Williams DK , Stokes C , Horenstein NA , Papke RL .

R01-GM057481 NIGMS NIH HHS , T32-AG000196 NIA NIH HHS , R01 GM057481 NIGMS NIH HHS , T32 AG000196 NIA NIH HHS

	Figure 1. Location of the L119 residue in a homology model of α7 (Celie et al., 2004). (A) The overview at left shows an α7-α7 homodimer and the location of the L119 residue in relation to the C-loop in the primary face of the agonist binding site. The image on the right shows the proximity at increased magnification. Images were created in Deep View (Swiss-PdbViewer; Guex and Peitsch, 1997) from the crystal structure model of the ACh binding protein (deposited in the Protein Data Bank under accession no. 1I9B; Brejc et al., 2001). (B) The effect of MTSEA treatment (2 mM for 60 s) on the ACh-evoked responses of oocytes expressing the α7L119C mutation in a cysteine-null (α7C116S) background. In this experiment, peak current responses to 300 µM ACh were reduced 99.4 ± 0.2%, and net charge was reduced by 99.7 ± 0.1% (n = 4). Responses to 3 mM ACh were reduced to a similar extent: 97.9 ± 0.3 and 95.9 ± 2.0% for peak current and net charge, respectively (n = 4).
	Figure 2. Coexpression of either L119C or Y188F with wild-type α7 subunits at varying ratios. (A) The probability for the distribution of mutant subunits based on RNA ratios. Each single subunit within a pentamer is either mutant or wild-type, and if we assume X = probability of being wild-type (actually α7C116S) and Y = probability of being mutant, then 1 = X + Y, and for combinations of five subunits, (X + Y)n = 1. The expansion of this binomial is X5 + 5X4Y + 10X3Y2 + 10X2Y3 + 5XY4 + Y5 = 1. We assume that receptors are functionally equivalent regardless of the subunit positions within the pentameric structure. (B) Data traces obtained from oocytes expressing the α7C116S/L119C MTSEA-sensitive mutant and the α7C116S cysteine-null pseudo wild-type at the ratios indicated. For each panel, the traces on the left are the 300-µM ACh control responses obtained before the MTSEA treatment, and the series of traces on the right are the responses to progressively greater concentrations of ACh obtained after the MTSEA treatment. (C) Average net charge values for oocytes expressing the α7C116S/L119C MTSEA-sensitive mutant and α7C116S cysteine-null pseudo wild-type at the ratios indicated after treatment with MTSEA, normalized to the 300-µM ACh control responses before MTSEA treatment. The open circles represent the 300-µM ACh control data obtained before the MTSEA treatment (i.e., the values to which the posttreatment data are normalized). The data plotted are the means ± SEM for at least five oocytes at each of the ratios tested. See Table I for curve fit values.
	Figure 3. Probing for an α7 C-loop mutation with selective and nonselective agonists. (A) Mutation of the α7 tyrosine 188 to phenylalanine reduces sensitivity to low concentrations of ACh, with little impact on sensitivity to the responses to the α7-selective agonist 4OH-GTS-21 (Horenstein et al., 2007). The upper traces are representative responses of oocytes expressing wild-type α7, for which 300 µM 4OH-GTS-21 evoked responses that are 57 ± 4% the magnitude of the responses evoked by 300 µM ACh, in net charge. In contrast, for oocytes expressing α7Y188F, 300-µM 4OH-GTS-21–evoked net charge responses that are 405 ± 55% of the magnitude of the responses evoked by 300 µM ACh. (B) Oocytes were injected with RNA for α7Y188F and wild-type α7 at (mutant/wild-type) 1:0, 5:1, 3:1, 1:1, and 0:1 ratios and then tested for their relative responses to 300 µM ACh and 300 µM 4OH-GTS-21. The values are plotted in relation to the fraction of α7Y188F RNA injected at each ratio and are the means ± SEM of at least four oocytes for every condition.
	Figure 4. The effect of MTSEA on muscle-type receptors with mutations homologous to α7L119C in muscle δ, γ, and ε subunits. Representative responses obtained before MTSEA treatments are shown as black lines, and responses obtained after MTSEA are shown as gray lines. The schematics to the right of the traces represent the subunit composition and disposition of the ACh binding sites for the different receptor subtypes. The asterisks represent the location of the mutations in the complementary face of the agonist binding site.
