Nicotinic acetylcholine receptors: Conventional and unconventional ligands and signaling

Abstract

Postsynaptic nAChRs in the peripheral nervous system are critical for neuromuscular and autonomic neurotransmission. Pre- and peri-synaptic nAChRs in the brain modulate neurotransmission and are responsible for the addictive effects of nicotine. Subtypes of nAChRs in lymphocytes and non-synaptic locations may modulate inflammation and other cellular functions. All AChRs that function as ligand-gated ion channels are formed from five homologous subunits organized to form a central cation channel whose opening is regulated by ACh bound at extracellular subunit interfaces. nAChR subtype subunit composition can range from α7 homomers to α4β2α6β2β3 heteromers. Subtypes differ in affinities for ACh and other agonists like nicotine and in efficiencies with which their channels are opened and desensitized. Subtypes also differ in affinities for antagonists and for positive and negative allosteric modulators. Some agonists are "silent" with respect to channel opening, and AChRs may be able to signal metabotropic pathways by releasing G-proteins independent of channel opening. Electrophysiological studies that can resolve single-channel openings and molecular genetic approaches have allowed characterization of the structures of ligand binding sites, the cation channel, and the linkages between them, as well as the organization of AChR subunits and their contributions to function. Crystallography and cryo-electron-microscopy are providing increasing insights into the structures and functions of AChRs. However, much remains to be learned about both AChR structure and function, the in vivo functional roles of some AChR subtypes, and the development of better pharmacological tools directed at AChRs to treat addiction, pain, inflammation, and other medically important issues. This article is part of the special issue on 'Contemporary Advances in Nicotine Neuropharmacology'.

Figure 1.

Structural models of nAChRs. A) Structure of an α4β2 nAChR homolog binding nicotine at α4/β2 subunit extracellular interfaces based on a recent X-ray crystal structure (Morales-Perez et al., 2016). Note that, although the structure contains the extracellular and transmembrane domains, the more flexibly structured ICD was largely removed in order to obtain the structure. B) An expanded view of an α4/β2 subunit dimer illustrating the key features of primary (left) and complementary (right) surfaces of the orthosteric ligand binding site. Also highlighted is the Cys loop.

Figure 2.

Activation schemes. A) A minimal model for ligand-gated ion channel activation, involving a single binding site. In the absence of agonist (A), nAChRs are exclusively in the unbound closed state (R). As a function of agonist concentration, nAChRs will occupy the agonist bound state (AR). Note that the forward binding rate (k⁺) is pseudo first order (M⁻¹s⁻¹); all other rates are first order (s⁻¹). With probability reflected in their relative rates, bound nAChRs will convert to the open state (AR*) or the desensitized state (AD). The model also permits conversion of nAChRs between the AR* and AD states. B) Dynamics of state interconversion as a function of time, the occupancy of specific states represented by the relative size of the red circles. The rate constant for state transitions are inversely proportional to the log of the activation energy barriers connecting the states. Assuming the rapid delivery of a large (saturating), concentration of agonist, at the moment of binding site saturation (t = 0 ms), all nAChRs will be in the AR state. Initially (t = 0.1 ms), nAChR will be most likely to populate the open state. Over time (t = 100 ms), most nAChRs will adopt the low energy desensitized state. C) An expansion of the model in A to account for the presence of two agonist binding sites and the experimental observation that nAChRs show two kinetically distinct open states. D) Collapse of the model in C after a covalent modification of the nAChR that inactivates one of the two ACh binding sites (Williams et al., 2011a).

Figure 3.

Concentration-dependent desensitization of α7 nAChR ACh-evoked responses. A) A family of ACh responses of α7 nAChRs expressed in a Xenopus oocyte. Shown in red is an estimation of the rate of solution exchange as measured by the change in junction potential for an open-tipped electrode placed in the recording chamber at the position of an oocyte. By calculating the timing of the peak current relative to the solution exchange profile and the maximum concentration for each application, it was possible to estimate that the ACh concentration at the time of the peak currents was only about 60 μM when concentrations greater that 30 μM were applied (Papke and Thinschmidt, 1998). B) Partial agonist site occupancy model for α7 activation and desensitization (Papke et al., 2000).

Figure 4.

