NMR structures of the transmembrane domains of the α4β2 nAChR

Abstract

The α4β2 nicotinic acetylcholine receptor (nAChR) is the predominant heteromeric subtype of nAChRs in the brain, which has been implicated in numerous neurological conditions. The structural information specifically for the α4β2 and other neuronal nAChRs is presently limited. In this study, we determined structures of the transmembrane (TM) domains of the α4 and β2 subunits in lauryldimethylamine-oxide (LDAO) micelles using solution NMR spectroscopy. NMR experiments and size exclusion chromatography–multi-angle light scattering (SEC-MALS) analysis demonstrated that the TM domains of α4 and β2 interacted with each other and spontaneously formed pentameric assemblies in the LDAO micelles. The Na+ flux assay revealed that α4β2 formed Na+ permeable channels in lipid vesicles. Efflux of Na+ through the α4β2 channels reduced intra-vesicle Sodium Green™ fluorescence in a time-dependent manner that was not observed in vesicles without incorporating α4β2. The study provides the structural insight into the TM domains of the α4β2 nAChR. It offers a valuable structural framework for rationalizing extensive biochemical data collected previously on the α4β2 nAChR and for designing new therapeutic modulators.

Side-chain conformation at the selectivity filter shapes the permeation free-energy landscape of an ion channel

“…The glutamates of the AChR's intermediate ring occur in the first turn of an α-helix (13,15), a region where four residues in a row-typically referred to as positions Ncap, N1, N2, and N3 (27, 28)-can form only one of the two i→i-4 (donor→acceptor) backbone-backbone hydrogen bonds that are possible at positions in the interior of α-helices. At these four positions, the backbone amide groups cannot form hydrogen bonds with backbone carbonyl oxygens; instead, nearby side chains often act as hydrogen-bond acceptors.…”

“…Thus, in an attempt to correct for these uncertainties while still taking advantage of the valuable insight provided by these data (indeed, the tubular crystals contained plasma-membrane embedded AChRs), we generated a homology model (Fig. 1) combining the latter with NMR-based models of the α4 and β2 AChR subunits (15). To optimize the model, we used side chainconformation prediction algorithms (16) and a variety of all-atom MD simulations (17,18), as indicated in detail in Materials and Methods.…”

“…To model the α1 subunit, we used an NMR-based structural model of the α4 AChR subunit (PDB ID code 2LLY; ref. 15), whereas to model the β1, δ, and e subunits, we used an NMR-based structural model of the β2 AChR subunit (PDB ID code 2LM2; ref. 15).…”

“…15), whereas to model the β1, δ, and e subunits, we used an NMR-based structural model of the β2 AChR subunit (PDB ID code 2LM2; ref. 15). Residue substitutions were introduced to the models of the α4 and β2 subunits so as to match the primary sequences of the mouse α1 (accession number: P04756), β1 (P09690), δ (P02716), and e (P20782) subunits.…”

Seeking Structural Specificity: Direct Modulation of Pentameric Ligand-Gated Ion Channels by Alcohols and General Anesthetics

“…The abundance and ease of purification of Torpedo receptors (Cohen et al, 1972) have made them a popular model system for studying ligand-gated ion channel structure, function, and modulation. Recent higher-resolution structures of the ligand-binding domain from mouse a1 (Dellisanti et al, 2007) and transmembrane domains of human nicotinic receptor subunits (Bondarenko et al, 2012), determined by X-ray crystallography and solutionphase NMR, respectively, promise further insights into structure and function in this family.…”