Direct Measurement of Single-Molecule Ligand-Receptor Interactions

Lam, K.-T.; Taylor, E. L.; Thompson, Andrew James; Ruepp, Marc-David; Lochner, Martin; Martinez, M. J.; Brozik, James A. (2020). Direct Measurement of Single-Molecule Ligand-Receptor Interactions. Journal of physical chemistry - B, 124(36), pp. 7791-7802. American Chemical Society 10.1021/acs.jpcb.0c05474

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Measuring the kinetics that govern ligand-receptor interactions is fundamental to our understanding of pharmacology. For ligand-gated ion channels, binding of an agonist triggers allosteric motions that open an integral ion-permeable pore. By mathematically modelling stochastic electrophysiological responses with high temporal resolution (ms), previous single channel studies have been able to infer the rate constants of ligands binding to these receptors. However, there are no reports of the direct measurement of the single-molecule binding events that are vital to how agonists exert their functional effects. For the first time, we report these direct measurements, the rate constants, and corresponding free energy changes, that describe the transitions between the different binding states. To achieve this, we use the super resolution technique: points accumulation for imaging in nanoscale topography (PAINT) to observe binding of ATP to orthosteric binding sites on the P2X1 receptor. Furthermore, an analysis of time-resolved single-molecule interactions is used to measure elementary rate constants and thermodynamic forces that drive the allosteric motions. These single-molecule measurements unequivocally establish the location of each binding states of the P2X1 receptor and the stochastic nature of the interaction with its native ligand. The analysis leads to the measurement of the forward and reverse rates from a weak ligand binding state to a strong ligand binding state that is linked to allosteric motion and ion pore formation. These rates (kα = 1.41 sec-1 and kβ = 0.32 sec-1) were then used to determine the free energy associated with this critical mechanistic step (3.7 kJ/mol). Importantly, the described methods can be readily applied to all ligand-gated ion channels, and more broadly to the molecular interactions of other classes of membrane proteins.

Item Type:

Journal Article (Original Article)


04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine
08 Faculty of Science > Departement of Chemistry and Biochemistry

UniBE Contributor:

Thompson, Andrew James; Ruepp, Marc-David and Lochner, Martin


500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health
500 Science > 540 Chemistry




American Chemical Society


[UNSPECIFIED] Air Force Office of Scientific Research




Martin Lochner

Date Deposited:

13 Aug 2020 09:08

Last Modified:

13 Sep 2020 02:51

Publisher DOI:





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