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ABSTRACT: The homomeric α7 nicotinic acetylcholine receptor is a well-studied therapeutic target, though its characteristically rapid desensitization complicates the development of drugs with specific agonist effects. Moreover, some experimental compounds such as GTS-21 (2,4diMeOBA), a derivative of the α7-selective partial agonist benzylidene anabaseine (BA), produce a prolonged residual desensitization (RD) in which the receptor remains non-activatable long after the drug has been removed from extracellular solution. In contrast, the desensitization caused by GTS-21's dihydroxy metabolite (2,4diOHBA) is relatively short-lived. RD is hypothetically due to stable binding of the ligand to the receptor in its desensitized state. We can attribute the reduction in RD to a single BA hydroxyl group on the 4' benzylidene position. Computational prediction derived from homology modeling showed the serine36 (S36) residue of α7 as a reasonable candidate for point-to-point interaction between BA compounds and the receptor. Through evaluating the activity of BA and simple derivatives on wild-type and mutant α7 receptors, it was observed that the drug-receptor pairs which were capable of hydrogen bonding at residue 36 exhibited significantly less stable desensitization. Further experiments involving the type II positive allosteric modulator (PAM) PNU-120596 showed that the various BA compounds' preference to induce either a PAM-sensitive (D(s)) or PAM-insensitive (D(i)) desensitized state is concentration dependent and suggested that both states are destabilized by S36 H-bonding. These results indicate that the fine-tuning of agonists for specific interaction with S36 can facilitate the development of therapeutics with targeted effects on ion channel desensitization properties and conformational state stability.
Biochemical pharmacology 01/2013; · 4.25 Impact Factor
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ABSTRACT: Drug development for nicotinic acetylcholine receptors (nAChR) is challenged by subtype diversity arising from variations in subunit composition. On-target activity for neuronal heteromeric receptors is typically associated with CNS receptors that contain α4 and other subunits, while off-target activity could be associated with ganglionic-type receptors containing α3β4 binding sites and other subunits, including β4, β2, α5, or α3 as a structural subunit in the pentamer. Additional interest in α3 β4 α5-containing receptors arises from genome-wide association studies linking these genes, and a single nucleotide polymorphism (SNP) in α5 in particular, to lung cancer and heavy smoking. While α3 and β4 readily form receptors in expression system such as the Xenopus oocyte, since α5 is not required for function, simple co-expression approaches may under-represent α5-containing receptors. We used a concatamer of human α3 and β4 subunits to form ligand-binding domains, and show that we can force the insertions of alternative structural subunits into the functional pentamers. These α3β4 variants differ in sensitivity to ACh, nicotine, varenicline, and cytisine. Our data indicated lower efficacy for varenicline and cytisine than expected for β4-containing receptors, based on previous studies of rodent receptors. We confirm that these therapeutically important α4 receptor partial agonists may present different autonomic-based side-effect profiles in humans than will be seen in rodent models, with varenicline being more potent for human than rat receptors and cytisine less potent. Our initial characterizations failed to find functional effects of the α5 SNP. However, our data validate this approach for further investigations.
Neuropharmacology 05/2012; 63(4):538-46. · 4.81 Impact Factor
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ABSTRACT: Zebrafish (Danio rerio) have been used to study multiple effects of nicotine, for example on cognition, locomotion, and stress responses, relying on the assumption that pharmacological tools will operate similarly upon molecular substrates in the fish and mammalian systems. We have cloned the zebrafish nicotinic acetylcholine receptor (nAChR) subunits and expressed key nAChR subtypes in Xenopus oocytes including neuronal (α4β2, α2β2, α3β4, and α7) and muscle (α1β1(b)ɛδ) nAChR. Consistent with studies of mammalian nAChR, nicotine was relatively inactive on muscle-type receptors, having both low potency and efficacy. It had high efficacy but low potency for α7 receptors, and the best potency and good efficacy for α4β2 receptors. Cytisine, a key lead compound for the development of smoking cessation agents, is a full agonist for both mammalian α7 and α3β4 receptors, but a full agonist only for the fish α7, with surprisingly low efficacy for α3β4. The efficacy of cytisine for α4β2 was somewhat greater than typically reported for mammalian α4β2. The ganglionic blocker mecamylamine was most potent for blocking α3β4 receptors, least potent for α7, and roughly equipotent for the muscle receptors and the β2-containing nAChR. However, the block of β2-containing receptors was slowly reversible, consistent with effective targeting of these CNS-type receptors in vivo. Three prototypical α7-selective agonists, choline, tropane, and 4OH-GTS-21, were tested, and these agents were observed to activate both fish α7 and α4β2 nAChR. Our data therefore indicate that while some pharmacological tools used in zebrafish may function as expected, others will not.
