Probing function in ligand-gated ion channels without measuring ion transport

Abstract

Although the functional properties of ion channels are most accurately assessed using electrophysiological approaches, a number of experimental situations call for alternative methods. Here, working on members of the pentameric ligand-gated ion channel (pLGIC) superfamily, we focused on the practical implementation of, and the interpretation of results from, equilibrium-type ligand-binding assays. Ligand-binding studies of pLGICs are by no means new, but the lack of uniformity in published protocols, large disparities between the results obtained for a given parameter by different groups, and a general disregard for constraints placed on the experimental observations by simple theoretical considerations suggested that a thorough analysis of this classic technique was in order. To this end, we present a detailed practical and theoretical study of this type of assay using radiolabeled α-bungarotoxin, unlabeled small-molecule cholinergic ligands, the human homomeric α7-AChR, and extensive calculations in the framework of a realistic five-binding-site reaction scheme. Furthermore, we show examples of the practical application of this method to tackle two longstanding questions in the field: our results suggest that ligand-binding affinities are insensitive to binding-site occupancy and that mutations to amino-acid residues in the transmembrane domain are unlikely to affect the channel’s affinities for ligands that bind to the extracellular domain.

An experimental test of the nicotinic hypothesis of COVID-19

“…2 A ). Indeed, the concentration of unlabeled toxin that displaced one-half of the bound [ 125 I]-α-BgTx was ∼2.30 nM ( Table 1 ), a value that is remarkably close to the theoretically expected value of ∼2 nM (∼2× K D,closed ) at equilibrium under the conditions of our experiments ( 43 ). On the other hand, the Hill coefficient (∼1.25) ( Table 1 ) was higher than the value of unity expected from an inverse agonist ( 43 ), such as α-BgTx ( 45 , 46 ).…”

“…Indeed, the concentration of unlabeled toxin that displaced one-half of the bound [ 125 I]-α-BgTx was ∼2.30 nM ( Table 1 ), a value that is remarkably close to the theoretically expected value of ∼2 nM (∼2× K D,closed ) at equilibrium under the conditions of our experiments ( 43 ). On the other hand, the Hill coefficient (∼1.25) ( Table 1 ) was higher than the value of unity expected from an inverse agonist ( 43 ), such as α-BgTx ( 45 , 46 ). It is likely that a somewhat longer incubation would have brought this system even closer to equilibrium, but in the context of this particular report, it is abundantly clear that 24-h incubations at 37 °C are long enough to detect the specific binding of short peptides to the α7-AChR.…”

“…A most direct way of testing the “nicotinic hypothesis” of COVID-19 ( 14 , 18 , 19 ) is by means of competition ligand-binding assays. To this end, we followed a recently developed protocol for an equilibrium-type approach ( 43 ). To account for the likely importance of the membrane environment in the proposed ligand–receptor interactions, we used cell-surface-expressed AChRs, and to exclude the additional molecular players present in native systems [such as the lynx prototoxins expressed in neurons and immune cells ( 44 ), for example], we expressed AChRs heterologously on HEK-293 cells; indeed, only different AChRs and snake neurotoxins were used in the sequence alignments and molecular simulations that led to this hypothesis.…”

“…The unlabeled and labeled ligands were added to the cells at nearly the same time. Enough toxin was used in the assays to ensure that the concentration of unbound (“free”) [ 125 I]-α-BgTx was ∼1 nM throughout each curve; this is the value of our estimate of the toxin’s dissociation equilibrium constant from the α7-AChR’s closed state at 37 °C ( K D,closed ) ( 43 ). We previously found that, under these experimental conditions, the mixture of cell-expressed α7-AChRs, [ 125 I]-α-BgTx, and small-molecule cholinergic ligands (such as carbamylcholine, choline, or nicotine) approaches equilibrium closely ( 43 ).…”

“…Enough toxin was used in the assays to ensure that the concentration of unbound (“free”) [ 125 I]-α-BgTx was ∼1 nM throughout each curve; this is the value of our estimate of the toxin’s dissociation equilibrium constant from the α7-AChR’s closed state at 37 °C ( K D,closed ) ( 43 ). We previously found that, under these experimental conditions, the mixture of cell-expressed α7-AChRs, [ 125 I]-α-BgTx, and small-molecule cholinergic ligands (such as carbamylcholine, choline, or nicotine) approaches equilibrium closely ( 43 ). In the absence of competing unlabeled ligand, a 1-nM concentration of free [ 125 I]-α-BgTx ensures that approximately one-half of the toxin-binding sites on the receptor are occupied.…”