Acid-sensing ion channels (ASICs) have emerged as important, albeit challenging therapeutic targets for pain, stroke, etc. One approach to developing therapeutic agents could involve the generation of functional antibodies against these channels. To select such antibodies, we used channels assembled in nanodiscs, such that the target ASIC1a has a configuration as close as possible to its natural state in the plasma membrane. This methodology allowed selection of functional antibodies that inhibit acid-induced opening of the channel in a dose-dependent way. In addition to regulation of pH, these antibodies block the transport of cations, including calcium, thereby preventing acid-induced cell death in vitro and in vivo. As proof of concept for the use of these antibodies to modulate ion channels in vivo, we showed that they potently protect brain cells from death after an ischemic stroke. Thus, the methodology described here should be general, thereby allowing selection of antibodies to other important ASICs, such as those involved in pain, neurodegeneration, and other conditions.

The human glucagon‐like peptide‐1 receptor (GLP‐1R) and the glucagon receptor (GCGR) are class B G protein‐coupled receptors (GPCRs) that are activated by interactions with, respectively, the glucagon‐like peptide‐1 (GLP‐1) and glucagon (GCG). These polypeptide hormones are involved in the regulation of lipid and cholic acid metabolism, and thus play an important role in the pathogenesis of glucose metabolism and diabetes mellitus, which attracts keen interest of these GPCRs as drug targets. GLP‐1R and GCGR have therefore been extensively investigated by X‐ray crystallography and cryo‐electron microscopy (cryo‐EM), so that their structures are well known. Here, we present the groundwork for using nuclear magnetic resonance (NMR) spectroscopy in solution to complement the molecular architectures with information on intramolecular dynamics and on the thermodynamics and kinetics of interactions with physiological ligands and extrinsic drug candidates. This includes the generation of novel, near‐wild‐type constructs of GLP‐1R and GCGR, optimization of the solution conditions for NMR studies in detergent micelles and in nanodiscs, post‐translational chemical introduction of fluorine‐19 NMR probes, and sequence‐specific assignments of the 19F‐labels attached to indigenous cysteines. Addition of the negative allosteric modulator (NAM) NNC0640 was critically important for obtaining the long‐time stability needed for our NMR experiments, and we report on novel insights into the allosteric effects arising from binding of NNC0640 to the transmembrane domain of GLP‐1R (GLP‐1R[TMD]).

Large membrane proteins such as G protein‐coupled receptors (GPCRs) are difficult for NMR study due to severe signal overlaps and unfavorable relaxation properties. We used a trimethylsilyl (TMS) group as a reporter group for 1H NMR study of conformational changes in proteins, utilizing high‐intensity 1H NMR signals near 0 p.p.m. The β2‐adrenergic receptor was labeled with TMS groups at two cysteines located at the cytoplasmic ends of helices VI and VII. Binding of various ligands led to changes in 1H NMR signals, which manifested that helix VI is sensitive to G protein‐specific activation, whereas helix VII is sensitive to β‐arrestin‐specific activation. Thus, the TMS group is a useful reporter group in NMR for studying conformational changes in membrane proteins such as GPCRs.

The amino acid 4-fluoro-l-phenylalanine (4F-Phe) was introduced at the positions of Phe6 and Phe22 in the 29-residue polypeptide hormone glucagon by expressing glucagon in E. coli in the presence of an excess of 4F-Phe. Glucagon regulates blood glucose homeostasis by interaction with the glucagon receptor (GCGR), a class B GPCR. By referencing to the 4F-Phe chemical shifts at varying D2O concentrations, the solvent exposure of the two Phe sites along the glucagon sequence was determined, showing that 4F-Phe6 was fully solvent exposed and 4F-Phe22 was only partially exposed. The incorporation of fluorine atoms in polypeptide hormones paves the way for novel studies of their interactions with membrane-spanning receptors, specifically by differentiating between effects on the solvent accessibility, the line shapes, and the chemical shifts from interactions with lipids, detergents and proteins. Studies of interactions of GCGR with ligands in solution is at this point of keen interest, given that recent crystallographic studies revealed that an apparent small molecule antagonist actually binds as an allosteric effector at a distance of ~20 Å from the orthosteric ligand binding site (Jazayeri et al., in Nature 533:274–277, 2016).

No abstract