Biochemical pharmacology 05/2012; 84(3):352-65. · 4.25 Impact Factor
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ABSTRACT: A series of arylidene anabaseines were synthesized to probe the functional impact of hydrogen bonding on human α7 nicotinic acetylcholine receptor (nAChR) activation and desensitization. The aryl groups were either hydrogen bond acceptors (furans), donors (pyrroles), or neither (thiophenes). These compounds were tested against a series of point mutants of the ligand-binding domain residue Gln-57, a residue hypothesized to be proximate to the aryl group of the bound agonist and a putative hydrogen bonding partner. Q57K, Q57D, Q57E, and Q57L were chosen to remove the dual hydrogen bonding donor/acceptor ability of Gln-57 and replace it with hydrogen bond donating, hydrogen bond accepting, or nonhydrogen bonding ability. Activation of the receptor was compromised with hydrogen bonding mismatches, for example, pairing a pyrrole with Q57K or Q57L, or a furan anabaseine with Q57D or Q57E. Ligand co-applications with the positive allosteric modulator PNU-120596 produced significantly enhanced currents whose degree of enhancement was greater for 2-furans or -pyrroles than for their 3-substituted isomers, whereas the nonhydrogen bonding thiophenes failed to show this correlation. Interestingly, the PNU-120596 agonist co-application data revealed that for wild-type α7 nAChR, the 3-furan desensitized state was relatively stabilized compared with that of 2-furan, a reversal of the relationship observed with respect to the barrier for entry into the desensitized state. These data highlight the importance of hydrogen bonding on the receptor-ligand state, and suggest that it may be possible to fine-tune features of agonists that mediate state selection in the nAChR.
Journal of Biological Chemistry 05/2012; 287(26):21957-69. · 4.77 Impact Factor
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ABSTRACT: We have identified a means by which agonist-evoked responses of nicotinic receptors can be conditionally eliminated. Modification of α7L119C mutants by the sulfhydryl reagent 2-aminoethyl methanethiosulfonate (MTSEA) reduces responses to acetylcholine (ACh) by more than 97%, whereas corresponding mutations in muscle-type receptors produce effects that depend on the specific subunits mutated and ACh concentration. We coexpressed α7L119C subunits with pseudo wild-type α7C116S subunits, as well as ACh-insensitive α7Y188F subunits with wild-type α7 subunits in Xenopus laevis oocytes using varying ratios of cRNA. When mutant α7 cRNA was coinjected at a 5:1 ratio with wild-type cRNA, net charge responses to 300 µM ACh were retained by α7L119C-containing mutants after MTSEA modification and by the ACh-insensitive Y188F-containing mutants, even though the expected number of ACh-sensitive wild-type binding sites would on average be fewer than two per receptor. Responses of muscle-type receptors with one MTSEA-sensitive subunit were reduced at low ACh concentrations, but much less of an effect was observed when ACh concentrations were high (1 mM), indicating that saturation of a single binding site with agonist can evoke strong activation of nicotinic ACh receptors. Single-channel patch clamp analysis revealed that the burst durations of fetal wild-type and α1β1γδL121C receptors were equivalent until the α1β1γδL121C mutants were exposed to MTSEA, after which the majority (81%) of bursts were brief (≤2 ms). The longest duration events of the receptors modified at only one binding site were similar to the long bursts of native receptors traditionally associated with the activation of receptors with two sites containing bound agonists.
