The Uniqueα4(+)/(−)α4 Agonist Binding Site in (α4)₃(β2)₂Subtype Nicotinic Acetylcholine Receptors Permits Differential Agonist Desensitization Pharmacology versus the (α4)₂(β2)₃Subtype

Abstract

Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (a4) 2 (b2) 3 ) or low-sensitivity (LS) (a4) 3 (b2) 2 ) isoforms of human a4b2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using 86 Rb1 efflux in a stably transfected SH-EP1-ha4b2 human epithelial cell line, and twoelectrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS a4b2-nAChR constructs (HSP and LSP). Unlike previously studied agonists, desensitization by the highly selective agonists A-85380 [3-(2(S)-azetidinylmethoxy)pyridine] and sazetidine-A (Saz-A) preferentially reduced a4b2-nAChR HS-phase versus LS-phase responses. The concatenated-nAChR experiments confirmed that approximately 20% of LS-isoform acetylcholine-induced function occurs in an HS-like phase, which is abolished by Saz-A preincubation. Six mutant LSPs were generated, each targeting a conserved agonist binding residue within the LS-isoform-only a4(1)/(2)a4 interface agonist binding site. Every mutation reduced the percentage of LS-phase function, demonstrating that this site underpins LS-phase function. Oocyte-surface expression of the HSP and each of the LSP constructs was statistically indistinguishable, as measured using b2-subunit-specific [ 125 I]mAb295 labeling. However, maximum function is approximately five times greater on a "per-receptor" basis for unmodified LSP versus HSP a4b2-nAChRs. Thus, recruitment of the a4(1)/(2)a4 site at higher agonist concentrations appears to augment otherwisesimilar function mediated by the pair of a4(1)/(2)b2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two a4b2-nAChR isoforms, and demonstrate that HS versus LS a4b2-nAChR activity can be selectively manipulated using pharmacological approaches. Since a4b2 nAChRs are the predominant neuronal subtype, these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development.

The Novel α7β2-Nicotinic Acetylcholine Receptor Subtype Is Expressed in Mouse and Human Basal Forebrain: Biochemical and Pharmacological Characterization

“…This is not surprising since the apparent time constants will likely reflect the relatively slow kinetics of agonist application in the apparatus, rather than the much faster kinetics of a 7 *-nAChR desensitization (Papke, 2010). Indeed, the apparent t fast values are very similar to those measured for solution exchange in our apparatus (Eaton et al, 2014). However, the t slow value calculated for the (a 7 ) 3 (b 2 ) 2 construct was significantly slower than those associated with the other groups.…”

“…The immunoreactive bands were quantified in four separate experiments for the mouse hippocampus and basal forebrain as previously described (Grady et al, 2009) Concatemeric a 7 *-nAChR Constructs Fully pentameric nAChR concatemers were constructed from human nAChR subunit sequences. cDNAs encoding concatemers were created using the same subunit layout we have previously employed to encode high-and low-agonist-sensitivity a 4 b 2 *-nAChR isoforms and a 3 b 4 (a 5 [D/ N])-nAChRs Eaton et al, 2014). Subunits were arranged in the order a 7 -a 7 -a 7 -a 7 -a 7 (a 7 homopentamer), a 7 -a 7 -b 2 -a 7 -a 7 , or a 7 -b 2 -a 7 -b 2 -a 7 .…”

Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligand-bound α2 homopentamer

“…For that reason, we constructed the α2 mutant W84A and coexpressed it in Xenopus oocytes with the β2 WT nAChR subunit in 10:1 or in 1:10 RNA ratios, thus expressing solely the LS or the HS subtype, respectively (27). Trp84 is a highly conserved residue on the complementary side of the ligand binding site that in the α2-Epi structure was found to interact with the ligand directly and its role in the binding site of other nAChRs has been evaluated in numerous studies (29,39,40) (Fig. 4 A and B).…”

An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors

“…Partial agonism of sazetidine arises from its exclusive and high affinity action at the ␣4/␤2 agonist sites; i.e. sazetidine is a primary SSAg (21,22). Cytisine is an intrinsic partial agonist that non-selectively binds to both the ␣4/␤2 and ␣4/␣4 agonist sites (20).…”