCCGTCTGCCCAGTTACCGCTTC CQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRF CGCTACCGCCGCCGCTCCCGCTCCAGCTCCCGAGGCTCCAGCGAGGCCTCGCCATCGCGGGATGCGTCTCCCGGCGGCCCCGGGGGCCCGGGCTTCGCC RYRRRSRSSSRGSSEASPSRDASPGGPGGPGFA TCCACGGACATCTCCATGTACACGCTCAGTCGCGACCCGTCCAAGGGCAGCGTGGCTGCGGGGCTGGCGAGCGCCGGGGGTGGAGGCGGCGGTGCCGGC STDISMYTLSRDPSKGSVAAGLASAGGGGGGAG GTGGGTGCCTACGGCGGGGCGGCCGGGGCAGCGGGGGGCGGCGGGACGGGCTCGGAGCGGGACCGAGGGAGCTCAGCGGGCTTCCTCACGCTGCACAAC VGAYGGAAGAAGGGGTGSERDRGSSAGFLTLHN GCCTTCCCCAAGGAGGCGGCGTCCGGCGTCACGGTCACGGTCACCGGACCGCCCGCTGCGCCGGCGCCCGCGCCGCCCGCTCCTGCAGCGCCCGCGCCC AFPKEAASGVTVTVTGPPAAPAPAPPAPAAPAP GGGACGCTGTCCAAAGAGGCCGCCGCGTCCAACACCAACACGCTCAACAGGAAAACCACGCCCGTGTAG GTLSKEAAASNTNTLNRKTTPV

5 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

Supplemental Table 4. Location of splice points used to generate chimeras of human TARPs 8, 4, and 2. The numbering is based upon the amino acid sequence for each protein.

   region start end start end start end full length 1 425 1 327 1 323 NT 1 18 1 7 1 7 TM1 19 40 8 29 8 29 EX1 41 129 30 108 30 105 TM2 130 152 109 131 106 128 IN1 153 157 132 136 129 133 TM3 158 181 137 160 134 157 EX2 182 205 161 183 158 178 TM4 206 229 184 208 179 203 CT 230 425 209 327 204 323

6 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

Supplemental Table 5. Primers and templates used for PCR reactions to generate TARP chimeric protein expression constructs. The chimeras in this table are labelled using a shorthand notation (C1-C21 and D1); see Supplemental Table 6 for the translation for the shorthand notation to the nine-digit chimera name. Chimeras C1-C13 were codon-optimized and synthesized in a pD2610v12 vector by DNA 2.0 (http://dna20.com, Menlo Park CA). Chimeras C20, C21, and D1 were codon-optimized and synthesized in-house.

5’ end 3’ end Full length PCR Chi Forward Reverse Template Forward Reverse Template Forward Reverse Template mera primer primer primer primer primer primer C14 P21 P43 CACNG4 P44 P24 CACNG8 P21 P24 5’end + 3’ end C15 P21 P45 CACNG4 P46 P24 CACNG8 P21 P24 5’end + 3’ end C16 P21 P47 CACNG4 P48 P24 CACNG8 P21 P24 5’end + 3’ end C17 P21 P49 CACNG4 P50 P24 CACNG8 P21 P24 5’end + 3’ end C18 P21 P51 CACNG4 P52 P24 CACNG8 P21 P24 5’end + 3’ end C19 P21 P53 CACNG4 P54 P24 CACNG8 P21 P24 5’end + 3’ end

Primer sequences (5’ to 3’) P21 ACGTCAGAATTCGCCACCATGCAATCGATTCCATGTATGAGC P22 AACCTCAACTCTTTATTTTTCTCAATATAGATATTGACGGCCAG P23 CTGGCCGTCAATATCTATATTGAGAAAAATAAAGAGTTGAGGTT P24 CTGTATCGGCCGCTCACACAGGGGTCGTCCGTCGGTTCAGCA P25 GAATTCGCCACCATGGTGCGATGCGACCGCGGGCTGCAGAT P26 GCAATGGGCTTCTCGACTGCGCTCAATGTAAATGTTTACAGCCA P27 TGGCTGTAAACATTTACATTGAGCGCAGTCGAGAAGCCCATTGC P28 ACGTCAGCGGCCGCTCAGACGGGTGTGGTTTTTCGGTTCAGT P29 ACAGAGCTCCAAAGTAAAAAGACCAGCCATAGGAGTAGTGGT P30 ACCACTACTCCTATGGCTGGTCTTTTTACTTTGGAGCTCTGT P31 GTCACCTGTGTTGCTGGAAATATAGACGATCACGCCAATGAT P32 ATCATTGGCGTGATCGTCTATATTTCCAGCAACACAGGTGAC P33 AAGAGGATGCCGGCACTGAGGACGATATTTCTTTTGGACTTGTACACCCGGCTTGCT G P34 CAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCGTCCTCAGTGCCGGCATCCTCT T P35 GAGGACGATGTTGTTCTTGCGGCTGTACACCCGGCTTGCTGCCACGCA P36 TGCGTGGCAGCAAGCCGGGTGTACAGCCGCAAGAACAACATCGTCCTC P37 TGGTGCTGAGGATGGGGAAGACGGAGCTAGCCCGGACCACTCGCA P38 TGCGAGTGGTCCGGGCTAGCTCCGTCTTCCCCATCCTCAGCACCA P39 GAGTACAGCCAGTAGTCGGTGGAAATAGCGATTGTCATCAGCCCAA P40 TTGGGCTGATGACAATCGCTATTTCCACCGACTACTGGCTGTACTC 7 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

P41 GTGGTCAGCAGCATCTGCAGTCCTTTTTCGCACCACAGCCCTC P42 GAGGGCTGTGGTGCGAAAAAGGACTGCAGATGCTGCTGACCAC P43 CCCACTGTGGTCAGCAGGACCTGCAGCCCGCGGTCGC P44 GCGACCGCGGGCTGCAGGTCCTGCTGACCACAGTGGG P45 GTATACAGCCAGTAGTCAGTGGAGATGGCGATGGCCATGAGCGAGA P46 TCTCGCTCATGGCCATCGCCATCTCCACTGACTACTGGCTGTATAC P47 CCAGAATGATATTTCTTTTGGACTTGTAGATCCTGCCAGCACCGATGC P48 GCATCGGTGCTGGCAGGATCTACAAGTCCAAAAGAAATATCATTCTGG P49 GCCGGCTCCCAGAATGATGTTGTTCTTGCGGCTGTAGA P50 TCTACAGCCGCAAGAACAACATCATTCTGGGAGCCGGC P51 GTCCGGGCTCCCCTGCATTGGCGCTAATGTAGACGATGATACCGATG P52 CATCGGTATCATCGTCTACATTAGCGCCAATGCAGGGGAGCCCGGAC P53 GATAAAAGACAGTCCCCCAAAGTAGAAAGACCAGCCGTAGTTGTAATGGTTCT P54 AGAACCATTACAACTACGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATC

8 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

Supplemental Table 6. The complete DNA coding regions and the protein sequences of the CACNG8/CACNG4 chimeras. construct DNA Sequence protein sequence C1 ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGAGTCCAAGTCCTGCTCACTACCGTGGGGGCTTTCGCCGCCTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CTCATGACCATTGCCATCTCCACCGACTACTGGCTGTACACCCGGGCCCTGATCTGCAATACCACTAACCTCACCGCGGGGGGGGACGACGGAACCCCG LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP (888888884) CACCGCGGCGGCGGAGGCGCTAGCGAAAAGAAGGACCCAGGCGGCTTGACCCATTCGGGCCTGTGGAGGATCTGCTGTCTGGAGGGGCTGAAGCGGGGT HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG GTCTGCGTGAAGATTAACCACTTCCCTGAGGACACTGACTACGATCACGACTCCGCCGAGTATCTTCTGCGCGTCGTGCGGGCCTCCTCGATTTTCCCC VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATCCTGTCCGCCATCCTGCTGCTCCTGGGTGGAGTGTGCGTGGCAGCGTCACGCGTGTACAAGTCCAAAAGGAACATCATCCTTGGTGCTGGAATCCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTTGTGGCCGCCGGGCTGTCCAACATTATTGGAGTGATTGTGTACATCAGCGCAAACGCCGGAGAGCCCGGCCCGAAGCGCGATGAAGAAAAGAAGAAC FVAAGLSNIIGVIVYISANAGEPGPKRDEEKKN CATTACAGCTACGGCTGGTCCTTCTACTTCGGGGGTCTGTCGTTCATCTTGGCGGAAGTCATCGGAGTGCTTGCCGTGAATATCTACATCGAGAAGAAC HYSYGWSFYFGGLSFILAEVIGVLAVNIYIEKN AAGGAGCTGCGGTTCAAGACCAAGCGGGAGTTTCTGAAGGCCTCCAGCAGCTCCCCATATGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGCTCA KELRFKTKREFLKASSSSPYARMPSYRYRRRRS AGATCATCCTCCCGCTCGACCGAAGCGTCCCCGAGCCGGGACGTGTCGCCCATGGGACTCAAGATTACTGGAGCCATCCCTATGGGCGAACTCAGCATG RSSSRSTEASPSRDVSPMGLKITGAIPMGELSM TACACTCTGTCAAGAGAGCCACTCAAAGTGACCACTGCCGCCTCCTACTCGCCGGATCAGGAGGCATCCTTCCTGCAAGTCCACGATTTCTTCCAACAA YTLSREPLKVTTAASYSPDQEASFLQVHDFFQQ GACCTGAAGGAAGGATTCCACGTGTCTATGCTCAACCGACGCACTACCCCCGTGTAGTGA DLKEGFHVSMLNRRTTPV.. C2 ATGGTCAGATGTGATAGAGGATTGCAAATGCTGCTCACTACCGCCGGGGCTTTCGCCGCCTTTTCACTCATGGCGATTGCCATCGGTACCGACTACTGG MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYW CTGTACTCCTCGGCCCACATCTGCAATGGCACTAACCTCACCATGGACGACGGACCCCCGCCACGCCGGGCTCGGGGCGACTTGACCCATTCGGGCCTG LYSSAHICNGTNLTMDDGPPPRRARGDLTHSGL (444444448) TGGAGGGTCTGCTGTATTGAGGGGATCTACAAGGGTCACTGCTTCCGGATTAACCACTTCCCTGAGGACAACGACTACGATCACGACTCCTCCGAGTAT WRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEY CTTCTGCGCATCGTGCGGGCCTCCTCGGTGTTCCCCATCCTGTCCACCATCCTGCTGCTCCTGGGTGGACTGTGCATTGGGGCGGGCCGCATCTACTCC LLRIVRASSVFPILSTILLLLGGLCIGAGRIYS CGCAAAAACAACATCGTGCTTTCGGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATTGGAATCATTGTGTACATCAGCAGCAACACCGGA RKNNIVLSAGILFVAAGLSNIIGIIVYISSNTG GATCCCTCAGACAAGCGCGATGAAGATAAGAAGAACCATTACAACTACGGCTGGTCCTTCTACTTCGGGGCACTGTCGTTCATCGTGGCGGAAACCGTC DPSDKRDEDKKNHYNYGWSFYFGALSFIVAETV GGAGTGCTTGCCGTGAATATCTACATCGAGAGGTCCAGAGAGGCACACTGCCAGTCCCGGAGCGACCTCCTGAAGGCCGGTGGAGGGGCCGGAGGCTCC GVLAVNIYIERSREAHCQSRSDLLKAGGGAGGS GGAGGCAGCGGCCCATCGGCCATTCTGCGCCTGCCGAGCTACAGGTTTCGGTATCGGCGGCGCTCAAGATCATCCTCCCGCTCGTCCGAACCCTCCCCG GGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSP AGCCGGGACGCCTCGCCCGGAGGACCGGGTGGCCCGGGATTCGCCTCCACCGACATCAGCATGTACACTCTGTCAAGAGATCCAAGCAAAGGAAGCGTC SRDASPGGPGGPGFASTDISMYTLSRDPSKGSV GCCGCGGGCCTCGCTGGCGCCGGAGGGGGCGGGGGTGGAGCAGTGGGAGCCTTCGGGGGCGCCGCTGGAGGAGCCGGGGGCGGTGGCGGAGGAGGCGGC AAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGG GGAGCCGGTGCCGAGCGCGATCGGGGGGGCGCATCCGGTTTCCTGACGCTGCACAACGCGTTCCCGAAGGAAGCGGGCGGAGGAGTGACCGTGACTGTG GAGAERDRGGASGFLTLHNAFPKEAGGGVTVTV ACCGGACCGCCTGCCCCGCCTGCGCCTGCGCCCCCTGCTCCTTCCGCGCCTGCCCCCGGAACTCTCGCCAAGGAGGCCGCCGCCTCTAACACCAACACA TGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNT CTCAACCGAAAGACTACCCCCGTGTAGTGA LNRKTTPV.. C3 ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGAGTCCAAGTCCTGCTCACTACCGTGGGGGCTTTCGCCGCCTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CTCATGACCATTGCCATCTCCACCGACTACTGGCTGTACACCCGGGCCCTGATCTGCAATACCACTAACCTCACCGCGGGGGGGGACGACGGAACCCCG LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP (888888844) CACCGCGGCGGCGGAGGCGCTAGCGAAAAGAAGGACCCAGGCGGCTTGACCCATTCGGGCCTGTGGAGGATCTGCTGTCTGGAGGGGCTGAAGCGGGGT HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG GTCTGCGTGAAGATTAACCACTTCCCTGAGGACACTGACTACGATCACGACTCCGCCGAGTATCTTCTGCGCGTCGTGCGGGCCTCCTCGATTTTCCCC VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATCCTGTCCGCCATCCTGCTGCTCCTGGGTGGAGTGTGCGTGGCAGCGTCACGCGTGTACAAGTCCAAAAGGAACATCATCCTTGGTGCTGGAATCCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTTGTGGCCGCCGGGCTGTCCAACATTATTGGAGTGATTGTGTACATCAGCGCAAACGCCGGAGAGCCCGGCCCGAAGCGCGATGAAGAAAAGAAGAAC FVAAGLSNIIGVIVYISANAGEPGPKRDEEKKN CATTACAGCTACGGCTGGTCCTCCTTCTACTTCGGGGCTCTGTCGTTCATCGTGGCGGAAACGGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAAG HYSYGWSSFYFGALSFIVAETVGVLAVNIYIEK AACAAGGAGCTGCGGTTCAAGACCAAGCGGGAGTTTCTGAAGGCCTCCAGCAGCTCCCCATATGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGC NKELRFKTKREFLKASSSSPYARMPSYRYRRRR TCAAGATCATCCTCCCGCTCGACCGAAGCGTCCCCGAGCCGGGACGTGTCGCCCATGGGACTCAAGATTACTGGAGCCATCCCTATGGGCGAACTCAGC SRSSSRSTEASPSRDVSPMGLKITGAIPMGELS ATGTACACTCTGTCAAGAGAGCCACTCAAAGTGACCACTGCCGCCTCCTACTCGCCGGATCAGGAGGCATCCTTCCTGCAAGTCCACGATTTCTTCCAA MYTLSREPLKVTTAASYSPDQEASFLQVHDFFQ CAAGACCTGAAGGAAGGATTCCACGTGTCTATGCTCAACCGACGCACTACCCCCGTGTAGTGA QDLKEGFHVSMLNRRTTPV.. C4 ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGAGTCCAAGTCCTGCTCACTACCGTGGGGGCTTTCGCCGCCTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CTCATGACCATTGCCATCTCCACCGACTACTGGCTGTACACCCGGGCCCTGATCTGCAATACCACTAACCTCACCGCGGGGGGGGACGACGGAACCCCG LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP (888888444) CACCGCGGCGGCGGAGGCGCTAGCGAAAAGAAGGACCCAGGCGGCTTGACCCATTCGGGCCTGTGGAGGATCTGCTGTCTGGAGGGGCTGAAGCGGGGT HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG GTCTGCGTGAAGATTAACCACTTCCCTGAGGACACTGACTACGATCACGACTCCGCCGAGTATCTTCTGCGCGTCGTGCGGGCCTCCTCGATTTTCCCC VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATCCTGTCCGCCATCCTGCTGCTCCTGGGTGGAGTGTGCGTGGCAGCGTCACGCGTGTACAAGTCCAAAAGGAACATCATCCTTGGTGCTGGAATCCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTTGTGGCCGCCGGGCTGTCCAACATTATTGGAGTGATTGTGTACATCAGCTCCAACACGGGAGATCCCAGCGACAAGCGCGATGAAGATAAGAAGAAC FVAAGLSNIIGVIVYISSNTGDPSDKRDEDKKN CATTACAACTACGGCTGGTCCTTCTACTTCGGGGCACTGTCGTTCATCGTGGCGGAAACCGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAAGAAC HYNYGWSFYFGALSFIVAETVGVLAVNIYIEKN AAGGAGCTGCGGTTCAAGACCAAGCGGGAGTTTCTGAAGGCCTCCAGCAGCTCCCCATATGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGCTCA KELRFKTKREFLKASSSSPYARMPSYRYRRRRS AGATCATCCTCCCGCTCGACCGAAGCGTCCCCGAGCCGGGACGTGTCGCCCATGGGACTCAAGATTACTGGAGCCATCCCTATGGGCGAACTCAGCATG RSSSRSTEASPSRDVSPMGLKITGAIPMGELSM TACACTCTGTCAAGAGAGCCACTCAAAGTGACCACTGCCGCCTCCTACTCGCCGGATCAGGAGGCATCCTTCCTGCAAGTCCACGATTTCTTCCAACAA YTLSREPLKVTTAASYSPDQEASFLQVHDFFQQ GACCTGAAGGAAGGATTCCACGTGTCTATGCTCAACCGACGCACTACCCCCGTGTAGTGA DLKEGFHVSMLNRRTTPV.. C5 ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGAGTCCAAGTCCTGCTCACTACCGTGGGGGCTTTCGCCGCCTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CTCATGACCATTGCCATCTCCACCGACTACTGGCTGTACACCCGGGCCCTGATCTGCAATACCACTAACCTCACCGCGGGGGGGGACGACGGAACCCCG LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP (888884444) CACCGCGGCGGCGGAGGCGCTAGCGAAAAGAAGGACCCAGGCGGCTTGACCCATTCGGGCCTGTGGAGGATCTGCTGTCTGGAGGGGCTGAAGCGGGGT HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG GTCTGCGTGAAGATTAACCACTTCCCTGAGGACACTGACTACGATCACGACTCCGCCGAGTATCTTCTGCGCGTCGTGCGGGCCTCCTCGATTTTCCCC VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATCCTGTCCGCCATCCTGCTGCTCCTGGGTGGAGTGTGCGTGGCAGCGTCACGCGTGTACAAGTCCAAAAGGAACATCGTGCTTTCCGCTGGAATCCTG ILSAILLLLGGVCVAASRVYKSKRNIVLSAGIL TTTGTGGCCGCCGGGCTGTCCAACATTATTGGAATCATTGTGTACATCAGCTCGAACACAGGAGATCCCTCCGACAAGCGCGATGAAGATAAGAAGAAC FVAAGLSNIIGIIVYISSNTGDPSDKRDEDKKN CATTACAACTACGGCTGGTCCTTCTACTTCGGGGCACTGTCGTTCATCGTGGCGGAAACCGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAAGAAC HYNYGWSFYFGALSFIVAETVGVLAVNIYIEKN AAGGAGCTGCGGTTCAAGACCAAGCGGGAGTTTCTGAAGGCCTCCAGCAGCTCCCCATATGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGCTCA KELRFKTKREFLKASSSSPYARMPSYRYRRRRS AGATCATCCTCCCGCTCGACCGAAGCGTCCCCGAGCCGGGACGTGTCGCCCATGGGACTCAAGATTACTGGAGCCATCCCTATGGGCGAACTCAGCATG RSSSRSTEASPSRDVSPMGLKITGAIPMGELSM TACACTCTGTCAAGAGAGCCACTCAAAGTGACCACTGCCGCCTCCTACTCGCCGGATCAGGAGGCATCCTTCCTGCAAGTCCACGATTTCTTCCAACAA YTLSREPLKVTTAASYSPDQEASFLQVHDFFQQ GACCTGAAGGAAGGATTCCACGTGTCTATGCTCAACCGACGCACTACCCCCGTGTAGTGA DLKEGFHVSMLNRRTTPV.. C6 ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGAGTCCAAGTCCTGCTCACTACCGTGGGGGCTTTCGCCGCCTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CTCATGACCATTGCCATCTCCACCGACTACTGGCTGTACACCCGGGCCCTGATCTGCAATACCACTAACCTCACCGCGGGGGGGGACGACGGAACCCCG LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP (888844444) CACCGCGGCGGCGGAGGCGCTAGCGAAAAGAAGGACCCAGGCGGCTTGACCCATTCGGGCCTGTGGAGGATCTGCTGTCTGGAGGGGCTGAAGCGGGGT HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG GTCTGCGTGAAGATTAACCACTTCCCTGAGGACACTGACTACGATCACGACTCCGCCGAGTATCTTCTGCGCGTCGTGCGGGCCTCCTCGATTTTCCCC VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATCCTGTCCGCCATCCTGCTGCTCCTGGGTGGAGTGTGCGTGGCAGCGTCACGCGTGTACTCCCGGAAAAACAACATCGTGCTTTCCGCTGGAATCCTG ILSAILLLLGGVCVAASRVYSRKNNIVLSAGIL TTTGTGGCCGCCGGGCTGTCCAACATTATTGGAATCATTGTGTACATCAGCTCGAACACAGGAGATCCCTCCGACAAGCGCGATGAAGATAAGAAGAAC FVAAGLSNIIGIIVYISSNTGDPSDKRDEDKKN CATTACAACTACGGCTGGTCCTTCTACTTCGGGGCTCTGTCGTTCATCGTGGCGGAAACGGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAAGAAC HYNYGWSFYFGALSFIVAETVGVLAVNIYIEKN AAGGAGCTGCGGTTCAAGACCAAGCGGGAGTTTCTGAAGGCCTCCAGCAGCTCCCCATATGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGCTCA KELRFKTKREFLKASSSSPYARMPSYRYRRRRS AGATCATCCTCCCGCTCGACCGAAGCGTCCCCGAGCCGGGACGTGTCGCCCATGGGACTCAAGATTACTGGAGCCATCCCTATGGGCGAACTCAGCATG RSSSRSTEASPSRDVSPMGLKITGAIPMGELSM TACACTCTGTCAAGAGAGCCACTCAAAGTGACCACTGCCGCCTCCTACTCGCCGGATCAGGAGGCATCCTTCCTGCAAGTCCACGATTTCTTCCAACAA YTLSREPLKVTTAASYSPDQEASFLQVHDFFQQ GACCTGAAGGAAGGATTCCACGTGTCTATGCTCAACCGACGCACTACCCCCGTGTAGTGA DLKEGFHVSMLNRRTTPV.. C7 ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGAGTCCAAGTCCTGCTCACTACCGTGGGGGCTTTCGCCGCCTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CTCATGACCATTGCCATCTCCACCGACTACTGGCTGTACACCCGGGCCCTGATCTGCAATACCACTAACCTCACCGCGGGGGGGGACGACGGAACCCCG LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP (888444444) CACCGCGGCGGCGGAGGCGCTAGCGAAAAGAAGGACCCAGGCGGCTTGACCCATTCGGGCCTGTGGAGGATCTGCTGTCTGGAGGGGCTGAAGCGGGGT HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG GTCTGCGTGAAGATTAACCACTTCCCTGAGGACACTGACTACGATCACGACTCCGCCGAGTATCTTCTGCGCGTCGTGCGGGCCTCCTCGGTGTTCCCC VCVKINHFPEDTDYDHDSAEYLLRVVRASSVFP ATCCTGTCCACCATCCTGCTGCTCCTGGGTGGACTGTGCATTGGCGCGGGTCGCATCTACTCCCGGAAAAACAACATCGTGCTTTCCGCTGGAATCCTG ILSTILLLLGGLCIGAGRIYSRKNNIVLSAGIL TTTGTGGCCGCCGGGCTGTCCAACATTATTGGAATCATTGTGTACATCAGCTCGAACACAGGAGATCCCTCCGACAAGCGCGATGAAGATAAGAAGAAC FVAAGLSNIIGIIVYISSNTGDPSDKRDEDKKN CATTACAACTACGGCTGGTCCTTCTACTTCGGGGCTCTGTCGTTCATCGTGGCGGAAACGGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAAGAAC HYNYGWSFYFGALSFIVAETVGVLAVNIYIEKN AAGGAGCTGCGGTTCAAGACCAAGCGGGAGTTTCTGAAGGCCTCCAGCAGCTCCCCATATGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGCTCA KELRFKTKREFLKASSSSPYARMPSYRYRRRRS AGATCATCCTCCCGCTCGACCGAAGCGTCCCCGAGCCGGGACGTGTCGCCCATGGGACTCAAGATTACTGGAGCCATCCCTATGGGCGAACTCAGCATG RSSSRSTEASPSRDVSPMGLKITGAIPMGELSM TACACTCTGTCAAGAGAGCCACTCAAAGTGACCACTGCCGCCTCCTACTCGCCGGATCAGGAGGCATCCTTCCTGCAAGTCCACGATTTCTTCCAACAA YTLSREPLKVTTAASYSPDQEASFLQVHDFFQQ GACCTGAAGGAAGGATTCCACGTGTCTATGCTCAACCGACGCACTACCCCCGTGTAGTGA DLKEGFHVSMLNRRTTPV..