	Figure 5. ACh concentration–response data for muscle-type single-subunit mutants before and after MTSEA treatment. Oocytes were stimulated alternately with control applications of 30 µM ACh and ACh at increasing concentrations: 1 µM, 10 µM, 100 µM, and 1 mM (i.e., the sequence of applications was 30, 1, 30, 10, 30, 100, 30, 1,000, and 30). Next, the oocytes were treated with 2 mM MTSEA for 60 s before being tested with the same sequence of ACh applications. All of the data were normalized to the individual oocytes’ average responses to the five 30-µM ACh applications given before the MTSEA treatment. Therefore, the 30-µM point in the pretreatment data is fixed at 1, and the SEM plotted for that point is the average SEM of the five 30-µM responses obtained from each cell. The 30-µM point in the post-MTSEA curve is based on the responses to 30 µM ACh obtained between the posttreatment 10-µM ACh and 100-µM ACh applications. The plots on the right represent the repeated 30-µM ACh responses obtained through the course of the entire experiments, normalized to the average pre-MTSEA 30-µM ACh responses from each cell. The arrowhead indicates the point at which MTSEA was applied. The values plotted are the means ± SEM of five, three, and eight oocytes for α1β1γδL121C, α1β1εL119Cδ, and α1β1εδL121C, respectively. Fit parameters are listed in Table II.
	Figure 6. The effect of MTSEA treatment on peak and NPopen responses from receptors expressed in BOSC 23 cells. (A) Example traces of outside-out patches from each condition. A rapid (≤0.7-ms) drug application system was used to apply ACh and MTSEA. (B) Summary of the effect of MTSEA treatment (5 mM for 60 s) on peak current and NPopen responses to 1 mM ACh shown as the average of post-MTSEA measurements relative to the pre-MTSEA measurements of each patch. The measurements are normalized to the average peak current and NPopen responses from eight rundown/desensitization control patches. Asterisks above the values for α1β1γL119CδL121C indicate statistical significance (P < 0.01) when compared with values from either α1β1γδL121C or wild-type. See Table III for values.
	Figure 7. Single-channel traces and fit burst–duration histograms from wild-type α1β1γδ and α1β1γδL121C receptors before and after MTSEA treatment (5 mM for 60 s) as indicated. Bursts from α1β1γδL121C receptors before and after MTSEA treatment (indicated by * or #) are shown on the bottom row in higher time resolution together with the closed-duration histogram from α1β1γδL121C (before MTSEA treatment) used to define bursts. Currents were sampled at 100 kHz and ultimately low-pass filtered to 5 kHz. Each histogram represents the data pooled from at least individual four patches recorded under identical conditions, except for the 10-nM ACh concentration histogram, where data were pooled from three patches. Fit parameters are listed in Table IV.

Amin, Insights into the activation mechanism of rho1 GABA receptors obtained by coexpression of wild type and activation-impaired subunits. 1996, Pubmed, Xenbase

Amin, Insights into the activation mechanism of rho1 GABA receptors obtained by coexpression of wild type and activation-impaired subunits. 1996, Pubmed , Xenbase
Barron, An allosteric modulator of alpha7 nicotinic receptors, N-(5-Chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)-urea (PNU-120596), causes conformational changes in the extracellular ligand binding domain similar to those caused by acetylcholine. 2009, Pubmed , Xenbase
Beato, The activation mechanism of alpha1 homomeric glycine receptors. 2004, Pubmed
Brejc, Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. 2001, Pubmed
Celie, Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. 2004, Pubmed
Colquhoun, Fast events in single-channel currents activated by acetylcholine and its analogues at the frog muscle end-plate. 1985, Pubmed
Colquhoun, Fluctuations in the microsecond time range of the current through single acetylcholine receptor ion channels. 1981, Pubmed
Dani, Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system. 2007, Pubmed
Descarries, Diffuse transmission by acetylcholine in the CNS. 1997, Pubmed
Franke, Liquid filament switch for ultra-fast exchanges of solutions at excised patches of synaptic membrane of crayfish muscle. 1987, Pubmed
Groebe, alpha-Conotoxins selectively inhibit one of the two acetylcholine binding sites of nicotinic receptors. 1995, Pubmed
Guex, SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. 1997, Pubmed
Hansen, Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations. 2005, Pubmed
Horenstein, Reversal of agonist selectivity by mutations of conserved amino acids in the binding site of nicotinic acetylcholine receptors. 2007, Pubmed , Xenbase