Effects of positive allosteric modulators. ACh-evoked responses of α7 receptors expressed in Xenopus oocytes (Gulsevin et al., 2019; Stokes et al., 2019). Each set of traces compares the averaged responses (black line) and the SEM of the averages (shaded areas) for 5 – 8 cells. Individual cell responses were normalized to the initial control responses to 60 μM ACh obtained from the same cells. Each 210 s trace represents 10,000 points acquired at 50 Hz and filtered at 5 Hz. A) Responses to 60 μM ACh co-applied with 10 μM NS-1738, compared to ACh responses before and after the co-application. The inset is a scaled overlay of the ACh control and the potentiated current to illustrate the similarity in kinetics. B) Responses to 10 μM PNU-120596 applied alone, compared to ACh responses before and after the co-application. Note that although there was no response to the PNU-120596, there was a small (p < 0.01) increase in the ACh response after PNU-120596. C) Responses to 60 μM ACh co-applied with 10 μM PNU-120596, compared to ACh responses before and after the co-application. The inset is a scaled overlay of the ACh control and the potentiated current to illustrate the difference in kinetics. D) Responses to 10 μM GAT107 (the active isomer of 4BP-TQS), compared to ACh responses before and after the ago-PAM application. GAT107 application evoked a large response and also produced a large potentiation of the subsequent ACh response. E) Responses to 60 μM ACh co-applied with 10 μM GAT107, compared to ACh responses before and after the co-application.

Figure 5.

NS6740 and GAT107 produce stable nonconducting states with opposite consequences for subsequent applications of ACh and a unique potential for interaction. A) Single applications of 30 μM NS6740 produce little channel activation but inhibit six subsequent responses to ACh applied at four minute intervals (P < 0.05). Following the applications of ACh, an application 10 μM PNU-120596 produced a response nearly 10-fold larger than the initial ACh controls (p <0.05). B) Currents return to baseline following a single application of 30 μM GAT107; however, receptors remained primed for generation of potentiated ACh-evoked responses over seven subsequent applications of ACh alone (p< 0.05). C) The combined effect of 30 μM NS6740 stable desenitization and the prolonged potentiation produced by 30 μM GAT107 generates persistent currents. The data are taken from Papke et al, 2017 (Papke et al., 2017). Each set of traces illustrates the averaged responses (black line) and the SEM of the averages (shaded areas) for 5 cells. Individual cell responses were normalized to the initial control responses to 60 μM ACh obtained from the same cells. Each 210 s trace represents 10,000 points acquired at 50 Hz and filtered at 5 Hz.

Figure 6.

Orthosteric and allosteric binding sites on an α7 nAChR.

Figure 7.

Single-channel currents of α7 nAChR. A) Representative single-channel currents from an outside-out patch from a cell expressing α7 and Ric-3 and stimulated with the rapid application of ACh. The data is taken from Williams et al, 2011 (Williams et al., 2011b). Single channel current appear as brief isolated events. B) The open time duration distribution was fit with a single time constant of 59 ms. C) Representative burst of single channel activity evoked by 10 μM GAT107, recorded in cell-attached patch-clamp configuration from a cell stably expressing α7 and RIC3 (Quadri et al., 2019). The lower trace displays the center segment of the burst at an expanded time scale in order to resolve the different conductance levels corresponding to the full open state (O’), and two subconductance states (O” and O’’’) (Quadri et al., 2019). There are also multiple intraburst closed states which could correspond to PAM-sensitive desensitized state (D_s) or the active intermediate “Flip” state (F) (Lape et al., 2008). D) A hypothetical energy landscape showing these interconvertable states. Although the absolute assignment of states in the model to conductance levels in the data is only hypothetical, the association of state with the lowest free energy (O’) to the open state with the longest dwell time is reasonable. Likewise, the briefer states, O’’ and O’’’ were assigned to states with higher free energy.

Figure 8.

ICDs of nAChR subunits illustrating the diversity in length and amino acid character. Intracellular sequences were aligned with Clustal Omega, and colored according to their method (Madeira et al., 2019). Included are the possible helices near TM3 and TM4 with Clustal consensus notations. The putative G-protein binding site (RMKR) of α7 (Kabbani et al., 2013; King et al., 2017) is indicated with a box.