The Journal of General Physiology 04/2011; 137(4):369-84. · 3.84 Impact Factor
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ABSTRACT: A large number of structurally diverse ligands have been produced to selectively target α7 nicotinic acetylcholine receptors (nAChRs). We applied the method of scanning cysteine accessibility mutations (SCAM) to the ligand-binding domain of the α7 nAChR to identify subdomains of particular importance to the binding and subsequent activation by select agonists. We evaluated the activity of four structurally distinct α7 agonists on wild-type human α7 and 44 targeted mutants expressed in Xenopus oocytes. Responses were measured prior and subsequent to the application of the sulfhydryl reagent methanethiosulfonate ethylammonium (MTSEA). One mutant (C116S) served as a Cys-null control, and the additional mutants were made in the C116S background. In many cases, the insertion of free cysteines into the agonist-binding site had a negative effect on function, with 12 of 44 mutants showing no detectable responses to ACh, and with only 19 of the 44 mutants showing sufficiently large responses to permit further study. Several of the cysteine mutations, including W55C, showed selectively reduced responses to the largest agonist tested, 2-methoxy,4-hydroxy-benzylidene anabaseine. Interestingly, although homology models suggest that most of the introduced cysteine mutations should have had good solvent accessibility, application of MTSEA had no effect or produced only modest changes in the agonist response profile of most mutants. Consistent with previous studies implicating W55 to play important roles in agonist activation, MTSEA treatment further decreased the functional responses of W55C to all the test agonists. While the cysteine mutation at L119 itself had relatively little effect on receptor function, treatment of L119C receptors with MTSEA or alternative cationic sulfhydryl reagents profoundly decreased activation by all agonists tested, suggesting a general block of gating. The homologous mutation in heteromeric nAChRs produced similar results, provided that the mutation was placed in the beta subunit complementary surface of the ligand-binding domain. Structural models locate the L119 residue directly across the subunit interface from the C-loop of the primary face of the binding domain. Our data suggest that a covalent modification of L119C by MTSEA or other cationic reagents might block the binding of even small agonists such as TMA through electrostatic interactions. Reaction of L119C with small non-polar reagents increases activation by small agonists but can block the access of large ligands such as benzylidene anabaseines to the ligand-binding domain.
Neuropharmacology 01/2011; 60(1):159-71. · 4.81 Impact Factor
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ABSTRACT: Homomeric α7 nicotinic acetylcholine receptors represent an important and complex pharmaceutical target. They can be activated by structurally diverse agonists and are highly likely to enter and remain in desensitized states at rates determined by the structures of the agonists. To identify structural elements regulating this function, we introduced reactive cysteines into the α7 ligand-binding domain allowing us to bind sulfhydryl-reactive (SH) agonist analogs or control reagents onto specific positions in the ligand binding domain. We identified four α7 mutants (S36C, L38C, W55C, and L119C) in which the tethering of the SH reagents blocked further acetylcholine-evoked activation of the receptor. However, after selective reaction with SH agonist analogs, the type II allosteric modulator N-(5-chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl-3-isoxazolyl)-urea (PNU-120596) could reactivate L119C and W55C mutants and receptors with a reduced or modified C-loop. Modified S36C and L38C mutants were insensitive to reactivation by PNU-120596, whether they were reacted with agonist analogs or alternative SH reagents. Molecular modeling showed that in the W55C and L119C mutants, the ammonium pharmacophore of the agonist analog methanethiosulfonate-ethyltrimethylammonium would be in a similar but nonidentical position underneath the C-loop. The orientation assumed by the ligand tethered to 119C was approximately 3-fold more sensitive to PNU-120596 than the alternative pose at 55C. Our results support the hypothesis that a single ligand can bind within the receptor in different ways and, depending on the specific binding pose, may variously promote activation or desensitization, or, alternatively, function as a competitive antagonist. This insight may provide a new approach for drug development.
