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Neuropharmacology
2015 Oct 01;97:75-85. doi: 10.1016/j.neuropharm.2015.05.006.
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The influence of allosteric modulators and transmembrane mutations on desensitisation and activation of α7 nicotinic acetylcholine receptors.
Chatzidaki A
,
D'Oyley JM
,
Gill-Thind JK
,
Sheppard TD
,
Millar NS
.
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Acetylcholine activates nicotinic acetylcholine receptors (nAChRs) by binding at an extracellular orthosteric site. Previous studies have described several positive allosteric modulators (PAMs) that are selective for homomeric α7 nAChRs. These include type I PAMs, which exert little or no effect on the rate of receptor desensitisation, and type II PAMs, which cause a dramatic loss of agonist-induced desensitisation. Here we report evidence that transmembrane mutations in α7 nAChRs have diverse effects on receptor activation and desensitisation by allosteric ligands. It has been reported previously that the L247T mutation, located toward the middle of the second transmembrane domain (at the 9' position), confers reduced levels of desensitisation. In contrast, the M260L mutation, located higher up in the TM2 domain (at the 22' position), does not show any difference in desensitisation compared to wild-type receptors. We have found that in receptors containing the L247T mutation, both type I PAMs and type II PAMs are converted into non-desensitising agonists. In contrast, in receptors containing the M260L mutation, this effect is seen only with type II PAMs. These findings, indicating that the M260L mutation has a selective effect on type II PAMs, have been confirmed both with previously described PAMs and also with a series of novel α7-selective PAMs. The novel PAMs examined in this study have close chemical similarity but diverse pharmacological properties. For example, they include compounds displaying effects on receptor desensitisation that are typical of classical type I and type II PAMs but, in addition, they include compounds with intermediate properties.
Fig. 1. Chemical structures of the allosteric modulators examined in this study.
Fig. 2. Potentiation of responses to acetylcholine on wild-type α7 nAChRs by TBS compounds. Representative traces are shown illustrating responses to acetylcholine (100 μM) together with responses from the same oocyte to acetylcholine (100 μM) after pre- and co-application of 10 μM TBS-346 (A), TBS-546 (B), TBS-345 (C), TBS-556 (D) or TBS-516 (E).
Fig. 3. Influence of TBS compounds on the recovery of α7 nAChRs from desensitisation. Representative traces showing prolonged exposure of α7 nAChRs to acetylcholine (100 μM), causing activation, followed by rapid desensitisation. In the continued presence of acetylcholine, application of (A) TBS-346 (10 μM) and (B) TBS-546 (10 μM) does not result in reactivation of the receptor. However, application of (C) TBS-345 (10 μM), (D) TBS-556 (10 μM) and (E) TBS-516 (10 μM) results in reactivation of desensitised receptors. Traces have been scaled to their response to acetylcholine.
Fig. 4. Competition radioligand binding. Equilibrium radioligand binding was performed with [3H]-α-bungarotoxin (1 nM) with mammalian tsA201 cells transiently transfected with human α7 nAChR subunit and with human RIC-3 cDNAs (1:1 ratio). TBS-345, TBS-346, TBS-516, TBS-546 and TBS-556 caused no significant displacement of [3H]-α-bungarotoxin binding, whereas MLA caused complete displacement of specific radioligand binding. Data points are means of triplicate samples (± SD) from a single experiment, and data are typical of three independent experiments.
Fig. 5. The influence of α7 nAChR mutations (L247T and M260L) on activation by acetylcholine. A) The location of L247T (9â²) and M260L (22â²) mutations in the α7 nAChR subunit transmembrane (TM2) domain. The transmembrane region of an α7 nAChR subunit homology model (Young et al., 2008) is shown viewed from the top (left hand image) and from the side (right hand image). The α-helical transmembrane regions are illustrated as ribbon structures with the side chains of the two mutated amino acids shown as space-filling models (L247 in red and M260 in blue). B) Representative traces are shown illustrating responses to maximal concentrations of acetylcholine on human wild-type (WT) α7 nAChRs and α7 nAChRs containing the M260L mutation (M260L) and the L247T mutation (L247T). Acetylcholine concentrations: 1 mM for WT and M260L and 10 μM for L247T. C) Acetylcholine concentration-response data are presented for wild-type α7 nAChRs (triangles) and for α7 nAChRs containing either the M260L mutation (circles) or the L247T mutation (diamonds). Data are means ± SEM of at least three independent experiments and are normalised to the respective maximum response obtained with each nAChR variant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. The influence of a type I (NS-1738) and a type II PAM (TQS) on wild-type and mutated (L247T or M260L) α7 nAChRs. A) Representative traces illustrating responses with wild-type α7 nAChRs to acetylcholine (100 μM) and after the pre- and co-application of either NS-1738 (10 μM; Left pair of traces) or TQS (30 μM; Right). B) Representative traces illustrating responses with mutated (L247T) α7 nAChRs to acetylcholine (10 μM) and to NS-1738 (10 μM; Left) or TQS (30 μM; Right). C) Representative traces illustrating responses with mutated (M260L) α7 nAChRs to acetylcholine (100 μM) and NS-1738 (10 μM; Left) or TQS (30 μM; Right).