9 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

C8 ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGAGTCCAAGTCCTGCTCACTACCGTGGGGGCTTTCGCCGCCTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CTCATGACCATTGCCATCTCCACCGACTACTGGCTGTACTCAAGCGCCCACATCTGCAATGGCACTAACCTCACCATGGACGACGGACCTCCGCCTCGC LMTIAISTDYWLYSSAHICNGTNLTMDDGPPPR (884444444) CGGGCTAGGGGCGACTTGACCCATTCGGGCCTGTGGAGGGTCTGCTGTATCGAGGGGATCTACAAGGGTCACTGCTTCCGCATTAACCACTTCCCTGAG RARGDLTHSGLWRVCCIEGIYKGHCFRINHFPE GACAACGACTACGATCACGACTCCTCCGAGTATCTTCTGCGCATTGTGCGGGCCTCCTCGGTGTTCCCCATCCTGTCCACCATCCTGCTGCTCCTGGGT DNDYDHDSSEYLLRIVRASSVFPILSTILLLLG GGACTGTGCATTGGCGCGGGTCGCATCTACTCCCGGAAAAACAACATCGTGCTTTCCGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATT GLCIGAGRIYSRKNNIVLSAGILFVAAGLSNII GGAATCATTGTGTACATCAGCTCGAACACAGGAGATCCCTCCGACAAGCGCGATGAAGATAAGAAGAACCATTACAACTACGGCTGGTCCTTCTACTTC GIIVYISSNTGDPSDKRDEDKKNHYNYGWSFYF GGGGCTCTGTCGTTCATCGTGGCGGAAACGGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAAGAACAAGGAGCTGCGGTTCAAGACCAAGCGGGAG GALSFIVAETVGVLAVNIYIEKNKELRFKTKRE TTTCTGAAGGCCTCCAGCAGCTCCCCATATGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGCTCAAGATCATCCTCCCGCTCGACCGAAGCGTCC FLKASSSSPYARMPSYRYRRRRSRSSSRSTEAS CCGAGCCGGGACGTGTCGCCCATGGGACTCAAGATTACTGGAGCCATCCCTATGGGCGAACTCAGCATGTACACTCTGTCAAGAGAGCCACTCAAAGTG PSRDVSPMGLKITGAIPMGELSMYTLSREPLKV ACCACTGCCGCCTCCTACTCGCCGGATCAGGAGGCATCCTTCCTGCAAGTCCACGATTTCTTCCAACAAGACCTGAAGGAAGGATTCCACGTGTCTATG TTAASYSPDQEASFLQVHDFFQQDLKEGFHVSM CTCAACCGACGCACTACCCCCGTGTAGTGA LNRRTTPV.. C9 ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGACTGCAAATGCTGCTCACTACCGCGGGGGCTTTCGCCGCCTTTTCC MESLKRWNEERGLWCEKGLQMLLTTAGAFAAFS CTCATGGCGATTGCCATCGGCACCGACTACTGGCTGTACAGCAGCGCCCACATCTGCAATGGCACTAACCTCACCATGGACGACGGACCTCCGCCGCGC LMAIAIGTDYWLYSSAHICNGTNLTMDDGPPPR (844444444) CGGGCTCGCGGCGACTTGACCCATTCGGGCCTGTGGAGGGTCTGCTGTATCGAGGGGATCTACAAGGGTCACTGCTTCCGGATTAACCACTTCCCTGAG RARGDLTHSGLWRVCCIEGIYKGHCFRINHFPE GACAACGACTACGATCACGACTCCTCCGAGTATCTTCTGCGCATTGTGCGGGCCTCCTCGGTGTTCCCCATCCTGTCCACCATCCTGCTGCTCCTGGGT DNDYDHDSSEYLLRIVRASSVFPILSTILLLLG GGACTGTGCATTGGCGCGGGTCGCATCTACTCCCGGAAAAACAACATCGTGCTTTCCGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATT GLCIGAGRIYSRKNNIVLSAGILFVAAGLSNII GGAATCATTGTGTACATCAGCTCGAACACAGGAGATCCCTCCGACAAGCGCGATGAAGATAAGAAGAACCATTACAACTACGGCTGGTCCTTCTACTTC GIIVYISSNTGDPSDKRDEDKKNHYNYGWSFYF GGGGCTCTGTCGTTCATCGTGGCGGAAACGGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAAGAACAAGGAGCTGCGGTTCAAGACCAAGCGGGAG GALSFIVAETVGVLAVNIYIEKNKELRFKTKRE TTTCTGAAGGCCTCCAGCAGCTCCCCATATGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGCTCAAGATCATCCTCCCGCTCGACCGAAGCGTCC FLKASSSSPYARMPSYRYRRRRSRSSSRSTEAS CCGAGCCGGGACGTGTCGCCCATGGGACTCAAGATTACTGGAGCCATCCCTATGGGCGAACTCAGCATGTACACTCTGTCAAGAGAGCCACTCAAAGTG PSRDVSPMGLKITGAIPMGELSMYTLSREPLKV ACCACTGCCGCCTCCTACTCGCCGGATCAGGAGGCATCCTTCCTGCAAGTCCACGATTTCTTCCAACAAGACCTGAAGGAAGGATTCCACGTGTCTATG TTAASYSPDQEASFLQVHDFFQQDLKEGFHVSM CTCAACCGACGCACTACCCCCGTGTAGTGA LNRRTTPV.. C10 ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGAGTCCAAGTCCTGCTCACTACCGTGGGGGCTTTCGCCGCCTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CTCATGACCATTGCCATCTCCACCGACTACTGGCTGTACTCCTCGGCCCACATCTGCAATGGCACTAACCTCACCATGGACGACGGACCCCCGCCACGC LMTIAISTDYWLYSSAHICNGTNLTMDDGPPPR (884888888) CGGGCTCGGGGCGACTTGACCCATTCGGGCCTGTGGAGGGTCTGCTGTATTGAGGGGATCTACAAGGGTCACTGCTTCCGGATTAACCACTTCCCTGAG RARGDLTHSGLWRVCCIEGIYKGHCFRINHFPE GACAACGACTACGATCACGACTCCTCCGAGTATCTTCTGCGCATCGTGCGGGCCTCCTCGATTTTCCCCATCCTGTCCGCCATCCTGCTGCTCCTGGGT DNDYDHDSSEYLLRIVRASSIFPILSAILLLLG GGAGTGTGCGTGGCAGCGTCACGCGTGTACAAGTCCAAAAGGAACATCATCCTTGGTGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATT GVCVAASRVYKSKRNIILGAGILFVAAGLSNII GGAGTGATTGTGTACATCAGCGCAAACGCCGGAGAGCCCGGCCCGAAGCGCGATGAAGAAAAGAAGAACCATTACAGCTACGGCTGGTCCTTCTACTTC GVIVYISANAGEPGPKRDEEKKNHYSYGWSFYF GGGGGCCTGTCGTTCATCCTGGCGGAAGTCATCGGAGTGCTTGCCGTGAATATCTACATCGAGAGGTCCAGAGAGGCACACTGCCAGTCCCGGAGCGAC GGLSFILAEVIGVLAVNIYIERSREAHCQSRSD CTCCTGAAGGCCGGTGGAGGGGCCGGAGGCTCCGGAGGCAGCGGCCCATCGGCCATTCTGCGCCTGCCGAGCTACAGGTTTCGGTATCGGCGGCGCTCA LLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRS AGATCATCCTCCCGCTCGTCCGAACCCTCCCCGAGCCGGGACGCCTCGCCCGGAGGACCGGGTGGCCCGGGATTCGCCTCCACCGACATCAGCATGTAC RSSSRSSEPSPSRDASPGGPGGPGFASTDISMY ACTCTGTCAAGAGATCCAAGCAAAGGAAGCGTCGCCGCGGGCCTCGCTGGCGCCGGAGGGGGCGGGGGTGGAGCAGTGGGAGCCTTCGGGGGCGCCGCT TLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAA GGAGGAGCCGGGGGCGGTGGCGGAGGAGGCGGCGGAGCCGGTGCCGAGCGCGATCGGGGGGGCGCATCCGGTTTCCTGACGCTGCACAACGCGTTCCCG GGAGGGGGGGGGAGAERDRGGASGFLTLHNAFP AAGGAAGCGGGCGGAGGAGTGACCGTGACTGTGACCGGACCGCCTGCCCCGCCTGCGCCTGCGCCCCCTGCTCCTTCCGCGCCTGCCCCCGGAACTCTC KEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTL GCCAAGGAGGCCGCCGCCTCTAACACCAACACACTCAACCGAAAGACTACCCCCGTGTAGTGA AKEAAASNTNTLNRKTTPV.. C11 ATGGTCAGATGTGATAGAGGATTGCAAATGCTGCTCACTACCGCCGGGGCTTTCGCCGCCTTTTCCCTCATGGCGATTGCCATCGGTACCGACTACTGG MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYW CTGTACACCCGGGCCCTGATCTGCAATACCACTAACCTCACCGCGGGGGGGGACGACGGAACCCCGCACCGCGGCGGCGGAGGCGCTAGCGAAAAGAAG LYTRALICNTTNLTAGGDDGTPHRGGGGASEKK (448444444) GACCCAGGCGGCTTGACCCATTCGGGCCTGTGGAGGATCTGCTGTCTGGAGGGGCTGAAGCGGGGTGTCTGCGTGAAGATTAACCACTTCCCTGAGGAC DPGGLTHSGLWRICCLEGLKRGVCVKINHFPED ACTGACTACGATCACGACTCCGCCGAGTATCTTCTGCGCGTCGTGCGGGCCTCCTCGGTGTTCCCCATCCTGTCCACCATCCTGCTGCTCCTGGGTGGA TDYDHDSAEYLLRVVRASSVFPILSTILLLLGG CTGTGCATTGGCGCGGGTCGCATCTACTCCCGGAAAAACAACATCGTGCTTTCCGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATTGGA LCIGAGRIYSRKNNIVLSAGILFVAAGLSNIIG ATCATTGTGTACATCAGCTCGAACACAGGAGATCCCTCCGACAAGCGCGATGAAGATAAGAAGAACCATTACAACTACGGCTGGTCCTTCTACTTCGGG IIVYISSNTGDPSDKRDEDKKNHYNYGWSFYFG GCTCTGTCGTTCATCGTGGCGGAAACGGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAAGAACAAGGAGCTGCGGTTCAAGACCAAGCGGGAGTTT ALSFIVAETVGVLAVNIYIEKNKELRFKTKREF CTGAAGGCCTCCAGCAGCTCCCCATATGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGCTCAAGATCATCCTCCCGCTCGACCGAAGCGTCCCCG LKASSSSPYARMPSYRYRRRRSRSSSRSTEASP AGCCGGGACGTGTCGCCCATGGGACTCAAGATTACTGGAGCCATCCCTATGGGCGAACTCAGCATGTACACTCTGTCAAGAGAGCCACTCAAAGTGACC SRDVSPMGLKITGAIPMGELSMYTLSREPLKVT ACTGCCGCCTCCTACTCGCCGGATCAGGAGGCATCCTTCCTGCAAGTCCACGATTTCTTCCAACAAGACCTGAAGGAAGGATTCCACGTGTCTATGCTC TAASYSPDQEASFLQVHDFFQQDLKEGFHVSML AACCGACGCACTACCCCCGTGTAGTGA NRRTTPV.. C12 ATGGTCAGATGTGATAGAGGATTGCAAATGCTGCTCACTACCGCCGGGGCTTTCGCCGCCTTTTCACTCATGGCGATTGCCATCGGTACCGACTACTGG MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYW CTGTACTCCTCGGCCCACATCTGCAATGGCACTAACCTCACCATGGACGACGGACCCCCGCCACGCCGGGCTCGGGGCGACTTGACCCATTCGGGCCTG LYSSAHICNGTNLTMDDGPPPRRARGDLTHSGL (444444484) TGGAGGGTCTGCTGTATTGAGGGGATCTACAAGGGTCACTGCTTCCGGATTAACCACTTCCCTGAGGACAACGACTACGATCACGACTCCTCCGAGTAT WRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEY CTTCTGCGCATCGTGCGGGCCTCCTCGGTGTTCCCCATCCTGTCCACCATCCTGCTGCTCCTGGGTGGACTGTGCATTGGGGCGGGCCGCATCTACTCC LLRIVRASSVFPILSTILLLLGGLCIGAGRIYS CGCAAAAACAACATCGTGCTTTCGGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATTGGAATCATTGTGTACATCAGCAGCAACACCGGA RKNNIVLSAGILFVAAGLSNIIGIIVYISSNTG GATCCCTCAGACAAGCGCGATGAAGATAAGAAGAACCATTACAACTACGGCTGGTTCTACTTCGGGGGCCTGTCGTTCATCCTGGCGGAAGTCATCGGA DPSDKRDEDKKNHYNYGWFYFGGLSFILAEVIG GTGCTTGCCGTGAATATCTACATCGAGAAGAACAAGGAGCTGCGGTTCAAGACGAAGCGGGAGTTTCTGAAGGCCTCCAGCAGCAGCCCATACGCCCGC VLAVNIYIEKNKELRFKTKREFLKASSSSPYAR ATGCCGAGCTACAGGTACCGGCGGCGGCGCTCAAGATCATCCTCCCGCTCGACTGAAGCATCCCCGAGCCGGGACGTGTCGCCCATGGGACTGAAGATT MPSYRYRRRRSRSSSRSTEASPSRDVSPMGLKI ACCGGAGCCATCCCTATGGGCGAACTGAGCATGTACACTCTGTCAAGAGAACCACTCAAAGTGACCACCGCCGCCTCCTACTCGCCGGATCAGGAAGCA TGAIPMGELSMYTLSREPLKVTTAASYSPDQEA TCCTTCCTGCAAGTCCACGACTTCTTCCAACAAGACCTGAAGGAAGGATTCCACGTGTCTATGCTCAACCGAAGGACTACCCCCGTGTAGTGA SFLQVHDFFQQDLKEGFHVSMLNRRTTPV.. C13 ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGAGTCCAAGTCCTGCTCACTACCGTGGGGGCTTTCGCCGCCTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CTCATGACCATTGCCATCTCCACCGACTACTGGCTGTACACCCGGGCCCTGATCTGCAATACCACTAACCTCACCGCGGGGGGGGACGACGGAACCCCG LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP (888888848) CACCGCGGCGGCGGAGGCGCTAGCGAAAAGAAGGACCCAGGCGGCTTGACCCATTCGGGCCTGTGGAGGATCTGCTGTCTGGAGGGGCTGAAGCGGGGT HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG GTCTGCGTGAAGATTAACCACTTCCCTGAGGACACTGACTACGATCACGACTCCGCCGAGTATCTTCTGCGCGTCGTGCGGGCCTCCTCGATTTTCCCC VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATCCTGTCCGCCATCCTGCTGCTCCTGGGTGGAGTGTGCGTGGCAGCGTCACGCGTGTACAAGTCCAAAAGGAACATCATCCTTGGTGCTGGAATCCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTTGTGGCCGCCGGGCTGTCCAACATTATTGGAGTGATTGTGTACATCAGCGCAAACGCCGGAGAGCCCGGCCCGAAGCGCGATGAAGAAAAGAAGAAC FVAAGLSNIIGVIVYISANAGEPGPKRDEEKKN CATTACAGCTACGGCTGGTCGTCCTTCTACTTCGGGGCGCTGTCGTTCATCGTGGCGGAAACCGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAGG HYSYGWSSFYFGALSFIVAETVGVLAVNIYIER TCCAGAGAGGCACACTGCCAGTCCCGGAGCGACCTCCTGAAGGCCGGTGGAGGGGCCGGAGGCTCCGGAGGCAGCGGCCCATCGGCCATTCTGCGCCTG SREAHCQSRSDLLKAGGGAGGSGGSGPSAILRL CCGAGCTACAGGTTTCGGTATCGGCGGCGCTCAAGATCATCCTCCCGCTCGTCCGAACCCTCCCCGAGCCGGGACGCCTCGCCCGGAGGACCGGGTGGC PSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGG CCGGGATTCGCCTCCACCGACATCAGCATGTACACTCTGTCAAGAGATCCAAGCAAAGGAAGCGTCGCCGCGGGCCTCGCTGGCGCCGGAGGGGGCGGG PGFASTDISMYTLSRDPSKGSVAAGLAGAGGGG GGTGGAGCAGTGGGAGCCTTCGGGGGCGCCGCTGGAGGAGCCGGGGGCGGTGGCGGAGGAGGCGGCGGAGCCGGTGCCGAGCGCGATCGGGGGGGCGCA GGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGA TCCGGTTTCCTGACGCTGCACAACGCGTTCCCGAAGGAAGCGGGCGGAGGAGTGACCGTGACTGTGACCGGACCGCCTGCCCCGCCTGCGCCTGCGCCC SGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAP CCTGCTCCTTCCGCGCCTGCCCCCGGAACTCTCGCCAAGGAGGCCGCCGCCTCTAACACCAACACACTCAACCGAAAGACTACCCCCGTGTAGTGA PAPSAPAPGTLAKEAAASNTNTLNRKTTPV.. C14 atggtgcgatgcgaccgcgggctgcagGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGG MVRCDRGLQVLLTTVGAFAAFGLMTIAISTDYW CTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAA LYTRALICNTTNLTAGGDDGTPHRGGGGASEKK (488888888) GATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGAC DPGGLTHSGLWRICCLEGLKRGVCVKINHFPED ACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGC TDYDHDSAEYLLRVVRASSIFPILSAILLLLGG GTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGC VCVAASRVYKSKRNIILGAGILFVAAGLSNIIG GTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGG VIVYISANAGEPGPKRDEEKKNHYSYGWSFYFG GGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTG GLSFILAEVIGVLAVNIYIERSREAHCQSRSDL CTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGA LKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSR TCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACA SSSRSSEPSPSRDASPGGPGGPGFASTDISMYT CTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGG LSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAG GGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAG GAGGGGGGGGGAGAERDRGGASGFLTLHNAFPK GAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCC EAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLA AAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA KEAAASNTNTLNRKTTPV C15 atggtgcgatgcgaccgcgggctgcagatgctgctgaccacggccggagccttcgccgccttctcgctcatggccatcgccatcTCCACTGACTACTGG MVRCDRGLQMLLTTAGAFAAFSLMAIAISTDYW CTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAA LYTRALICNTTNLTAGGDDGTPHRGGGGASEKK (448888888) GATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGAC DPGGLTHSGLWRICCLEGLKRGVCVKINHFPED ACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGC TDYDHDSAEYLLRVVRASSIFPILSAILLLLGG GTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGC VCVAASRVYKSKRNIILGAGILFVAAGLSNIIG GTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGG VIVYISANAGEPGPKRDEEKKNHYSYGWSFYFG GGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTG GLSFILAEVIGVLAVNIYIERSREAHCQSRSDL CTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGA LKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSR TCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACA SSSRSSEPSPSRDASPGGPGGPGFASTDISMYT CTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGG LSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAG GGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAG GAGGGGGGGGGAGAERDRGGASGFLTLHNAFPK GAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCC EAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLA AAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA KEAAASNTNTLNRKTTPV