Molecular pharmacology 12/2010; 78(6):1012-25. · 4.53 Impact Factor
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ABSTRACT: OpusXpress is a semi-automated system for high throughput voltage clamp recording from Xenopus oocytes. We participated in the development process for this system and were the only laboratory to field test a prototype. Subsequently, we obtained an early production model that we have used on a regular basis for the last seven years, conducting many thousands of experiments, publishing extensively, and carrying out collaborative research in drug discovery. In this article, we relate our experience with the OpusXpress recording system and large volume oocyte handling. We provide our standard operating procedures and outline the organization of our successful team. Some of our advice is specific to researchers fortunate enough to have access to an OpusXpress system, but most of it is applicable to any group using Xenopus oocytes for the heterologous expression of ion channels.
Methods 05/2010; 51(1):121-33. · 4.01 Impact Factor
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ABSTRACT: We have shown previously that a highly conserved Tyr in the nicotinic acetylcholine receptor (nAChR) ligand-binding domain (LBD) (alpha7 Tyr188 or alpha4 Tyr195) differentially regulates the activity of acetylcholine (ACh) and the alpha7-selective agonist 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21) in alpha4beta2 and alpha7 nAChR. In this study, we mutated two highly conserved LBD Trp residues in human alpha7 and alpha4beta2 and expressed the receptors in Xenopus laevis oocytes. Alpha7 receptors with Trp55 mutated to Gly or Tyr became less responsive to 4OH-GTS-21, whereas mutation of the homologous Trp57 in beta2 to Gly, Tyr, Phe, or Ala resulted in alpha4beta2 receptors that showed increased responses to 4OH-GTS-21. Mutation of alpha7 Trp55 to Val resulted in receptors for which the partial agonist 4OH-GTS-21 became equally efficacious as ACh, whereas alpha4beta2 receptors with the homologous mutation remained nonresponsive to 4OH-GTS-21. In contrast to the striking alterations in agonist activity profiles that were observed with mutations of alpha7 Trp55 and beta2 Trp57, mutations of alpha7 Trp149 or alpha4 Trp154 universally resulted in receptors with reduced function. Our data support the hypothesis that some conserved residues in the nAChR LBD differentially regulate receptor activation by subtype-selective agonists, whereas other equally well conserved residues play fundamental roles in receptor activation by any agonist. Residues like alpha7 Trp149 (alpha4 Trp154) may be considered pillars upon which basic receptor function depends, whereas alpha7 Trp55 (beta2 Trp57) and alpha7 Tyr188 (alpha4 Tyr195) may be fulcra upon which agonists may operate differentially in specific receptor subtypes, consistent with the hypothesis that ACh and 4OH-GTS-21 are able to activate nAChR in distinct ways.
Journal of Pharmacology and Experimental Therapeutics 05/2009; 330(1):40-53. · 3.83 Impact Factor
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Journal of Medicinal Chemistry 02/2009; · 4.80 Impact Factor
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ABSTRACT: Extensive molecular docking, molecular dynamics simulations, and binding free energy calculations have been performed to understand how alpha7-specific agonists of nicotinic acetylcholine receptor (nAChR), including AR-R17779 (1), GTS-21 (4), and 4-OH-GTS-21 (5), interact with the alpha7 receptor, leading to important new insights into the receptor-agonist binding. In particular, the cationic head of 4 and 5 has favorable hydrogen bonding and cation-pi interactions with residue Trp149. The computational results have also led us to better understand the roles of Gln117 and other residues in the receptor binding with agonists. The computational predictions are supported by data obtained from wet experimental tests. The new insights into the binding and structure-activity relationship obtained from this study should be valuable for future rational design of more potent and selective agonists of the alpha7 receptor.