Fig. 7. Potentiation and agonist effects of TBS compounds on wild-type and mutated α7 nAChRs. A) Representative traces are shown illustrating responses from wild-type α7 nAChRs to acetylcholine (100 μM), together with responses to acetylcholine (100 μM) after pre- and co-application of TBS compounds (10 μM). To illustrate differences in the rate of desensitsation, all responses on wild-type α7 nAChRs have been normalised to their peak response. B) Representative traces are shown illustrating agonist responses from α7 nAChRs containing the L247T (9â²) mutation to either acetylcholine (10 μM) or TBS compounds (10 μM). C) Representative traces are shown illustrating agonist responses from α7 nAChRs containing the M260L (22â²) mutation to either acetylcholine (100 μM) or TBS compounds (10 μM).
Fig. 8. Antagonism of agonist responses to acetylcholine and TQS responses on M260L and L247T α7 nAChRs. A) Representative traces with M260L α7 nAChRs illustrating initial agonist responses with acetylcholine (1 mM) or TQS (10 μM) (left), antagonism by pre- and co-application of MLA (1 μM) (middle) and recovery in the absence of MLA (right). B) Representative traces with L247T α7 nAChRs illustrating initial agonist responses with acetylcholine (10 μM) or TQS (3 μM) (left), antagonism by pre- and co-application of MLA (1 μM) (middle) and recovery in the absence of MLA (right).
Fig. 9. Potentiation of acetylcholine responses by allosteric modulators on M260L α7 nAChRs. Representative traces are shown illustrating responses to acetylcholine (100 μM) (left) together with responses from the same oocyte after pre- and co-application of an allosteric modulator (10 μM) (right). Representative traces are shown for NS-1738 (A), TQS (B), TBS-346 (C), TBS-546 (D), TBS-345 (E), TBS-556 (F) and TBS-516 (G).
Fig. 10. Concentrationâresponse curves for the wild-type and mutated α7 nAChRs. Data are shown from wild-type α7 nAChRs (A), α7 nAChRs containing the L247T (9â²) mutation (B) and α7 nAChRs containing the M260L (22â²) mutation (C). Data are presented for a range of concentrations of acetylcholine (circles), the allosteric agonist 4BP-TQS (hexagons), the type II PAMs, TQS (squares) and TBS-516 (inverted triangles), and the type I PAMs, NS-1738 (triangles) and TBS-346 (diamonds). Data are means ± SEM of at least three independent experiments and are normalised to the maximum acetylcholine response.
Fig. 11. Type I PAMs block agonist activity of TQS and TBS-516 on α7 nAChRs containing the M260L mutation. Representative traces are shown, obtained by two-electrode voltage-clamp recording in oocytes expressing α7 nAChRs containing the M260L mutation, in which a type I PAM (NS-1738 or TBS-346) was pre applied for 10 s and then co-applied with a type II PAM (TQS or TBS-516) (AâD). A) NS-1738 (10 μM) completely blocked responses to TQS (10 μM). B) TBS-346 (10 μM) blocked responses to TQS (10 μM) by 95.7 ± 1.1% (n = 3). C) NS-1738 (10 μM) blocked responses to TBS-516 (10 μM) by 92.7 ± 2.3% (n = 3). D) TBS-346 (10 μM) blocked responses to TBS-516 (10 μM) by 83.4 ± 4.1% (n = 3). E) The agonist concentration-response curve for TQS on α7 nAChRs containing the M260L mutation was shifted to the right in the presence of NS-1738 (2 μM, pre-applied for 10 s and then co-applied with TQS). The antagonism by NS-1738 was surmountable at high concentrations of TQS.
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