10 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

C16 atggtgcgatgcgaccgcgggctgcagatgctgctgaccacggccggagccttcgccgccttctcgctcatggccatcgccatcggcaccgactactgg MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYW ctgtactccagcgcgcacatctgcaacggcaccaacctgaccatggacgacgggcccccgccccgccgcgcccgcggcgacctcacccactctggtctg LYSSAHICNGTNLTMDDGPPPRRARGDLTHSGL (444488888) tggcgggtgtgctgcatcgaagggatctataaagggcactgcttccggatcaatcacttcccagaggacaatgactacgaccacgacagctcggagtac WRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEY ctcctccgcatcgtgcgagcctccagcgtcttccccatcctcagcaccatcctgctcctgctgggtggcctgtgcatcggtgctggcaggatctacagc LLRIVRASSVFPILSTILLLLGGLCIGAGRIYK cgcaagaacaacATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGG SKRNIILGAGILFVAAGLSNIIGVIVYISANAG GAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATT EPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVI GGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCC GVLAVNIYIERSREAHCQSRSDLLKAGGGAGGS GGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCC GGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSP AGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTG SRDASPGGPGGPGFASTDISMYTLSRDPSKGSV GCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGG AAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGG GGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTG GAGAERDRGGASGFLTLHNAFPKEAGGGVTVTV ACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACA TGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNT CTGAACCGAAAAACCACACCCGTCTGA LNRKTTPV C17 atggtgcgatgcgaccgcgggctgcagatgctgctgaccacggccggagccttcgccgccttctcgctcatggccatcgccatcggcaccgactactgg MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYW ctgtactccagcgcgcacatctgcaacggcaccaacctgaccatggacgacgggcccccgccccgccgcgcccgcggcgacctcacccactctggtctg LYSSAHICNGTNLTMDDGPPPRRARGDLTHSGL (444448888) tggcgggtgtgctgcatcgaagggatctataaagggcactgcttccggatcaatcacttcccagaggacaatgactacgaccacgacagctcggagtac WRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEY ctcctccgcatcgtgcgagcctccagcgtcttccccatcctcagcaccatcctgctcctgctgggtggcctgtgcatcggtgctggcaggatctacagc LLRIVRASSVFPILSTILLLLGGLCIGAGRIYS cgcaagaacaacATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGG RKNNIILGAGILFVAAGLSNIIGVIVYISANAG GAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATT EPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVI GGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCC GVLAVNIYIERSREAHCQSRSDLLKAGGGAGGS GGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCC GGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSP AGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTG SRDASPGGPGGPGFASTDISMYTLSRDPSKGSV GCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGG AAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGG GGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTG GAGAERDRGGASGFLTLHNAFPKEAGGGVTVTV ACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACA TGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNT CTGAACCGAAAAACCACACCCGTCTGA LNRKTTPV C18 atggtgcgatgcgaccgcgggctgcagatgctgctgaccacggccggagccttcgccgccttctcgctcatggccatcgccatcggcaccgactactgg MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYW ctgtactccagcgcgcacatctgcaacggcaccaacctgaccatggacgacgggcccccgccccgccgcgcccgcggcgacctcacccactctggtctg LYSSAHICNGTNLTMDDGPPPRRARGDLTHSGL (444444888) tggcgggtgtgctgcatcgaagggatctataaagggcactgcttccggatcaatcacttcccagaggacaatgactacgaccacgacagctcggagtac WRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEY ctcctccgcatcgtgcgagcctccagcgtcttccccatcctcagcaccatcctgctcctgctgggtggcctgtgcatcggtgctggcaggatctacagc LLRIVRASSVFPILSTILLLLGGLCIGAGRIYS cgcaagaacaacatcgtcctcagtgccggcatcctcttcgtggctgcaggcctcagtaacatcatcggtatcatcgtctacattAGCGCCAATGCAGGG RKNNIVLSAGILFVAAGLSNIIGIIVYISANAG GAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATT EPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVI GGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCC GVLAVNIYIERSREAHCQSRSDLLKAGGGAGGS GGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCC GGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSP AGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTG SRDASPGGPGGPGFASTDISMYTLSRDPSKGSV GCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGG AAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGG GGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTG GAGAERDRGGASGFLTLHNAFPKEAGGGVTVTV ACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACA TGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNT CTGAACCGAAAAACCACACCCGTCTGA LNRKTTPV C19 Atggtgcgatgcgaccgcgggctgcagatgctgctgaccacggccggagccttcgccgccttctcgctcatggccatcgccatcggcaccgactactgg MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYW ctgtactccagcgcgcacatctgcaacggcaccaacctgaccatggacgacgggcccccgccccgccgcgcccgcggcgacctcacccactctggtctg LYSSAHICNGTNLTMDDGPPPRRARGDLTHSGL (444444488) tggcgggtgtgctgcatcgaagggatctataaagggcactgcttccggatcaatcacttcccagaggacaatgactacgaccacgacagctcggagtac WRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEY ctcctccgcatcgtgcgagcctccagcgtcttccccatcctcagcaccatcctgctcctgctgggtggcctgtgcatcggtgctggcaggatctacagc LLRIVRASSVFPILSTILLLLGGLCIGAGRIYS cgcaagaacaacatcgtcctcagtgccggcatcctcttcgtggctgcaggcctcagtaacatcatcggtatcatcgtctacatttccagcaacacaggt RKNNIVLSAGILFVAAGLSNIIGIIVYISSNTG gacccgagtgacaagcgggacgaagacaaaaagaaccattacaactacggctggtctTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATT DPSDKRDEDKKNHYNYGWSFYFGGLSFILAEVI GGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCC GVLAVNIYIERSREAHCQSRSDLLKAGGGAGGS GGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCC GGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSP AGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTG SRDASPGGPGGPGFASTDISMYTLSRDPSKGSV GCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGG AAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGG GGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTG GAGAERDRGGASGFLTLHNAFPKEAGGGVTVTV ACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACA TGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNT CTGAACCGAAAAACCACACCCGTCTGA LNRKTTPV C20 atggtgcgatgcgaccgcgggctgcagatgctgctgaccacggccggagccttcgccgccttctcgctcatggccatcgccatcggcaccgactactgg MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYW ctgtactccagcgcgcacatctgcaacggcaccaacctgaccatggacgacgggcccccgccccgccgcgcccgcggcgacctcacccactctggtctg LYSSAHICNGTNLTMDDGPPPRRARGDLTHSGL (444448484) tggcgggtgtgctgcatcgaagggatctataaagggcactgcttccggatcaatcacttcccagaggacaatgactacgaccacgacagctcggagtac WRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEY ctcctccgcatcgtgcgagcctccagcgtcttccccatcctcagcaccatcctgctcctgctgggtggcctgtgcatcggtgctggcaggatctacagc LLRIVRASSVFPILSTILLLLGGLCIGAGRIYS cgcaagaacaacATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTtccagcaacacaggt RKNNIILGAGILFVAAGLSNIIGVIVYISSNTG gacccgagtgacaagcgggacgaagacaaaaagaaccattacaactacggctggtctttttactttggaGGACTGTCTTTTATCCTGGCAGAAGTCATT DPSDKRDEDKKNHYNYGWSFYFGGLSFILAEVI ggcgtcctggctgtaaacatttacattgagaaaaataaagagttgaggtttaagaccaaacgggaattccttaaggcgtcttcctcttctccttatgcc GVLAVNIYIEKNKELRFKTKREFLKASSSSPYA aggatgccgagctacaggtaccggcgacggcgctcgaggtccagctcaaggtccaccgaggcctcgccctccagggacgtgtcgcccatgggcctgaag RMPSYRYRRRRSRSSSRSTEASPSRDVSPMGLK atcacaggggccatccccatgggggagctgtccatgtacacgctgtccagggagcccctcaaggtgaccaccgcagccagctacagccccgaccaggag ITGAIPMGELSMYTLSREPLKVTTAASYSPDQE gccagcttcctgcaggtgcatgactttttccagcaggacctgaaggaaggtttccacgtcagcatgctgaaccgacggacgacccctgtgtga ASFLQVHDFFQQDLKEGFHVSMLNRRTTPV C21 atggtgcgatgcgaccgcgggctgcagatgctgctgaccacggccggagccttcgccgccttctcgctcatggccatcgccatcggcaccgactactgg MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYW ctgtactccagcgcgcacatctgcaacggcaccaacctgaccatggacgacgggcccccgccccgccgcgcccgcggcgacctcacccactctggtctg LYSSAHICNGTNLTMDDGPPPRRARGDLTHSGL (444448444) tggcgggtgtgctgcatcgaagggatctataaagggcactgcttccggatcaatcacttcccagaggacaatgactacgaccacgacagctcggagtac WRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEY ctcctccgcatcgtgcgagcctccagcgtcttccccatcctcagcaccatcctgctcctgctgggtggcctgtgcatcggtgctggcaggatctacagc LLRIVRASSVFPILSTILLLLGGLCIGAGRIYS cgcaagaacaacATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTtccagcaacacaggt RKNNIILGAGILFVAAGLSNIIGVIVYISSNTG gacccgagtgacaagcgggacgaagacaaaaagaaccattacaactacggctggtctttttactttggagctctgtctttcattgtggctgagaccgtg DPSDKRDEDKKNHYNYGWSFYFGALSFIVAETV ggcgtcctggctgtaaacatttacattgagaaaaataaagagttgaggtttaagaccaaacgggaattccttaaggcgtcttcctcttctccttatgcc GVLAVNIYIEKNKELRFKTKREFLKASSSSPYA aggatgccgagctacaggtaccggcgacggcgctcgaggtccagctcaaggtccaccgaggcctcgccctccagggacgtgtcgcccatgggcctgaag RMPSYRYRRRRSRSSSRSTEASPSRDVSPMGLK atcacaggggccatccccatgggggagctgtccatgtacacgctgtccagggagcccctcaaggtgaccaccgcagccagctacagccccgaccaggag ITGAIPMGELSMYTLSREPLKVTTAASYSPDQE gccagcttcctgcaggtgcatgactttttccagcaggacctgaaggaaggtttccacgtcagcatgctgaaccgacggacgacccctgtgtga ASFLQVHDFFQQDLKEGFHVSMLNRRTTPV D1 ATGGGTCTGTTCGACAGAGGTGTGCAGGTGCTGCTGACTACTGTTGGAGCTTTTGCAGCTTTTGGACTGATGACCATTGCTATCAGCACTGACTACTGG MGLFDRGVQVLLTTVGAFAAFGLMTIAISTDYW CTGTACAGCAGAGGAGTGTGCAAGACCAAGAGTGTGAGTGAGAACGAGACCAGCAAGAAGAACGAGGAGGTGATGACTCACAGTGGTCTGTGGAGAACC LYSRGVCKTKSVSENETSKKNEEVMTHSGLWRT (282828282) TGTTGTCTGGAAGGTAACTTCAAAGGTCTGTGCAAGCAGATCGACCACTTTCCAGAGGACGCTGACTACGAGGCTGACACTGCAGAGTACTTCCTGAGA CCLEGNFKGLCKQIDHFPEDADYEADTAEYFLR GCTGTGAGAGCTAGTAGCATCTTTCCAATCCTGAGTGCTATCCTGCTGCTGCTTGGAGGTGTGTGTGTTGCAGCTAGCAGAGTGTACAAGACCAGACAC AVRASSIFPILSAILLLLGGVCVAASRVYKTRH AACATCATCCTTGGAGCTGGAATCCTGTTCGTTGCAGCTGGACTTAGCAACATCATTGGAGTGATCGTGTACATCAGTGCTAATGCAGGTGATCCAAGC NIILGAGILFVAAGLSNIIGVIVYISANAGDPS AAGAGTGACAGCAAGAAGAACAGCTACAGCTATGGTTGGAGCTTCTACTTTGGAGGTCTGAGCTTCATCCTTGCTGAGGTGATTGGTGTTCTTGCTGTG KSDSKKNSYSYGWSFYFGGLSFILAEVIGVLAV AACATCTACATCGAGAGACACAAGCAGCTGAGAGCTACTGCTAGAGCTACTGACTACCTGCAAGCTAGTGCTATCACCAGAATCCCTAGCTACAGATAC NIYIERHKQLRATARATDYLQASAITRIPSYRY AGATACCAGAGAAGAAGCAGAAGCAGTAGCAGAAGTACTGAACCTAGTCACAGCAGAGATGCCAGTCCTGTAGGTATCAAAGGTTTCAACACTCTTCCA RYQRRSRSSSRSTEPSHSRDASPVGIKGFNTLP AGCACTGAGATCAGCATGTACACTCTGAGCAGAGATCCTCTGAAAGCTGCAACCACTCCTACTGCAACCTACAACAGCGACAGAGACAACAGCTTCCTG STEISMYTLSRDPLKAATTPTATYNSDRDNSFL CAGGTGCACAACTGCATCCAGAAGGAGAACAAGGACAGTCTGCACAGCAACACTGCTAACAGAAGAACCACTCCAGTGTGA QVHNCIQKENKDSLHSNTANRRTTPV

