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

Nicotinic Activity of Arecoline, the Psychoactive Element of "Betel Nuts", Suggests a Basis for Habitual Use and Anti-Inflammatory Activity.

Habitual chewing of "betel nut" preparations constitutes the fourth most common human self-administration of a psychoactive substance after alcohol, caffeine, and nicotine. The primary active ingredient in these preparations is arecoline, which comes from the areca nut, the key component of all such preparations. Arecoline is known to be a relatively non-selective muscarinic partial agonist, accounting for many of the overt peripheral and central nervous system effects, but not likely to account for the addictive properties of the drug. We report that arecoline has activity on select nicotinic acetylcholine receptor (nAChR) subtypes, including the two classes of nAChR most related to the addictive properties of nicotine: receptors containing α4 and β2 subunits and those which also contain α6 and β3 subunits. Arecoline is a partial agonist with about 6-10% efficacy for the α4* and α6* receptors expressed in Xenopus oocytes. Additionally, arecoline is a silent agonist of α7 nAChR; while it does not activate α7 receptors when applied alone, it produces substantial activation when co-applied with the positive allosteric modulator PNU-120696. Some α7 silent agonists are effective inhibitors of inflammation, which might account for anti-inflammatory effects of arecoline. Arecoline's activity on nAChR associated with addiction may account for the habitual use of areca nut preparations in spite of the well-documented risk to personal health associated with oral diseases and cancer. The common link between betel and tobacco suggests that partial agonist therapies with cytisine or the related compound varenicline may also be used to aid betel cessation attempts

Sulfonium Ligands of the α7 nAChR

The α7 nicotinic acetylcholine receptor (nAChR) is an important target given its role in cognitive function as well as in the cholinergic anti-inflammatory pathway, where ligands that are effective at stabilizing desensitized states of the receptor are of particular interest. The typical structural element associated with a good desensitizer is the ammonium pharmacophore, but recent work has identified that a trivalent sulfur, in the positively charged sulfonium form, can substitute for the nitrogen in the ammonium pharmacophore. However, the breadth and scope of employing the sulfonium group is largely unexplored. In this work, we have surveyed a disparate group of sulfonium compounds for their functional activity with α7 as well as other nAChR subtypes. Amongst them, we found that there is a wide range of ability to induce α7 desensitization, with 4-hydroxyphenyldimethylsulfonium and suplatast sulfonium salts being the most desensitizing. The smallest sulfonium compound, trimethylsulfonium, was a partial agonist for α7 and other neuronal nAChR. Molecular docking into the α7 receptor extracellular domain revealed preferred poses in the orthosteric binding site for all but one compound, with typical cation–pi interactions as seen with traditional ammonium compounds. A number of the compounds tested may serve as useful platforms for further development of α7 desensitizing ability and for receptor subtype selectivity

Differential Regulation of Receptor Activation and Agonist Selectivity by Highly Conserved Tryptophans in the Nicotinic Acetylcholine Receptor Binding Site

We have shown previously that a highly conserved Tyr in the nicotinic acetylcholine receptor (nAChR) ligand-binding domain (LBD) (α7 Tyr188 or α4 Tyr195) differentially regulates the activity of acetylcholine (ACh) and the α7-selective agonist 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21) in α4β2 and α7 nAChR. In this study, we mutated two highly conserved LBD Trp residues in human α7 and α4β2 and expressed the receptors in Xenopus laevis oocytes. α7 Receptors with Trp55 mutated to Gly or Tyr became less responsive to 4OH-GTS-21, whereas mutation of the homologous Trp57 in β2 to Gly, Tyr, Phe, or Ala resulted in α4β2 receptors that showed increased responses to 4OH-GTS-21. Mutation of α7 Trp55 to Val resulted in receptors for which the partial agonist 4OH-GTS-21 became equally efficacious as ACh, whereas α4β2 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 α7 Trp55 and β2 Trp57, mutations of α7 Trp149 or α4 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 α7 Trp149 (α4 Trp154) may be considered pillars upon which basic receptor function depends, whereas α7 Trp55 (β2 Trp57) and α7 Tyr188 (α4 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

Arecoline activation of other nAChR subtypes.

<p>100 μM arecoline was applied to cells expressing the nAChR subunits indicated. Responses of human α3β4, α7, and mouse muscle (α1β1εδ) subunits were barely at the threshold of detection, less than 1% the ACh maximum, extrapolated from comparisons to ACh controls and ACh concentration-response studies conducted previously. The responses of cells expressing α4β2 or a concatamer containing α6 and β3 in addition to α4 and β2 were substantially larger and well above the threshold of detection.</p

Modulation of HS α4β2 receptors with a low concentration of arecoline.

<p>(A) Partial agonists for α4β2 nAChR such as varenicline and cytisine [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140907#pone.0140907.ref035" target="_blank">35</a>] modulate the sensitivity of the receptors to the endogenous activator ACh through pre-desensitization for prolonged periods, even when present at very low concentrations. They can also stimulate low levels of tonic activation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140907#pone.0140907.ref035" target="_blank">35</a>]. After obtaining initial control responses to ACh, a steady flow of 3 μM arecoline was applied to the bath. After 8 minutes the responses to a control application of ACh was reduced approximately 50%. (B) The perfusion of 3 μM arecoline was continued, and 100 μM mecamylamine was applied along with the arecoline to reveal the mecamylamine-sensitive steady-state current. The decrease in inward current shown is the averaged response of seven cells. Normalized relative to initial ACh controls and adjusted for ACh maximum, these currents indicated steady-state activation of approximately 1% ACh maximum.</p

Effects 100 μM arecoline (structure illustrated) on oocytes expressing α7 nAChR.

<p>Cells were tested for their responses to control applications of 60 μM ACh prior to the application of the test solution. The second of two such control applications is shown. After a 4 minute wash period, 100 μM arecoline ± 10 μM PNU-120596 was applied (0.4 ml over 12 seconds) followed by another application of 60 μM ACh, as shown. Prior to the calculation of the multi-cell averages, each single cell response was normalized to the average of the two initial controls obtained from that cell. The cells were voltage clamped at -60 mV, and the traces shown represent the average of the normalized responses (black line) ± the S.E.M. (shaded band) calculated for each of the 10,500 points in the 210 s traces (acquired at 50 Hz). For arecoline alone (n = 8), and for arecoline plus PNU-120596 (n = 7). In order to allow for comparison between experiments, the data for the responses to the nut infusion plus PNU-120596 shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140907#pone.0140907.g002" target="_blank">Fig 2</a> were also normalized to their respective controls and are displayed along with the arecoline plus PNU-120596 data in the insert.</p