Inhibition of Human α7 Nicotinic Acetylcholine Receptors by Cyclic Monoterpene Carveol

Cyclic monoterpenes are a group of phytochemicals with antinociceptive, local anesthetic, and anti-inflammatory actions. Effects of cyclic monoterpenes including vanilin, pulegone, eugenole, carvone, carvacrol, carveol, thymol, thymoquinone, menthone, and limonene were investigated on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes. Monoterpenes inhibited the α7 nicotinic acetylcholine receptor in the order carveol\u3ethymoquinone\u3ecarvacrol\u3ementhone\u3ethymol\u3elimonene\u3eeugenole\u3epulegone≥carvone≥vanilin. Among the monoterpenes, carveol showed the highest potency on acetylcholine-induced responses, with IC50 of 8.3 µM. Carveol-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. In line with functional experiments, docking studies indicated that cyclic monoterpenes such as carveol may interact with an allosteric site located in the α7 transmembrane domain. Our results indicate that cyclic monoterpenes inhibit the function of human α7 nicotinic acetylcholine receptors, with varying potencies

The <it>Drosophila</it> nicotinic acetylcholine receptor subunits Dα5 and Dα7 form functional homomeric and heteromeric ion channels

<p>Abstract</p> <p>Background</p> <p>Nicotinic acetylcholine receptors (nAChRs) play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. In insects, nAChRs are the site of action of commercially important insecticides and, as a consequence, there is considerable interest in examining their functional properties. However, problems have been encountered in the successful functional expression of insect nAChRs, although a number of strategies have been developed in an attempt to overcome such difficulties. Ten nAChR subunits have been identified in the model insect <it>Drosophila melanogaster</it> (Dα1-Dα7 and Dβ1-Dβ3) and a similar number have been identified in other insect species. The focus of the present study is the Dα5, Dα6 and Dα7 subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate α7 nAChR subunit.</p> <p>Results</p> <p>A full-length cDNA clone encoding the <it>Drosophila</it> nAChR Dα5 subunit has been isolated and the properties of Dα5-, Dα6- and Dα7-containing nAChRs examined in a variety of cell expression systems. We have demonstrated the functional expression, as homomeric nAChRs, of the Dα5 and Dα7 subunits in <it>Xenopus</it> oocytes by their co-expression with the molecular chaperone RIC-3. Also, using a similar approach, we have demonstrated the functional expression of a heteromeric ‘triplet’ nAChR (Dα5 + Dα6 + Dα7) with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. In addition, specific cell-surface binding of [¹²⁵I]-α-bungarotoxin was detected in both <it>Drosophila</it> and mammalian cell lines when Dα5 was co-expressed with Dα6 and RIC-3. In contrast, co-expression of additional subunits (including Dα7) with Dα5 and Dα6 prevented specific binding of [¹²⁵I]-α-bungarotoxin in cell lines, suggesting that co-assembly with other nAChR subunits can block maturation of correctly folded nAChRs in some cellular environments.</p> <p>Conclusion</p> <p>Data are presented demonstrating the ability of the <it>Drosophila</it> Dα5 and Dα7 subunits to generate functional homomeric and also heteromeric nAChRs.</p

Electrophysiological characterization of α7^L247T nAChRs expressed in <i>Xenopus</i> oocytes in response to acetylcholine.

<p>Bar charts illustrate responses (mean ± SEM) from α7^L247T nAChRs expressed in <i>Xenopus</i> oocytes in response to a maximal (30 µM) and <i>EC</i>₅₀ (0.4 µM) concentration of acetylcholine (A and B, respectively) at room temperature (RT; 21°C), higher temperature (37°C) and lower temperature (4°C). Data are means of 5–9 responses, each from a different oocyte, in which responses obtained at either 4°C or 37°C are normalized to responses obtained from the same oocyte at RT. C) Representative traces illustrating responses obtained at RT (upper trace) and 4°C (lower trace) from a single oocyte. D) Representative traces illustrating responses obtained at RT (upper trace) and 37°C (lower trace) from a single oocyte.</p

Electrophysiological characterization of nAChRs expressed in <i>Xenopus</i> oocytes examined at different temperatures.

<p>Representative traces are shown illustrating responses obtained at RT (black), 4°C (blue) and 37°C (red). Current traces obtained at each temperature have been normalized to the same peak response. In each case, the response showing the fastest rate of desensitization was observed at 37°C and the slowest rate of desensitization was observed at 4°C. Responses are from α7 nAChRs with 3 mM acetylcholine (A), α4β2 nAChRs with 1 mM acetylcholine in calcium-containing saline (B), α7^L247T nAChRs with 30 µM acetylcholine (C) and α7 nAChRs with 10 µM 4BP-TQS (D). Rates of receptor deactivation after removal of agonist were also influenced in a consistent manner by changes in temperature (faster at 37°C and slower at 4°C). Representative traces from α7^L247T nAChRs with 30 µM acetylcholine are illustrated (E) and are typical of results from all receptor/agonist combinations studied (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032073#pone-0032073-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032073#pone-0032073-t002" target="_blank">2</a> for details).</p

Amplitude and desensitization of nAChR responses examined at different temperatures.

†, ††<p>Data for desensitization of all receptor and agonist combinations (with the exception of wild-type α7, activated by ACh) are expressed as the percentage of decay from the peak response in 5 seconds. Due to the rapid rate of desensitization for wild-type α7 activated by ACh, these values are expressed as the time required for the response to decay to 50% of the peak response.</p><p>Data are means ± SEM. Significant differences to responses recorded at RT are indicated (* = <i>P</i><0.05, ** = <i>P</i><0.01, *** = <i>P</i><0.001).</p

Electrophysiological characterization of α7 nAChRs expressed in <i>Xenopus</i> oocytes in response to acetylcholine.

<p>Bar charts illustrate responses (mean ± SEM) from α7 nAChRs expressed in <i>Xenopus</i> oocytes in response to a maximal (3 mM) and <i>EC</i>₅₀ (100 µM) concentration of acetylcholine (A and B, respectively) at room temperature (RT; 21°C), higher temperature (37°C) and lower temperature (4°C). Data are means of 7–11 responses, each from a different oocyte, in which responses obtained at either 4°C or 37°C are normalized to responses obtained from the same oocyte at RT. C) Representative traces illustrating responses obtained at RT (upper trace) and 4°C (lower trace) from a single oocyte. D) Representative traces illustrating responses obtained at RT (upper trace) and 37°C (lower trace) from a single oocyte.</p

Electrophysiological characterization of α7 nAChRs expressed in <i>Xenopus</i> oocytes in response to 4BP-TQS.

<p>A) A bar chart illustrates responses (mean ± SEM) from α7 nAChRs expressed in <i>Xenopus</i> oocytes in response to a maximal (10 µM) concentration of the allosteric agonist 4BP-TQS at room temperature (RT; 21°C), higher temperature (37°C) and lower temperature (4°C). Data are means of 5–22 responses, each from a different oocyte, in which responses obtained at either 4°C or 37°C are normalized to responses obtained from the same oocyte at RT. B) Representative traces illustrating responses obtained at RT (upper trace) and 4°C (lower trace) from a single oocyte. C) Representative traces illustrating responses obtained at RT (upper trace) and 37°C (lower trace) from a single oocyte.</p