Clare Stokes

Extending the analysis of nicotinic receptor antagonists with the study of α6 nicotinic receptor subunit chimeras

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.

Antagonist activities of mecamylamine and nicotine show reciprocal dependence on beta subunit sequence in the second transmembrane domain

We show that a portion of the TM2 domain regulates the sensitivity of beta subunit‐containing rat neuronal nicotinic AChR to the ganglionic blocker mecamylamine, such that the substitution of 4 amino acids of the muscle beta subunit sequence into the neuronal beta4 sequence decreases the potency of mecamylamine by a factor of 200 and eliminates any long‐term effects of this drug on receptor function. The same exchange of sequence that decreases inhibition by mecamylamine produces a comparable potentiation of long‐term inhibition by nicotine. Inhibition by mecamylamine is voltage‐dependent, suggesting a direct interaction of mecamylamine with sequence elements within the membrane field. We have previously shown that sensitivity to TMP (tetramethylpiperidine) inhibitors is controlled by the same sequence elements that determine mecamylamine sensitivity. However, inhibition by bis‐TMP compounds is independent of voltage. Our experiments did not show any influence of voltage on the inhibition of chimeric receptors by nicotine, suggesting that the inhibitory effects of nicotine are mediated by binding to a site outside the membranes electric field. An analysis of point mutations indicates that the residues at the 6′ position within the beta subunit TM2 domain may be important for determining the effects of both mecamylamine and nicotine in a reciprocal manner. Single mutations at the 10′ position are not sufficient to produce effects, but 6′ 10′ double mutants show more effect than do the 6′ single mutants.

The effective opening of nicotinic acetylcholine receptors with single agonist binding sites

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.

Working with OpusXpress: Methods for high volume oocyte experiments

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.

Reversal of agonist selectivity by mutations of conserved amino acids in the binding site of nicotinic acetylcholine receptors.

Homomeric α7 and heteromeric α4β2 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 α7 receptors for which the α7-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 α4 receptors co-expressed with β2 resulted in α4β2 receptors for which ACh potency was relatively unchanged, although the efficacy of the α7-selective agonist 4OH-GTS-21 was increased greatly relative to that of ACh. We also investigated the significance of a conserved lysine (145 in α7), proposed to form a stable salt bridge with Asp-197 in the resting state of the receptor. Mutations of this residue in both α7 and α4 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 α4Y190F-binding site, large hydrophobic benzylidene anabaseines may close the C loop and initiate channel gating more effectively than the polar agonist ACh.

Looking below the surface of nicotinic acetylcholine receptors

The amino acid sequences of nicotinic acetylcholine receptors (nAChRs) from diverse species can be compared across extracellular, transmembrane, and intracellular domains. The intracellular domains are most divergent among subtypes, yet relatively consistent among species. The diversity indicates that each nAChR subtype has a unique language for communication with its host cell. The conservation across species also suggests that the intracellular domains have defining functional roles for each subtype. Secondary structure prediction indicates two relatively conserved alpha helices within the intracellular domains of all nAChRs. Among all subtypes, the intracellular domain of α7 nAChR is one of the most well conserved, and α7 nAChRs have effects in non-neuronal cells independent of generating ion currents, making it likely that the α7 intracellular domain directly mediates signal transduction. There are potential phosphorylation and protein-binding sites in the α7 intracellular domain, which are conserved and may be the basis for α7-mediated signal transduction.

The nicotinic acetylcholine receptors of zebrafish and an evaluation of pharmacological tools used for their study

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.

Modeling Binding Modes of α7 Nicotinic Acetylcholine Receptor with Ligands: The Roles of Gln117 and Other Residues of the Receptor in Agonist Binding

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.

Use of an α3β4 nicotinic acetylcholine receptor subunit concatamer to characterize ganglionic receptor subtypes with specific subunit composition reveals species-specific pharmacologic properties

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.

Cysteine accessibility analysis of the human alpha7 nicotinic acetylcholine receptor ligand-binding domain identifies L119 as a gatekeeper.

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.