11 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

Supplemental Table 7. The primers used for generation of the point mutations of CACNG8 and CACNG4. 5’ end 3’ end Full length mutations Forward Reverse Template Forward Reverse Template Forward Reverse Template primer primer primer primer primer primer CACNG8- P61 P62 CACNG8 P63 P64 CACNG8 P61 P64 5’ end + G210A 3’ end CACNG8- P61 P65 CACNG8 P66 P64 CACNG8 P61 P64 5’ end + L215V 3’ end CACNG8- P61 P67 CACNG8 P68 P64 CACNG8 P61 P64 5’ end + V218T 3’ end CACNG8- P61 P69 CACNG8 P70 P64 CACNG8 P61 P64 5’ end + I219V 3’ end CACNG8- P61 P71 CACNG8 P72 P64 CACNG8 P61 P64 5’ end + I159V 3’ end CACNG8- P61 P73 CACNG8 P74 P64 CACNG8 P61 P64 5’ end + G161S 3’ end CACNG8- P61 P75 CACNG8 P76 P64 CACNG8 P61 P64 5’ end + V177I 3’ end CACNG8- P61 P77 CACNG8 P78 P64 CACNG8 P61 P64 5’ end + N173A 3’ end CACNG8- P61 P79 CACNG8 P80 P64 CACNG8 P61 P64 5’ end + I174A 3’ end CACNG8- P61 P81 CACNG8 P82 P64 CACNG8 P61 P64 5’ end + I175A 3’ end CACNG8- P61 P83 CACNG8 P84 P64 CACNG8 P61 P64 5’ end + G176A 3’ end CACNG8- P61 P87 CACNG8 P88 P64 CACNG8 P61 P64 5’ end + G209A 3’ end CACNG8- P61 P89 CACNG8 P90 P64 CACNG8 P61 P64 5’ end + L211A 3’ end CACNG8- P61 P91 CACNG8 P92 P64 CACNG8 P61 P64 5’ end + S212A 3’ end CACNG8- P61 P93 CACNG8 P94 P64 CACNG8 P61 P64 5’ end + F213A 3’ end CACNG8- P61 P95 CACNG8 P96 P64 CACNG8 P61 P64 5’ end + I214A 3’ end CACNG8- P61 P62 CACNG8- P63 P64 CACNG8- P61 P64 5’ end + V177I, V177I V177I 3’ end G210A CACNG4- P97 P98 CACNG4 P99 P100 CACNG4 P97 P100 5’ end + A189G 3’ end CACNG4- P97 P101 CACNG4- P102 P100 CACNG4- P97 P100 5’ end + I156V, A189G A189G 3’ end A189A

Primer sequences (5’ TO 3’) P61 ACGTCAGAATTCGCCACCATGGAGAGCCTGAAGCGGTGGAACGA P62 ACTTCTGCCAGGATAAAAGACAGAGCCCCAAAGTAGAAAGACCA P63 TGGTCTTTCTACTTTGGGGCTCTGTCTTTTATCCTGGCAGAAGT 12 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

P64 ACGTCAGCGGCCGCTCAGACGGGTGTGGTTTTTCGGTTCAGTGTATTGG P65 CAGCACCCCAATGACTTCTGCTACGATAAAAGACAGTCC P66 GGACTGTCTTTTATCGTAGCAGAAGTCATTGGGGTGCTG P67 ATTGACGGCCAGCACCCCAATAGTTTCTGCCAGGATAAAAGAC P68 GTCTTTTATCCTGGCAGAAACTATTGGGGTGCTGGCCGTCAAT P69 ATAGATATTGACGGCCAGCACCCCGACGACTTCTGCCAGGATAAAAGA P70 TCTTTTATCCTGGCAGAAGTCGTCGGGGTGCTGGCCGTCAATATCTAT P71 GAACAGAATGCCGGCTCCCAGAACGATATTTCTTTTGGACTTGTA P72 TACAAGTCCAAAAGAAATATCGTTCTGGGAGCCGGCATTCTGTTC P73 CACGAACAGAATGCCGGCACTCAGAATGATATTTCTTTTGGA P74 TCCAAAAGAAATATCATTCTGAGTGCCGGCATTCTGTTCGTG P75 CATTGGCGCTAATATAGACGATAATGCCAATGATGTTTGACAGT P76 ACTGTCAAACATCATTGGCATTATCGTCTATATTAGCGCCAATG P77 AATATAGACGATCACGCCAATGATTGCTGACAGTCCTGCA P78 TGCAGGACTGTCAGCAATCATTGGCGTGATCGTCTATATT P79 ATAGACGATCACGCCAATTGCGTTTGACAGTCCTGCA P80 TGCAGGACTGTCAAACGCAATTGGCGTGATCGTCTAT P81 CTAATATAGACGATCACGCCTGCGATGTTTGACAGTCCT P82 AGGACTGTCAAACATCGCAGGCGTGATCGTCTATATTAG P83 CTAATATAGACGATCACTGCAATGATGTTTGACAGTC P84 GACTGTCAAACATCATTGCAGTGATCGTCTATATTAG P85 TTGGCGCTAATATAGACTGCCACGCCAATGATGTTTGAC P86 GTCAAACATCATTGGCGTGGCAGTCTATATTAGCGCCAA P87 CAGGATAAAAGACAGTCCTGCAAAGTAGAAAGACCA P88 TGGTCTTTCTACTTTGCAGGACTGTCTTTTATCCTG P89 CTTCTGCCAGGATAAAAGATGCTCCCCCAAAGTAGAAAG P90 CTTTCTACTTTGGGGGAGCATCTTTTATCCTGGCAGAAG P91 GACTTCTGCCAGGATAAATGCCAGTCCCCCAAAGTAG P92 CTACTTTGGGGGACTGGCATTTATCCTGGCAGAAGTC P93 CAATGACTTCTGCCAGGATTGCAGACAGTCCCCCAAAG P94 CTTTGGGGGACTGTCTGCAATCCTGGCAGAAGTCATTG P95 CAATGACTTCTGCCAGTGCAAAAGACAGTCCCCCAAA P96 TTTGGGGGACTGTCTTTTGCACTGGCAGAAGTCATTG P97 ACGTCAGAATTCGCCACCATGGTGCGATGCGACCGCGGGCTGCAGA P98 CAGCCACAATGAAAGACAGTCCTCCAAAGTAAAAAGACCAG P99 CTGGTCTTTTTACTTTGGAGGACTGTCTTTCATTGTGGCTG P100 ATCTGTGCGGCCGCTCACACAGGGGTCGTCCGTCGGTTCAGCAT P101 GGAAATGTAGACGATGACACCGATGATGTTACTGAG P102 CTCAGTAACATCATCGGTGTCATCGTCTACATTTCC

13 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

Supplemental Table 8. DNA and protein sequences for CACNG8 and CACNG4 point mutants. construct DNA Sequence protein sequence ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CACNG8- CTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCT LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP CACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGC HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG G210A GTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCT VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAAC FVAAGLSNIIGVIVYISANAGEPGPKRDEEKKN CACTACTCCTATGGCTGGTCTTTCTACTTTGGGgctCTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGT HYSYGWSFYFGALSFILAEVIGVLAVNIYIERS CGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCC REAHCQSRSDLLKAGGGAGGSGGSGPSAILRLP TCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCA SYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGP GGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGA GFASTDISMYTLSRDPSKGSVAAGLAGAGGGGG GGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGC GAVGAFGGAAGGAGGGGGGGGGAGAERDRGGAS GGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCA GFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPP GCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA APSAPAPGTLAKEAAASNTNTLNRKTTPV ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CACNG8- CTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCT LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP CACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGC HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG L215V GTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCT VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAAC FVAAGLSNIIGVIVYISANAGEPGPKRDEEKKN CACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCgtaGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGT HYSYGWSFYFGGLSFIVAEVIGVLAVNIYIERS CGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCC REAHCQSRSDLLKAGGGAGGSGGSGPSAILRLP TCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCA SYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGP GGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGA GFASTDISMYTLSRDPSKGSVAAGLAGAGGGGG GGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGC GAVGAFGGAAGGAGGGGGGGGGAGAERDRGGAS GGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCA GFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPP GCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA APSAPAPGTLAKEAAASNTNTLNRKTTPV ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CACNG8- CTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCT LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP CACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGC HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG V218T GTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCT VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAAC FVAAGLSNIIGVIVYISANAGEPGPKRDEEKKN CACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAactATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGT HYSYGWSFYFGGLSFILAETIGVLAVNIYIERS CGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCC REAHCQSRSDLLKAGGGAGGSGGSGPSAILRLP TCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCA SYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGP GGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGA GFASTDISMYTLSRDPSKGSVAAGLAGAGGGGG GGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGC GAVGAFGGAAGGAGGGGGGGGGAGAERDRGGAS GGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCA GFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPP GCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA APSAPAPGTLAKEAAASNTNTLNRKTTPV ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CACNG8- CTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCT LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP CACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGC HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG I219V GTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCT VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAAC FVAAGLSNIIGVIVYISANAGEPGPKRDEEKKN CACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCgtcGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGT HYSYGWSFYFGGLSFILAEVVGVLAVNIYIERS CGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCC REAHCQSRSDLLKAGGGAGGSGGSGPSAILRLP TCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCA SYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGP GGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGA GFASTDISMYTLSRDPSKGSVAAGLAGAGGGGG GGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGC GAVGAFGGAAGGAGGGGGGGGGAGAERDRGGAS GGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCA GFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPP GCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA APSAPAPGTLAKEAAASNTNTLNRKTTPV ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CACNG8- CTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCT LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP CACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGC HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG I159V GTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCT VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCgttCTGGGAGCCGGCATTCTG ILSAILLLLGGVCVAASRVYKSKRNIVLGAGIL TTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAAC FVAAGLSNIIGVIVYISANAGEPGPKRDEEKKN CACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGT HYSYGWSFYFGGLSFILAEVIGVLAVNIYIERS CGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCC REAHCQSRSDLLKAGGGAGGSGGSGPSAILRLP TCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCA SYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGP GGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGA GFASTDISMYTLSRDPSKGSVAAGLAGAGGGGG GGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGC GAVGAFGGAAGGAGGGGGGGGGAGAERDRGGAS GGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCA GFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPP GCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA APSAPAPGTLAKEAAASNTNTLNRKTTPV ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CACNG8- CTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCT LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP CACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGC HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG G161S GTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCT VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGagtGCCGGCATTCTG ILSAILLLLGGVCVAASRVYKSKRNIILSAGIL TTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAAC FVAAGLSNIIGVIVYISANAGEPGPKRDEEKKN CACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGT HYSYGWSFYFGGLSFILAEVIGVLAVNIYIERS CGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCC REAHCQSRSDLLKAGGGAGGSGGSGPSAILRLP TCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCA SYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGP GGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGA GFASTDISMYTLSRDPSKGSVAAGLAGAGGGGG GGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGC GAVGAFGGAAGGAGGGGGGGGGAGAERDRGGAS GGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCA GFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPP GCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA APSAPAPGTLAKEAAASNTNTLNRKTTPV ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CACNG8- CTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCT LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP CACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGC HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG V177I GTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCT VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTCGTGGCTGCAGGACTGTCAAACATCATTGGCattATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAAC FVAAGLSNIIGIIVYISANAGEPGPKRDEEKKN CACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGT HYSYGWSFYFGGLSFILAEVIGVLAVNIYIERS CGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCC REAHCQSRSDLLKAGGGAGGSGGSGPSAILRLP TCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCA SYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGP GGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGA GFASTDISMYTLSRDPSKGSVAAGLAGAGGGGG GGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGC GAVGAFGGAAGGAGGGGGGGGGAGAERDRGGAS GGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCA GFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPP GCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA APSAPAPGTLAKEAAASNTNTLNRKTTPV 14 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CACNG8- CTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCT LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP CACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGC HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG N173A GTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCT VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTCGTGGCTGCAGGACTGTCAgcaATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAAC FVAAGLSAIIGVIVYISANAGEPGPKRDEEKKN CACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGT HYSYGWSFYFGGLSFILAEVIGVLAVNIYIERS CGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCC REAHCQSRSDLLKAGGGAGGSGGSGPSAILRLP TCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCA SYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGP GGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGA GFASTDISMYTLSRDPSKGSVAAGLAGAGGGGG GGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGC GAVGAFGGAAGGAGGGGGGGGGAGAERDRGGAS GGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCA GFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPP GCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA APSAPAPGTLAKEAAASNTNTLNRKTTPV ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CACNG8- CTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCT LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP CACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGC HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG I174A GTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCT VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTCGTGGCTGCAGGACTGTCAAACgcaATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAAC FVAAGLSNAIGVIVYISANAGEPGPKRDEEKKN CACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGT HYSYGWSFYFGGLSFILAEVIGVLAVNIYIERS CGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCC REAHCQSRSDLLKAGGGAGGSGGSGPSAILRLP TCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCA SYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGP GGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGA GFASTDISMYTLSRDPSKGSVAAGLAGAGGGGG GGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGC GAVGAFGGAAGGAGGGGGGGGGAGAERDRGGAS GGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCA GFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPP GCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA APSAPAPGTLAKEAAASNTNTLNRKTTPV ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CACNG8- CTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCT LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP CACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGC HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG I175A GTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCT VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTCGTGGCTGCAGGACTGTCAAACATCgcaGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAAC FVAAGLSNIAGVIVYISANAGEPGPKRDEEKKN CACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGT HYSYGWSFYFGGLSFILAEVIGVLAVNIYIERS CGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCC REAHCQSRSDLLKAGGGAGGSGGSGPSAILRLP TCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCA SYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGP GGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGA GFASTDISMYTLSRDPSKGSVAAGLAGAGGGGG GGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGC GAVGAFGGAAGGAGGGGGGGGGAGAERDRGGAS GGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCA GFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPP GCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA APSAPAPGTLAKEAAASNTNTLNRKTTPV ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CACNG8- CTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCT LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP CACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGC HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG G176A GTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCT VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTCGTGGCTGCAGGACTGTCAAACATCATTgcaGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAAC FVAAGLSNIIAVIVYISANAGEPGPKRDEEKKN CACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGT HYSYGWSFYFGGLSFILAEVIGVLAVNIYIERS CGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCC REAHCQSRSDLLKAGGGAGGSGGSGPSAILRLP TCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCA SYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGP GGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGA GFASTDISMYTLSRDPSKGSVAAGLAGAGGGGG GGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGC GAVGAFGGAAGGAGGGGGGGGGAGAERDRGGAS GGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCA GFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPP GCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA APSAPAPGTLAKEAAASNTNTLNRKTTPV ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CACNG8- CTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCT LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP CACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGC HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG G209A GTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCT VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAAC FVAAGLSNIIGVIVYISANAGEPGPKRDEEKKN CACTACTCCTATGGCTGGTCTTTCTACTTTgcaGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGT HYSYGWSFYFAGLSFILAEVIGVLAVNIYIERS CGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCC REAHCQSRSDLLKAGGGAGGSGGSGPSAILRLP TCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCA SYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGP GGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGA GFASTDISMYTLSRDPSKGSVAAGLAGAGGGGG GGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGC GAVGAFGGAAGGAGGGGGGGGGAGAERDRGGAS GGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCA GFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPP GCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA APSAPAPGTLAKEAAASNTNTLNRKTTPV ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CACNG8- CTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCT LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP CACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGC HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG L211A GTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCT VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAAC FVAAGLSNIIGVIVYISANAGEPGPKRDEEKKN CACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGAgcaTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGT HYSYGWSFYFGGASFILAEVIGVLAVNIYIERS CGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCC REAHCQSRSDLLKAGGGAGGSGGSGPSAILRLP TCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCA SYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGP GGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGA GFASTDISMYTLSRDPSKGSVAAGLAGAGGGGG GGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGC GAVGAFGGAAGGAGGGGGGGGGAGAERDRGGAS GGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCA GFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPP GCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA APSAPAPGTLAKEAAASNTNTLNRKTTPV ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CACNG8- CTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCT LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP CACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGC HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG S212A GTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCT VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAAC FVAAGLSNIIGVIVYISANAGEPGPKRDEEKKN CACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGgcaTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGT HYSYGWSFYFGGLAFILAEVIGVLAVNIYIERS CGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCC REAHCQSRSDLLKAGGGAGGSGGSGPSAILRLP TCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCA SYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGP GGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGA GFASTDISMYTLSRDPSKGSVAAGLAGAGGGGG GGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGC GAVGAFGGAAGGAGGGGGGGGGAGAERDRGGAS GGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCA GFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPP GCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA APSAPAPGTLAKEAAASNTNTLNRKTTPV