Journal of Medicinal Chemistry 11/2008; 51(20):6293-302. · 4.80 Impact Factor
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ABSTRACT: Heterologous expression systems have increased the feasibility of developing selective ligands to target nicotinic acetylcholine receptor (nAChR) subtypes. However, the alpha6 subunit, a component in nAChRs that mediates some of the reinforcing effects of nicotine, is not easily expressed in systems such as the Xenopus oocyte. Certain aspects of alpha6-containing receptor pharmacology have been studied by using chimeric subunits containing the alpha6 ligand-binding domain. However, these chimeras would not be sensitive to an alpha6-selective channel blocker; therefore we developed an alpha6 chimera (alpha4/6) that has the transmembrane and intracellular domains of alpha6 and the extracellular domain of alpha4. We examined the pharmacological properties of alpha4/6-containing receptors and other important nAChR subtypes, including alpha7, alpha4beta2, alpha4beta4, alpha3beta4, alpha3beta2, and alpha3beta2beta3, as well as receptors containing alpha6/3 and alpha6/4 chimeras. Our data show that the absence or presence of the beta4 subunit is an important factor for sensitivity to the ganglionic blocker mecamylamine, and that dihydro-beta-erythroidine is most effective on subtypes containing the alpha4 subunit extracellular domain. Receptors containing the alpha6/4 subunit are sensitive to alpha-conotoxin PIA, while receptors containing the reciprocal alpha4/6 chimera are insensitive. In experiments with novel antagonists of nicotine-evoked dopamine release, the alpha4/6 chimera indicated that structural rigidity was a key element of compounds that could result in selectivity for noncompetitive inhibition of alpha6-containing receptors. Our data extend the information available on prototypical nAChR antagonists, and establish the alpha4/6 chimera as a useful new tool for screening drugs as selective nAChR antagonists.
Neuropharmacology 07/2008; 54(8):1189-200. · 4.81 Impact Factor
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ABSTRACT: Homomeric alpha7 and heteromeric alpha4beta2 nicotinic acetylcholine receptors (nAChR) can be distinguished by their pharmacological properties, including agonist specificity. We introduced point mutations of conserved amino acids within the C loop, a region of the receptor critical for agonist binding, and we examined the expression of the mutant receptors in Xenopus oocytes. Mutation of either a conserved C loop tyrosine (188) to phenylalanine or a nearby conserved aspartate (197) to alanine resulted in alpha7 receptors for which the alpha7-selective agonist 3-(4-hydroxy, 2-methoxybenzylidene) anabaseine (4OH-GTS-21) had roughly the same potency as for wild-type receptors, whereas the physiologic agonist acetylcholine (ACh) showed drastically reduced potency for these mutant receptors. Corresponding mutations in alpha4 receptors co-expressed with beta2 resulted in alpha4beta2 receptors for which ACh potency was relatively unchanged, although the efficacy of the alpha7-selective agonist 4OH-GTS-21 was increased greatly relative to that of ACh. We also investigated the significance of a conserved lysine (145 in alpha7), proposed to form a stable salt bridge with Asp-197 in the resting state of the receptor. Mutations of this residue in both alpha7 and alpha4 resulted in receptors that were largely unresponsive to both ACh and 4OH-GTS-21. Our results suggest that initiation of gating depends both on specific interactions between residues in the C loop domain and, depending on receptor subtype, the physiochemical properties of the agonist, so that in the altered environment of the alpha4Y190F-binding site, large hydrophobic benzylidene anabaseines may close the C loop and initiate channel gating more effectively than the polar agonist ACh.
