Molecular function of α7 nicotinic receptors as drug targets

Abstract

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels involved in many physiological and pathological processes. In vertebrates, there are seventeen different nAChR subunits that combine to yield a variety of receptors with different pharmacology, function, and localization. The homomeric α7 receptor is one of the most abundant nAChRs in the nervous system and it is also present in non-neuronal cells. It plays important roles in cognition, memory, pain, neuroprotection, and inflammation. Its diverse physiological actions and associated disorders have made of α7 an attractive novel target for drug modulation. Potentiation of the α7 receptor has emerged as a novel therapeutic strategy for several neurological diseases, such as Alzheimer's and Parkinson's diseases, and inflammatory disorders. In contrast, increased α7 activity has been associated with cancer cell proliferation. The presence of different drug target sites offers a great potential for α7 modulation in different pathological contexts. In particular, compounds that target allosteric sites offer significant advantages over orthosteric agonists due to higher selectivity and a broader spectrum of degrees and mechanisms of modulation. Heterologous expression of α7, together with chaperone proteins, combined with patch clamp recordings have provided important advances in our knowledge of the molecular basis of α7 responses and their potential modulation for pathological processes. This review gives a synthetic view of α7 and its molecular function, focusing on how its unique activation and desensitization features can be modified by pharmacological agents. This fundamental information offers insights into therapeutic strategies.

Keywords: Cys-loop receptors; nicotinic receptors; patch clamp.

Figure 1. Cladogram of vertebrate nAChR subunits

Adapted from Le Novère et al. (2002).

Figure 2. Structure of nAChR

A, side view of the α4β2 receptor (PDB code: 5KXI) (Morales‐Perez et al. 2016). The receptor is formed by the combination of five subunits arranged around a central pore. Most of the ICD is not shown since it was removed to obtain well‐diffracting crystals (Morales‐Perez et al. 2016). B, schematic representation of the ACh binding site at the interface between two adjacent subunits in the ECD. The principal face is formed by loops A, B and C from one subunit, and the complementary face is formed by loops D, E and F from the adjacent subunit. Two key residues for ACh activation are shown. C, cartoon representation of the top view of the TMD. Each subunit shows four transmembrane segments (M1–M4), where the M2 segment of each of the five subunits form the channel pore.

Figure 3. Single channel and macroscopic recordings of human α7

The receptor was expressed on mammalian BOSC‐23 cells, which are modified HEK293 cells. Top, schematic representations of patch clamp configurations used to obtain the electrophysiological recordings. Bottom left, macroscopic recordings obtained by fast perfusion of ACh or 4BP‐TQS (black traces) and ACh in the presence of PAMs (red traces). Bottom right, single channel recordings from human α7 receptors activated by ACh in the absence and presence of positive allosteric modulators (PAMs, type I: NS‐1738 and type II: PNU‐120596), and by 4BP‐TQS (allosteric agonist). Openings are shown as upward deflections. Membrane potential −70 mV.

Figure 4. Scheme of modulation of α7 by different types of allosteric ligands

Cartoon representation of the α7 receptor and the sites of action of different compounds. The response elicited by the natural agonist (ACh) can be increased, decreased or remain unaffected by the binding of the different allosteric modulators PAMs, NAMs and SAMs, respectively.

Figure 5. Scheme showing two different ways to determine the functional stoichiometry of α7

One strategy uses concatemeric receptors that allow the introduction of changes in any of the five identical subunits. The other corresponds to the electrical fingerprinting strategy that combines two subunits, one of which contains a reporter triple mutation (Q428R, E432R, S436R) that decreases the amplitude of single channel events to undetectable levels under the present recording conditions without affecting receptor kinetics. The mutant subunit is called α7 low conductance (α7LC). By co‐expressing α7LC with α7 subunits, receptors of different stoichiometries are formed. The amplitude of each single‐channel opening is the signature of the stoichiometry of the receptor that originated that event. Typical single‐channel traces are shown for each strategy. Channels were recorded from α7 activated by 100 μm ACh in the presence of PNU‐120596. Openings are shown as upward deflections. Membrane potential −70 mV.