15 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CACNG8- CTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCT LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP CACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGC HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG F213A GTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCT VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAAC FVAAGLSNIIGVIVYISANAGEPGPKRDEEKKN CACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTgcaATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGT HYSYGWSFYFGGLSAILAEVIGVLAVNIYIERS CGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCC REAHCQSRSDLLKAGGGAGGSGGSGPSAILRLP TCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCA SYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGP GGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGA GFASTDISMYTLSRDPSKGSVAAGLAGAGGGGG GGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGC GAVGAFGGAAGGAGGGGGGGGGAGAERDRGGAS GGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCA GFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPP GCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA APSAPAPGTLAKEAAASNTNTLNRKTTPV ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CACNG8- CTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCT LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP CACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGC HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG I214A GTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCT VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP ATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAAC FVAAGLSNIIGVIVYISANAGEPGPKRDEEKKN CACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTgcaCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGT HYSYGWSFYFGGLSFALAEVIGVLAVNIYIERS CGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCC REAHCQSRSDLLKAGGGAGGSGGSGPSAILRLP TCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCA SYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGP GGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGA GFASTDISMYTLSRDPSKGSVAAGLAGAGGGGG GGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGC GAVGAFGGAAGGAGGGGGGGGGAGAERDRGGAS GGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCA GFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPP GCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA APSAPAPGTLAKEAAASNTNTLNRKTTPV ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGG MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFG CACNG8- CTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCT LMTIAISTDYWLYTRALICNTTNLTAGGDDGTP CACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGC HRGGGGASEKKDPGGLTHSGLWRICCLEGLKRG V177I, GTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCT VCVKINHFPEDTDYDHDSAEYLLRVVRASSIFP G210A ATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTG ILSAILLLLGGVCVAASRVYKSKRNIILGAGIL TTCGTGGCTGCAGGACTGTCAAACATCATTGGCattATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAAC FVAAGLSNIIGIIVYISANAGEPGPKRDEEKKN CACTACTCCTATGGCTGGTCTTTCTACTTTGGGgcaCTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGT HYSYGWSFYFGALSFILAEVIGVLAVNIYIERS CGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCC REAHCQSRSDLLKAGGGAGGSGGSGPSAILRLP TCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCA SYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGP GGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGA GFASTDISMYTLSRDPSKGSVAAGLAGAGGGGG GGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGC GAVGAFGGAAGGAGGGGGGGGGAGAERDRGGAS GGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCA GFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPP GCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA APSAPAPGTLAKEAAASNTNTLNRKTTPV atggtgcgatgcgaccgcgggctgcagatgctgctgaccacggccggagccttcgccgccttctcgctcatggccatcgccatcggcaccgactactgg MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYW CACNG4- ctgtactccagcgcgcacatctgcaacggcaccaacctgaccatggacgacgggcccccgccccgccgcgcccgcggcgacctcacccactctggtctg LYSSAHICNGTNLTMDDGPPPRRARGDLTHSGL tggcgggtgtgctgcatcgaagggatctataaagggcactgcttccggatcaatcacttcccagaggacaatgactacgaccacgacagctcggagtac WRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEY A189G ctcctccgcatcgtgcgagcctccagcgtcttccccatcctcagcaccatcctgctcctgctgggtggcctgtgcatcggtgctggcaggatctacagc LLRIVRASSVFPILSTILLLLGGLCIGAGRIYS cgcaagaacaacatcgtcctcagtgccggcatcctcttcgtggctgcaggcctcagtaacatcatcggtatcatcgtctacatttccagcaacacaggt RKNNIVLSAGILFVAAGLSNIIGIIVYISSNTG gacccgagtgacaagcgggacgaagacaaaaagaaccattacaactacggctggtctttttactttggaGGActgtctttcattgtggctgagaccgtg DPSDKRDEDKKNHYNYGWSFYFGGLSFIVAETV ggcgtcctggctgtaaacatttacattgagaaaaataaagagttgaggtttaagaccaaacgggaattccttaaggcgtcttcctcttctccttatgcc GVLAVNIYIEKNKELRFKTKREFLKASSSSPYA aggatgccgagctacaggtaccggcgacggcgctcgaggtccagctcaaggtccaccgaggcctcgccctccagggacgtgtcgcccatgggcctgaag RMPSYRYRRRRSRSSSRSTEASPSRDVSPMGLK atcacaggggccatccccatgggggagctgtccatgtacacgctgtccagggagcccctcaaggtgaccaccgcagccagctacagccccgaccaggag ITGAIPMGELSMYTLSREPLKVTTAASYSPDQE gccagcttcctgcaggtgcatgactttttccagcaggacctgaaggaaggtttccacgtcagcatgctgaaccgacggacgacccctgtgtga ASFLQVHDFFQQDLKEGFHVSMLNRRTTPV atggtgcgatgcgaccgcgggctgcagatgctgctgaccacggccggagccttcgccgccttctcgctcatggccatcgccatcggcaccgactactgg MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYW CACNG4- ctgtactccagcgcgcacatctgcaacggcaccaacctgaccatggacgacgggcccccgccccgccgcgcccgcggcgacctcacccactctggtctg LYSSAHICNGTNLTMDDGPPPRRARGDLTHSGL tggcgggtgtgctgcatcgaagggatctataaagggcactgcttccggatcaatcacttcccagaggacaatgactacgaccacgacagctcggagtac WRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEY I156V, ctcctccgcatcgtgcgagcctccagcgtcttccccatcctcagcaccatcctgctcctgctgggtggcctgtgcatcggtgctggcaggatctacagc LLRIVRASSVFPILSTILLLLGGLCIGAGRIYS A189A cgcaagaacaacatcgtcctcagtgccggcatcctcttcgtggctgcaggcctcagtaacatcatcggtGTCatcgtctacatttccagcaacacaggt RKNNIVLSAGILFVAAGLSNIIGVIVYISSNTG gacccgagtgacaagcgggacgaagacaaaaagaaccattacaactacggctggtctttttactttggaGGActgtctttcattgtggctgagaccgtg DPSDKRDEDKKNHYNYGWSFYFGGLSFIVAETV ggcgtcctggctgtaaacatttacattgagaaaaataaagagttgaggtttaagaccaaacgggaattccttaaggcgtcttcctcttctccttatgcc GVLAVNIYIEKNKELRFKTKREFLKASSSSPYA aggatgccgagctacaggtaccggcgacggcgctcgaggtccagctcaaggtccaccgaggcctcgccctccagggacgtgtcgcccatgggcctgaag RMPSYRYRRRRSRSSSRSTEASPSRDVSPMGLK atcacaggggccatccccatgggggagctgtccatgtacacgctgtccagggagcccctcaaggtgaccaccgcagccagctacagccccgaccaggag ITGAIPMGELSMYTLSREPLKVTTAASYSPDQE gccagcttcctgcaggtgcatgactttttccagcaggacctgaaggaaggtttccacgtcagcatgctgaaccgacggacgacccctgtgtga ASFLQVHDFFQQDLKEGFHVSMLNRRTTPV

16 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

Supplemental Table 9. Selectivity of JNJ-55511118 and JNJ-56022486 against a panel of receptors using radioligand binding assays. Inhibition of binding by less than 10% is indicated by a dash. Data were generated by Cerep SA (http://www.cerep.fr/). inhibition of radioligand binding (% control) 56022486 55511118 Assay Reference Compound (1 µM) (1µM) A1 (h) (antagonist radioligand) DPCPX 12 39 A2A (h) (agonist radioligand) NECA 18 ‐ A3 (h) (agonist radioligand) IB‐MECA ‐ 11 alpha 1 (non‐selective) (antagonist radioligand) prazosin ‐ ‐ alpha 2 (non‐selective) (antagonist radioligand) yohimbine ‐ ‐ beta 1 (h) (agonist radioligand) atenolol ‐ ‐ AT1 (h) (antagonist radioligand) saralasin ‐ ‐ BZD (central) (agonist radioligand) diazepam ‐ ‐ B2 (h) (agonist radioligand) NPC 567 ‐ ‐ CCK1 (CCKA) (h) (agonist radioligand) CCK‐8s ‐ ‐ D1 (h) (antagonist radioligand) SCH 23390 12 ‐ D2S (h) (antagonist radioligand) (+)butaclamol ‐ ‐ ETA (h) (agonist radioligand) endothelin‐1 ‐ ‐ GABA (non‐selective) (agonist radioligand) GABA ‐ ‐ GAL2 (h) (agonist radioligand) galanin ‐ ‐ CXCR2 (IL‐8B) (h) (agonist radioligand) IL‐8 ‐ ‐ CCR1 (h) (agonist radioligand) MIP‐1a ‐ ‐ H1 (h) (antagonist radioligand) pyrilamine ‐ ‐ H2 (h) (antagonist radioligand) cimetidine ‐ ‐ MC4 (h) (agonist radioligand) NDP‐a‐MSH ‐ ‐ MT1 (ML1A) (h) (agonist radioligand) melatonin 57 19 M1 (h) (antagonist radioligand) pirenzepine ‐ ‐ M2 (h) (antagonist radioligand) methoctramine ‐ ‐ M3 (h) (antagonist radioligand) 4‐DAMP ‐ ‐ NK2 (h) (agonist radioligand) [Nleu10]‐NKA (4‐10) ‐ ‐ NK3 (h) (antagonist radioligand) SB 222200 ‐ ‐ Y1 (h) (agonist radioligand) NPY ‐ ‐ Y2 (h) (agonist radioligand) NPY ‐ ‐ NTS1 (NT1) (h) (agonist radioligand) neurotensin ‐ ‐ delta 2 (DOP) (h) (agonist radioligand) DPDPE ‐ ‐ kappa (KOP) (agonist radioligand) U 50488 ‐ ‐ mu (MOP) (h) (agonist radioligand) DAMGO ‐ ‐ NOP (ORL1) (h) (agonist radioligand) nociceptin ‐ ‐ 5‐HT1A (h) (agonist radioligand) 8‐OH‐DPAT ‐ ‐ 5‐HT1B (antagonist radioligand) serotonin ‐ ‐ 5‐HT2A (h) (antagonist radioligand) ketanserin ‐ 20 5‐HT2B (h) (agonist radioligand) (±)DOI ‐ 78 5‐HT3 (h) (antagonist radioligand) MDL 72222 ‐ ‐ 5‐HT5a (h) (agonist radioligand) serotonin ‐ 13 5‐HT6 (h) (agonist radioligand) serotonin ‐ ‐ 5‐HT7 (h) (agonist radioligand) serotonin ‐ ‐ sst (non‐selective) (agonist radioligand) somatostatin‐14 ‐ ‐ VPAC1 (VIP1) (h) (agonist radioligand) VIP ‐ ‐ V1a (h) (agonist radioligand) [d(CH2)51,Tyr(Me)2]‐AVP ‐ ‐ Ca2+ channel (L, verapamil site) (antagonist radioligand) D 600 ‐ ‐ KV channel (antagonist radioligand) a‐dendrotoxin ‐ ‐ SKCa channel (antagonist radioligand) apamin ‐ ‐ Na+ channel (site 2) (antagonist radioligand) veratridine ‐ ‐ Cl‐ channel (GABA‐gated) (antagonist radioligand) picrotoxinin ‐ ‐ norepinephrine transporter (h) (antagonist radioligand) protriptyline ‐ ‐ dopamine transporter (h) (antagonist radioligand) BTCP ‐ ‐ 5‐HT transporter (h) (antagonist radioligand) imipramine ‐ 10

17 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

Supplemental Table 10. Summary of pharmacokinetic parameters. ‘n.d.’ indicates that this parameter was not determined.

parameter JNJ‐55511118 JNJ‐56022486 rat mouse rat mouse free fraction (brain) 0.24% n.d. 2.07% n.d. free fraction (plasma) 1.48% 0.88% 7.59% 7.06% oral bioavailability 133% n.d. 93% n.d. clearance (mL/min/kg) 4.8 n.d. 11.4 n.d.