Journal of Biological Chemistry 03/2007; 282(8):5899-909. · 4.77 Impact Factor
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ABSTRACT: An alpha7 nicotinic acetylcholine receptor sequence was cloned from Rhesus monkey (Macaca mulatta). This clone differs from the mature human alpha7 nicotinic acetylcholine receptor in only four amino acids, two of which are in the extracellular domain. The monkey alpha7 nicotinic receptor was characterized in regard to its functional responses to acetylcholine, choline, cytisine, and the experimental alpha7-selective agonists 4OH-GTS-21, TC-1698, and AR-R17779. For all of these agonists, the EC(50) for activation of monkey receptors was uniformly higher than for human receptors. In contrast, the potencies of mecamylamine and MLA for inhibiting monkey and human alpha7 were comparable. Acetylcholine and 4OH-GTS-21 were used to probe the significance of the single point differences in the extracellular domain. Mutants with the two different amino acids in the extracellular domain of the monkey receptor changed to the corresponding sequence of the human receptor had responses to these agonists that were not significantly different in EC(50) from wild-type human alpha7 nicotinic receptors. Monkey alpha7 nicotinic receptors have a serine at residue 171, while the human receptors have an asparagine at this site. Monkey S171N mutants were more like human alpha7 nicotinic receptors, while mutations at the other site (K186R) had relatively little effect. These experiments point toward the basic utility of the monkey receptor as a model for the human alpha7 nicotinic receptor, albeit with the caveat that these receptors will vary in their agonist concentration dependency. They also point to the potential importance of a newly identified sequence element for modeling the specific amino acids involved with receptor activation.
European Journal of Pharmacology 12/2005; 524(1-3):11-8. · 2.52 Impact Factor
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ABSTRACT: The alpha7 nAChR-selective partial agonist 3-(2,4-dimethoxybenzylidene)anabaseine (GTS-21) is more efficacious and potent for rat receptors than for human alpha7 receptors. Four single amino acid differences exist between human and rat alpha7 in the agonist binding site, two in the C loop, and one each in the E and F loops. Reciprocal mutations were made in these three domains and evaluated in Xenopus laevis oocytes. Mutations in the C and F loops significantly increased the efficacy of GTS-21 for the human receptor mutants but not to the level of the wild-type, and reciprocal mutations in rat alpha7 did not decrease responses to GTS-21. Whereas mutations in the E loop alone were without effect, the E- and F-loop mutations together increased GTS-21 efficacy and potency for human receptors, but the EF mutations in the rat receptors decreased the GTS-21 potency without changing the efficacy. The only mutants that showed a full reversal of the efficacy differences between human and rat alpha7 contained complete exchange of all four sites in the C, E, and F loops or just the sites in the C and F loops. However, the reversal of the potency ratio seen with the EF mutants was not evident in the CEF mutants. Our data therefore indicate that the pharmacological differences between rat and human alpha7 receptors are caused by reciprocal differences in sites within and around the binding site. Specific features in the agonist molecule itself are also identified that interact with these structural features of the receptor agonist binding site.
Molecular Pharmacology 08/2004; 66(1):14-24. · 4.88 Impact Factor
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ABSTRACT: Heterologous expression systems have increased the feasibility of developing selective ligands to target nicotinic acetylcholine receptor (nAChR) subtypes. However, the α6 subunit, a component in nAChRs that mediates some of the reinforcing effects of nicotine, is not easily expressed in systems such as the Xenopus oocyte. Certain aspects of α6-containing receptor pharmacology have been studied by using chimeric subunits containing the α6 ligand-binding domain. However, these chimeras would not be sensitive to an α6-selective channel blocker; therefore we developed an α6 chimera (α4/6) that has the transmembrane and intracellular domains of α6 and the extracellular domain of α4. We examined the pharmacological properties of α4/6-containing receptors and other important nAChR subtypes, including α7, α4β2, α4β4, α3β4, α3β2, and α3β2β3, as well as receptors containing α6/3 and α6/4 chimeras. Our data show that the absence or presence of the β4 subunit is an important factor for sensitivity to the ganglionic blocker mecamylamine, and that dihydro-β-erythroidine is most effective on subtypes containing the α4 subunit extracellular domain. Receptors containing the α6/4 subunit are sensitive to α-conotoxin PIA, while receptors containing the reciprocal α4/6 chimera are insensitive. In experiments with novel antagonists of nicotine-evoked dopamine release, the α4/6 chimera indicated that structural rigidity was a key element of compounds that could result in selectivity for noncompetitive inhibition of α6-containing receptors. Our data extend the information available on prototypical nAChR antagonists, and establish the α4/6 chimera as a useful new tool for screening drugs as selective nAChR antagonists.
Neuropharmacology.