Jackson, Kinetics of unliganded acetylcholine receptor channel gating. 1986, Pubmed
Jackson, Spontaneous openings of the acetylcholine receptor channel. 1984, Pubmed
Jaramillo, Kinetic differences between embryonic- and adult-type acetylcholine receptors in rat myotubes. 1988, Pubmed
Jha, Acetylcholine receptor channels activated by a single agonist molecule. 2010, Pubmed
Jope, Seizures increase acetylcholine and choline concentrations in rat brain regions. 1991, Pubmed
KATZ, A study of the desensitization produced by acetylcholine at the motor end-plate. 1957, Pubmed
Karlin, The affinity-labeling of partially purified acetylcholine receptor from electric tissue of Electrophorus. 1973, Pubmed
Labarca, The occurrence of long openings in the purified cholinergic receptor channel increases with acetylcholine concentration. 1985, Pubmed
Li, Characterization of the neuroprotective and toxic effects of alpha7 nicotinic receptor activation in PC12 cells. 1999, Pubmed , Xenbase
Mishina, Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. , Pubmed , Xenbase
Mortensen, Single-channel recording of ligand-gated ion channels. 2007, Pubmed
Mott, Open probability of homomeric murine 5-HT3A serotonin receptors depends on subunit occupancy. 2001, Pubmed , Xenbase
Numa, Molecular structure of the nicotinic acetylcholine receptor. 1983, Pubmed
Palma, Neuronal nicotinic alpha 7 receptor expressed in Xenopus oocytes presents five putative binding sites for methyllycaconitine. 1996, Pubmed , Xenbase
Papke, Relationships of agonist properties to the single channel kinetics of nicotinic acetylcholine receptors. 1988, Pubmed
Papke, Comparative pharmacology of rat and human alpha7 nAChR conducted with net charge analysis. 2002, Pubmed , Xenbase
Papke, An evaluation of neuronal nicotinic acetylcholine receptor activation by quaternary nitrogen compounds indicates that choline is selective for the alpha 7 subtype. 1996, Pubmed , Xenbase
Papke, alpha7 receptor-selective agonists and modes of alpha7 receptor activation. 2000, Pubmed
Papke, Mechanisms of noncompetitive inhibition of acetylcholine-induced single-channel currents. 1989, Pubmed
Papke, Cysteine accessibility analysis of the human alpha7 nicotinic acetylcholine receptor ligand-binding domain identifies L119 as a gatekeeper. 2011, Pubmed , Xenbase
Papke, The correction of alpha7 nicotinic acetylcholine receptor concentration-response relationships in Xenopus oocytes. 1998, Pubmed , Xenbase
Rayes, Number and locations of agonist binding sites required to activate homomeric Cys-loop receptors. 2009, Pubmed
Sakmann, Role of acetylcholine receptor subunits in gating of the channel. , Pubmed , Xenbase
Schuetze, Developmental regulation of nicotinic acetylcholine receptors. 1987, Pubmed
Scremin, Time-dependent changes in cerebral choline and acetylcholine induced by transient global ischemia in rats. 1991, Pubmed
Sine, Identification of equivalent residues in the gamma, delta, and epsilon subunits of the nicotinic receptor that contribute to alpha-bungarotoxin binding. 1997, Pubmed
Sine, The nicotinic receptor ligand binding domain. 2002, Pubmed
Sine, Activation of a nicotinic acetylcholine receptor. 1984, Pubmed
Sine, Activation of acetylcholine receptors on clonal mammalian BC3H-1 cells by low concentrations of agonist. 1986, Pubmed
Sine, The relationship between agonist occupation and the permeability response of the cholinergic receptor revealed by bound cobra alpha-toxin. 1980, Pubmed
Sine, Activation of acetylcholine receptors on clonal mammalian BC3H-1 cells by high concentrations of agonist. 1987, Pubmed
Sine, Agonists block currents through acetylcholine receptor channels. 1984, Pubmed
Sivilotti, Acetylcholine receptors: too many channels, too few functions. 1995, Pubmed
Stokes, The structural basis for GTS-21 selectivity between human and rat nicotinic alpha7 receptors. 2004, Pubmed , Xenbase
Suzuki, Microglial alpha7 nicotinic acetylcholine receptors drive a phospholipase C/IP3 pathway and modulate the cell activation toward a neuroprotective role. 2006, Pubmed
Takeda, A patch-clamp study of the partial agonist actions of tubocurarine on rat myotubes. 1984, Pubmed
Uteshev, Regulation of neuronal function by choline and 4OH-GTS-21 through alpha 7 nicotinic receptors. 2003, Pubmed
Uteshev, Activation and inhibition of native neuronal alpha-bungarotoxin-sensitive nicotinic ACh receptors. 2002, Pubmed
Wang, Tethered agonist analogs as site-specific probes for domains of the human α7 nicotinic acetylcholine receptor that differentially regulate activation and desensitization. 2010, Pubmed , Xenbase
Wang, Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. 2003, Pubmed
de Jonge, The alpha7 nicotinic acetylcholine receptor as a pharmacological target for inflammation. 2007, Pubmed