Vss (L/kg) 1.8 n.d. 1.8 n.d.

t1/2 (min) 260 n.d. 109 n.d. BBB 2.2 1.8 0.41 n.d. Kpuu 0.35 0.49 0.11 n.d.

Tmax (10mg/kg, p.o.) (min) 120 60 120 n.d.

Cmax (10mg/kg, p.o.) (ng/mL) 1678 3678 1819 n.d. maximum occupancy (%) 73% 83% 41% n.d.

18 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

Supplemental Figures

Supplemental Figure 1. Synthesis of JNJ-55511118. To a solution of (2-chloro-6- (trifluoromethoxy)phenyl)boronic acid (4.5g, 19 mmol) and 5-bromo-1H-benzo[d]imidazol-2(3H)-one (2.0 g, 9.4 mmol) in 4:1 dioxane:water (20 ml) were added potassium phosphate (4.0 g, 19 mmol) and 1,1’- bis(di-tert-butylphosphino)ferrocene palladium (II) chloride (612 mg, 0.94 mmol). The mixture was degassed with nitrogen and then heated at 100 °C for 16h. After cooling to room temperature, the reaction mixture was filtered through celite and washed successively with EtOAc and DCM. The filtrate was combined with silica gel, concentrated in vacuo, and dry-loaded onto an 80g silica gel cartridge. Purification by flash chromatography (0-10% MeOH in DCM) afforded a solid. The solid was dissolved in hot EtOH (60 °C), and water was added to initiate precipitation. After cooling to room temperature, the precipitate was filtered and dried under high vacuum for 4h to provide the desired product as a white solid (918 mg, 30% yield). 1H NMR (400 MHz, DMSO-d6): δ 10.75 (s, 1H), 10.69 (s, 1H), 7.69 – 7.60 (m, 1H), 7.59 – 7.43 (m, 2H), 7.08 – 6.98 (m, 1H), 6.86 – 6.77 (m, 2H). 13C NMR (150 MHz, DMSO-d6): δ 155.3, 146.9, 134.52, 134.47, 129.9, 129.7, 129.5, 128.6, 125.1, 122.1, 119.9, 119.8 (q, J = 257 Hz), 109.6, 108.2. Anal. Calc’d. for C14H18ClF3N2O2: C, 51.16; H, 2.45; N, 8.52. Found: C, 51.34; H, 2.07; N, 8.41.

19 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

O H B O N O N a Br b CN H I I I c Cl Cl Cl

CN CN CN

H H H N d N e N O O O Cl Cl Cl N Br N T N H H H

o a) NBS, ACBN, CCl4 90 C o b) KCN, DMF/H2O40 C c) cat PdCl2(dppf)-CH2Cl2,K3PO4 o dioxane/H2O 100 C d) NBS, TFA 3 3 e) H-H ,Pd/C,MeOH

Supplemental Figure 2. Synthesis and tritiation of JNJ-56022486. (a) 1-(bromomethyl)-3-chloro-2- iodobenzene. To a solution of 1-chloro-2-iodo-3-methylbenzene (4.0 g, 16 mmol) in CCl4 (12 mL), were added N-bromosuccinimide (5.6 g, 32 mmol) and 1,1'-azobis(cyclohexanecarbonitrile) (3.9 g, 16 mmol). The mixture was degassed with nitrogen and then heated at 90 °C for 1h. After cooling to room temperature, silica gel was added, and the solvent was removed in vacuo. Purification by flash column chromatography (SiO2; 0 - 5% EtOAc /hexanes) provided the title compound as an oil (3.7 g, 70% yield). 1 H NMR (400 MHz, CDCl3): δ 7.37 (m, 2H), 7.29 – 7.23 (m, 1H), 4.65 (s, 2H). (b) 2-(3-chloro-2- iodophenyl)acetonitrile. To a solution of 1-(bromomethyl)-3-chloro-2-iodobenzene (1.0 g, 3.0 mmol) in DMF (13 mL) was added a solution of potassium cyanide (236 mg, 3.6 mmol) in water (1.3 mL). After stirring at 40 °C for 1h, the reaction mixture was cooled to room temperature, diluted with water, and extracted with EtOAc (2x). The combined organic extracts were dried over Na2SO4, and concentrated to 1 obtain the product as a white solid (770 mg, 92% yield). H NMR (500 MHz, CDCl3): δ 7.44 (m, 2H), 7.36 – 7.33 (m, 1H), 3.93 – 3.83 (m, 2H). (c) 2-[3-chloro-2-(2-oxo-1,3-dihydrobenzimidazol-5- yl)phenyl]acetonitrile. To a solution of 2-(3-chloro-2-iodophenyl)acetonitrile (448 mg, 1.6 mmol) and 5- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazol-2(3H)-one (350 mg, 1.4 mmol) in 4:1 dioxane:water (3.5 ml) were added potassium phosphate (571 mg, 2.7 mmol) and [1,1’- bis(diphenylphosphino)ferrocene]dichloropalladium (II) dichloromethane complex (98 mg, 0.13 mmol). The mixture was degassed with nitrogen and then heated at 100 °C for 16h. After cooling to room temperature, the reaction mixture was diluted with water and extracted with DCM (x3). The combined organic extracts were dried over Na2SO4 and filtered. Silica gel was added to the filtrate, and the solvent was removed in vacuo. The resulting solid was dry-loaded onto a 40g silica gel cartridge and purified by flash chromatography (0-50% 2M NH3 in MeOH/ DCM) to afford the desired product as a solid (211 mg, + 1 55% yield). MS (ESI): mass calcd. for C15H10ClN3O, 283.1; m/z found, 284.1 [M+H] . H NMR (400 MHz, DMSO-d6): δ 10.74 (s, 1H), 10.72 (s, 1H), 7.56 (dd, J = 7.9, 1.4 Hz, 1H), 7.53 – 7.49 (m, 1H), 7.48 – 20 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

7.40 (m, 1H), 7.06 – 7.00 (m, 1H), 6.78 – 6.72 (m, 2H), 3.69 (s, 2H). 13C NMR (150 MHz, DMSO-d6): δ 155.3, 140.2, 133.8, 132.3, 129.9, 129.5, 129.3, 128.9, 128.4, 127.6, 121.3, 118.6, 109.1, 108.6, 22.0. (d) 2-[2-(6-bromo-2-oxo-1,3-dihydrobenzimidazol-5-yl)-3-chloro-phenyl]acetonitrile. To a solution of 2-[3- chloro-2-(2-oxo-1,3-dihydrobenzimidazol-5-yl)phenyl]acetonitrile (210 mg, 0.74 mmol) in TFA (7.4 mL) was added N-bromosuccinimide (132 mg, 0.74 mmol). After stirring at room temperature for 24h, the solvent was removed in vacuo. Residual TFA was removed by trituration with DCM and concentration under reduced pressure. The resulting solid was diluted with saturated aqueous NaHCO3 and extracted twice with EtOAc. The combined organic extracts were dried over Na2SO4 and concentrated in vacuo. Purification by reverse-phase HPLC (0.05M NH4OH in water/MeCN) afforded the desired product as a white solid (107 mg, 40% yield). MS (ESI): mass calcd. for C15H9BrClN3O, 361.0; m/z found, 362.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 10.92 (bs, 2H), 7.63 – 7.42 (m, 3H), 7.32 – 7.21 (s, 1H), 6.85 – 6.74 (s, 1H), 3.67 – 3.57 (m, 2H). (e) 2-[3-chloro-2-(2-oxo-6-tritio-1,3-dihydrobenzimidazol-5- yl)phenyl]acetonitrile. This step was performed by Moravek Biochemicals, Inc. (Brea, CA). A 2 cc round- bottom flask was charged with 2-[2-(6-bromo-2-oxo-1,3-dihydrobenzimidazol-5-yl)-3-chloro- phenyl]acetonitrile (5 mg), 10 wt% Pd/C (5 mg), MeOH (0.5 ml), and tritium gas (10 Ci). The mixture was stirred for 6h at room temperature. The crude product was dissolved in EtOH and filtered. The labile tritium was exchanged as the EtOH was removed in vacuo. This was repeated 2 additional times. The crude product was purified by reverse-phase HPLC (Gemini 5 μm C-18 column, 35% aq acetonitrile containing 0.01% NH4OH) to afford the title compound. The specific activity was determined to be 22.3 Ci/mmol, and the product was stored at -20 °C in EtOH at a concentration of 1.0 mCi/mL. MS (FTMS + c NSI SIM): + mass calcd. for C15H9TClN3O, 285.1; m/z found, 286.1 [M+H] .

21 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

8 C1 C3 C4 C5 C6 C7 C8 C9 888888888 888888884 888888844 888888444 888884444 888844444 888444444 884444444 844444444

 C2 C14 C15 C16 C17 C18 C19 444444444 444444448 488888888 448888888 444888888 444488888 444448888 444444888

C10 C11 C12 C13 C20 C21 2 D1 884888888 448444444 444444484 888888848 444448484 444448444 222222222 282828282

Supplemental Figure 3. Schematic drawings of the chimeric proteins. Chimeras are labelled by their shorthand notation, with the ‘C’ prefix designating a chimera of TARP-8 and -4 and the ‘D’ prefix designating a chimera of TARP-8 and -2. Also shown is the nine-digit notation; each digit represents the TARP sequence for that section of the protein with the sequence NT, TM1, EX1, TM2, IN1, TM3, EX2, TM4, CT. The actual splice points used are listed in Supplemental Table 4. The drawing represent the predicted topology of the protein, with the colors representing the TARP sequence used for that segment: TARP-8 (blue), TARP-4 (red), TARP-2 (green).

22 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

120

100

80

60

40 peak current (%) peak current

20 2 8 2 8     GluA1o hippo (-/-) hippo hippo (+/+) hippo cerebellum GluA1i + GluA1i GluA1i + GluA1i GluA1o - - GluA1o GluA1o - - GluA1o

Supplemental Figure 4. Inhibition of peak currents by 1 µM JNJ-55511118 in cells expressing AMPA receptors (N = 4-8 per group). The box plots indicate the median value (line), the mean (square), the 25th– 75th percentiles (box) and the minimum-maximum (whiskers).

23 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

control control A JNJ-55511118 (1 µM) B JNJ-55511118 (1uM)

50 pA

100 pA 20 ms

20 ms C D control 3.0 55511118 control 2.5 JNJ-55511118 50 pA

2.0

1.5 200 ms 1.0 paired-pulse ratio

0.5

P2/P1 P3/P2 P4/P3 P5/P4 Pulse pairs

Supplemental Figure 5. Activity of JNJ-55511118 in hippocampal slices in whole-cell mode. (A) Representative eEPSC recordings prior to (black) and after (red) bath application of 1 µM JNJ-55511118. These traces are the average of five consecutive sweeps. Holding potential was -70 mV to isolate the AMPAR currents. (B) AMPAR-mediated eEPSCs in response to 50 Hz burst stimulation show reduced summation in the presence of 1 µM JNJ-55511118. (C) NMDAR-mediated currents were isolated by holding the cell ay +40mV; these currents were unaffected by 1 µM JNJ-55511118. (D) Paired-pulse ratio (ratio of the response to a pulse, divided by the response to the immediately preceding pulse) calculated from the data in (B). The paired-pulse ratio was unaffected by 1 µM JNJ-55511118, indicating a post- synaptic site of action for the drug.

24 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

Glu A CTZ

100 pA control JNJ-55511118

100 ms

B 1.0

0.8

0.6

0.4

0.2 (JNJ-55511118/control) I

0.0 peak steady-state plus CTZ

Supplemental Figure 6. Activity of JNJ-55511118 in GluA1i + TARP-8 with and without cyclothiazide (CTZ; 100 µM). (A) Time courses of the current in outside-out patches from HEK cells transiently transfected with GluA1i + TARP-8, in the continuous presence of cyclothiazide to remove desensitization. Solid lines are the average of five recordings prior to (black) and after (blue) exposure to 1 µM JNJ-55511118. (B) Average glutamate-evoked peak (black) and steady-state (red) currents in six patches, normalized to the corresponding current prior to the addition of 1 µM JNJ-55511118. Also shown are the steady-state currents from patches exposed to cyclothiazide (blue). The box plots show the median (line), mean (square), 25th and 75th percentiles (box), minimum/maximum (whiskers), and individual data (solid circles).

25 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

10000 100 A1000 rat B rat

80

1000 100 plasma 60 brain occupancy

10 40 p.o. (5 mg/kg) 100 i.v. (1 mg/kg) occupancy (percent) occupancy

concentration (ng/mL) concentration (ng/mL) concentration 20 1

10 0 0 6 12 18 24 0 6 12 18 24 time (hr) time (hr)

Supplemental Figure 7. Pharmacokinetics and target occupancy for JNJ-56022486 in rat. (A) Plasma concentration as a function of time in Sprague-Dawley rats following oral (5 mg/kg p.o.) or intravenous (1 mg/kg i.v.) administration. (B) Brain and plasma concentrations in rat after a 10 mg/kg p.o. dose. Target occupancy was determined by autoradiography (ARG) of brain slices.

26 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712 A

B

C

D E

Supplemental Figure 8. Dose response effects of JNJ-55511118 (1, 3, and 10 mg/kg, p.o.) on sleep parameters in rats. (A) Wake duration, (B) NREM duration, (C) REM duration, (D) NREM latency, and (E) REM latency were determined for the 8-hour period after compound or vehicle administration. Data are expressed in minutes and are represented as means ± SEM of the same 8 animals per dose. Oral administration of JNJ-55511118 in rats induced a dose-dependent increase in wake duration [F(9, 54) = 2.79, 27 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712 p = 0.009] and a decrease in the duration of both NREM [F(9, 54) = 2.27, p = 0.031] and REM [F(9, 54) = 1.95, p = 0.064] sleep. In addition, a dose-dependent increase in NREM latency [F(3, 18) = 9.61, p < 0.001] and in REM latency [F(3, 18) = 5.67, p = 0.007] was observed. At 10 mg/kg, JNJ-55511118 produced a significant wake-promoting effect for 4 hours and reduced the time spent in NREM sleep for 2 hours and in REM sleep for 6 hours following the treatment, associated with a significant delay to the onset of both NREM and REM sleep. * p < 0.05 ** p < 0.01 and *** p < 0.001 versus vehicle, based on one-way or two-way ANOVA followed by Dunnett’s multiple comparison test.

28 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712 A B

C D

E F

G H

Supplemental Figure 9. EEG effects JNJ-55511118 in sleep states. (A-D) Dose response effects of JNJ-55511118 (1, 3, and 10 mg/kg, p.o.) on EEG power activity during NREM sleep in rats. JNJ- 55511118 produced a dose-dependent decrease in EEG power in the entire 1-30 Hz frequency range during NREM sleep. Delta (1-4 Hz), theta (4-10 Hz), sigma (10-15 Hz) and beta (15-30 Hz) EEG oscillations were significantly reduced from the dose of 1 mg/kg onwards. (E-H) Dose response effects of JNJ-55511118 (1, 3, and 10 mg/kg, p.o.) on EEG power activity during REM sleep in rats. During REM sleep, delta (1-4 Hz), theta (4-10 Hz) and beta (15-30 Hz) EEG oscillations were significantly reduced from the dose of 1 mg/kg onwards. In contrast, EEG oscillations in the sigma frequency range (10-15 Hz) were minimally affected, except for a small increase at the dose of 10 mg/kg. * p < 0.05 ** p < 0.01 and *** p < 0.001 versus vehicle, based on one-way or two-way ANOVA followed by Dunnett’s multiple comparison test.

29 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

200

150

100

50 afterdischarge duration (s) 0 vehicle 10mg/kg

Supplemental Figure 10. Effects of JNJ-55511118 on amygdala kindling in rat. The box plot shows the median (line), mean (square), 25th and 75th percentiles (box), minimum/maximum (whiskers), and individual data (solid circles).

30 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

A Pathlength 0mg/kg 0.63mg/kg 2.5mg/kg 10mg/kg

1800 1600 1400 1200 ** (cm) * ** 1000 * 800 600 * Distance 400 * 200 ** 0 123 123 123 123 D1D2D3D4 D1 D2 D3 D4 Average per Day % time in periphery B 0mg/kg 0.63mg/kg 2.5mg/kg 10mg/kg 100

90 80 * 70 * 60 %

50

Time 40 30

20 10 0 123 123 123 123 D1D2D3D4 D1 D2 D3 D4 Average per Day

Swim speed

C 35 0mg/kg 0.63mg/kg 2.5mg/kg 10mg/kg

30 * * * * 25 * * * * * 20 (cm/s) 15 10 Velocity 5

0 123 123 123 123 D1D2D3D4 D1 D2 D3 D4 Average per Day

Supplemental Figure 11. Additional measures in the Morris Water Maze test. In each case, the points are the mean and SEM of N=12 animals for each of the three trials on the four training days. The panels on the right indicate the daily average for all trials. (A) Pathlength (total distance travelled before finding the platform). (B) Time in periphery (percentage of the total time spent in an incorrect quadrant). (C) Swim speed.

31 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

The effects of =JNJ55511118 The effects of JNJ55511118 A on omission percentage in DNMTP B on sample press latency in DNMTP

100 8 Veh Veh 1 mg/kg 1 mg/kg 80 3 mg/kg 6 3 mg/kg 10 mg/kg 10 mg/kg 60 4 *** 40 *

2 20 *** Sample pressSample latency (s) percentageOmission (%) * *** 0 0

The effects of JNJ55511118 The effects of JNJ55511118 on collection latency in DNMTP C on choice latency in DNMTP D

6 3 Veh Veh 1 mg/kg 1 mg/kg 3 mg/kg 3 mg/kg 4 10 mg/kg 2 10 mg/kg

*** *** *** 2 1 Collect latency (s) latency Collect Choice latency latency (s)Choice

0 0

Supplemental Figure 12. Secondary measures of the effects of JNJ-55511118 in the DNMTP assay. All data are represented as mean ± SEM (N=15), with statistical significance contrast relative to vehicle: *p<0.05, **p<0.01, ***p < 0.001. All tested doses increased the percent of trials omitted, and the two highest doses tested induced a significant increase on latency measures. The magnitudes of these effects were generally modest. (A) Percentage total omissions: omitted trials / total trials started. The analysis used a within-subject design, logistic regression model where the probability of omission is given as a function of treatment, with a random animal effect; statistically significant contrasts relative to vehicle. (B) Sample Press latency: duration from the extension of the sample lever until it is pressed. Within-subject design, repeated-measures ANOVA model comparing the various latencies by dosage group, with a random animal effect. (C) Choice latency: duration from the extension of the levers at choice phase until a selection. Within-subject design, repeated-measures ANOVA model comparing the various latencies by dosage group, with a random animal effect. (D) Collection latency: duration between the delivery of a reward pellet and its collection. Within-subject design, repeated-measures ANOVA model comparing the various latencies by dosage group, with a random animal effect.

32 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712 A gene code species IN1 TM3 EX2 CCG8_HUMAN Homo sapiens 153 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 184 CCG8_mouse Mus musculus 152 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 183 CCG8_rat Rattus norvegicus 152 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 183 J9NW64_CANFA Canis familiaris 152 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 183 F1RNJ2_PIG Sus scrofa 152 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 183 H2R5V2_PANTR Pan troglodytes 122 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 153 H0XKI8_OTOGA Otolemur garnettii 120 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 151 Q0VFK5_XENTR Xenopus tropicalis 133 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 164 H2STY8_TAKRU Takifugu rubripes 135 KSKRNIILGGGILFVAAGLSNIIGVIVYISAA 166 G3P8Z8_GASAC Gasterosteus aculeatus 145 KSKRNIILGAGILFVAAGLSNIIGVIVYISAA 176 H9GR85_ANOCA Anolis carolinensis 133 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 164 F1MV40_BOVIN Bos taurus 144 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 175 *********.*********************

B gene code species IN1 TM4 EX2 CCG8_HUMAN Homo sapiens 203 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 234 CCG8_mouse Mus musculus 202 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 233 CCG8_rat Rattus norvegicus 202 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 233 J9NW64_CANFA Canis familiaris 202 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 233 F1RNJ2_PIG Sus scrofa 202 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 233 H2R5V2_PANTR Pan troglodytes 172 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 203 H0XKI8_OTOGA Otolemur garnettii 170 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 201 Q0VFK5_XENTR Xenopus tropicalis 183 GWSFYFGGLSFIIAEMIGVLAVNIYIEKNREA 214 H2STY8_TAKRU Takifugu rubripes 185 GWSFYFGGLSFILAEMVGVLAVNIYIEKNKEL 216 G3P8Z8_GASAC Gasterosteus aculeatus 195 GWSFYFGGLSFIMAEMVGVLAVNIYIEKNKEL 226 H9GR85_ANOCA Anolis carolinensis 183 GWSFYFGGLSFILAEMIGVLAVNIYIEKNREA 214 F1MV40_BOVIN Bos taurus 194 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 225 ************:**::**********:.:*

Supplemental Figure 13. Sequence alignment of TARP-8 across species in the (A) TM3 and (B) TM4 regions. Data and alignments were retrieved from the Uniprot database for genes identified as CACNG8, from a selection of species. Predicted locations for TM3 and TM4 are designated with boxes. The key selectivity residues V177 and G210 (numbering from the human full-length sequence) are highlighted in gray. The transmembrane domains of TARP-8 are very highly conserved across vertebrates, including mammals (human, rat, mouse, monkey, etc.), reptile and amphibian (anole, frog), and fish (fugu, Gasterosteus).

33 Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

A CACNG2 gene code species IN1 TM3 EX2 IN1 TM4 EX2 CCG8_HUMAN Homo sapiens 153 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 184 203 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 234 CCG2_HUMAN Homo sapiens 129 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 208 CCG2_MOUSE Mus musculus 129 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 208 CCG2_RAT Rattus norvegicus 129 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 208 J9P237_CANFA Canis familiaris 129 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 208 F1SKJ6_PIG Sus scrofa 62 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 93 110 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 141 H2QLK9_PANTR Pan troglodytes 58 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 89 106 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 137 H0WLV7_OTOGA Otolemur garnettii 129 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 208 A4IIM6_XENTR Xenopus tropicalis 129 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 208 H2RJG2_TAKRU Takifugu rubripes 129 KSRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIDRHRQL 208 G3PNM3_GASAC Gasterosteus aculeatus 129 KSRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIMAEMVGVLAVHMFIDRHRQL 208 G1KSE5_ANOCA Anolis carolinensis 129 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 208 A0JNG9_BOVIN Bos taurus 129 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 208 *:::****.***:**:********:******* *******.****:**::*****.::*:* :

B CACNG3 gene code species IN1 TM3 EX2 IN1 TM4 EX2 CCG8_HUMAN Homo sapiens 153 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 184 203 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 234 CCG3_HUMAN Homo sapiens 129 RSRHNVILSAGIFFVSAGLSNIIGIIVYISAN 160 176 GWSFYFGAFSFIIAEIVGVVAVHIYIEKHQQL 207 CCG3_MOUSE Mus musculus 129 RSRHSVILSAGIFFVSAGLSNIIGIIVYISAN 160 176 GWSFYFGAFSFIIAEIVGVVAVHIYIEKHQQL 207 CCG3_RAT Rattus norvegicus 129 RSRHSVILSAGIFFVSAGLSNIIGIIVYISAN 160 176 GWSFYFGAFSFIIAEIVGVVAVHIYIEKHQQL 207 E2RKC2_CANFA Canis familiaris 72 RSRHNVILSAGIFFVSAGLSNIIGIIVYISAN 103 119 GWSFYFGAFSFIIAEIVGVVAVHIYIEKHQQL 150 F1RFC9_PIG Sus scrofa 72 RSRHNVILSAGIFFVSAGLSNIIGIIVYISAN 103 119 GWSFYFGAFSFIIAEIVGVVAVHIYIEKHQQL 150 H2QAS8_PANTR Pan troglodytes 129 RSRHNVILSAGIFFVSAGLSNIIGIIVYISAN 160 176 GWSFYFGAFSFIIAQIVGMIAVHIYIEKHQQL 207 H0XZP0_OTOGA Otolemur garnettii 72 RSRHNVILSAGIFFVSAGLSNIIGIIVYISAN 103 119 GWSFYFGAFSFIIAEIVGVVAVHIYIEKHQQL 150 F7BS52_XENTR Xenopus tropicalis 129 KNKHNVILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIEKHRQI 208 CCG3_BOVIN Bos taurus 129 RSRHNVILSAGIFFVSAGLSNIIGIIVYISAN 160 176 GWSFYFGAFSFIIAEIVGVVAVHIYIEKHQQL 207 :.::.:**.***:**:********:******* *******.:***:*:::*::**.::**: ::

C CACNG4 gene code species IN1 TM3 EX2 IN1 TM4 EX2 CCG8_HUMAN Homo sapiens 153 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 184 203 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 234 CCG4_HUMAN Homo sapiens 132 SRKNNIVLSAGILFVAAGLSNIIGIIVYISSN 163 182 GWSFYFGALSFIVAETVGVLAVNIYIEKNKEL 213 CCG4_MOUSE Mus musculus 132 SRKNNIVLSAGILFVAAGLSNIIGIIVYISSN 163 182 GWSFYFGALSFIVAETVGVLAVNIYIEKNKEL 213 CCG4_RAT Rattus norvegicus 132 SRKNNIVLSAGILFVAAGLSNIIGIIVYISSN 163 182 GWSFYFGALSFIVAETVGVLAVNIYIEKNKEL 213 F1RV12_PIG Sus scrofa 132 SRKNNIVLSAGILFVAAGLSNIIGIIVYISSN 163 182 GWSFYFGALSFIVAETVGVLAVNIYIEKNKEL 213 H2RDC7_PANTR Pan troglodytes 58 SRKNNIVLSAGILFVAAGLSNIIGIIVYISSN 89 108 GWSFYFGALSFIVAETVGVLAVNIYIEKNKEL 139 H0X9H7_OTOGA Otolemur garnettii 128 SRKNNIVLSAGILFVAAGLSNIIGIIVYISSN 159 178 GWSFYFGALSFIVAETVGVLAVNIYIEKNKEL 209 F7APU6_XENTR Xenopus tropicalis 133 SRRNNIILSAGILFVAAERRRAGRIEAKKEGE 164 183 GWSFYFGALSFIVAETIGVLXVNIYIERNKEL 214 H9G3D4_ANOCA Anolis carolinensis 113 SSKNNIILSAGILFVAAGLSNIIGIIVYISSN 144 163 GWSFYFGALSFIVAETVGVLAVNIYIEKNKEL 194 E1BFD7_BOVIN Bos taurus 129 SRKNNIVLSAGILFVAAGLSNIIGIIVYISSN 160 179 GWSFYFGALSFIVAETVGVLAVNIYIEKNKEL 210 . :.**:*.******** . : . ..: *******.****:**.:*** ******:.:*

Supplemental Figure 14. Sequence alignment of TARPs-2, -3, and -4 across species in the TM3 and TM4 regions. Data and alignments were retrieved from the Uniprot database (UniProt Consortium, 2015) for the genes identified as (A) CACNG2, (B) CACNG3, and (C) CACNG4 for the same species as shown in Supplemental Figure 13. For comparison and alignment, TARP-8 is also shown. The transmembrane domains are very highly conserved across vertebrates. In particular, the amino acids corresponding to V177 and G210 in CACNG8 are isoleucine and alanine, respectively, in all other species examined.

34