Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel

Vishal R. Patel, Arturo M. Salinas, Darong Qi, Shipra Gupta, David J. Sidote and Marcel P. Goldschen-Ohm ()
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Vishal R. Patel: The University of Texas at Austin
Arturo M. Salinas: The University of Texas at Austin
Darong Qi: The University of Texas at Austin
Shipra Gupta: The University of Texas at Austin
David J. Sidote: The University of Texas at Austin
Marcel P. Goldschen-Ohm: The University of Texas at Austin

Nature Communications, 2021, vol. 12, issue 1, 1-13

Abstract: Abstract Ligand binding to membrane proteins is critical for many biological signaling processes. However, individual binding events are rarely directly observed, and their asynchronous dynamics are occluded in ensemble-averaged measures. For membrane proteins, single-molecule approaches that resolve these dynamics are challenged by dysfunction in non-native lipid environments, lack of access to intracellular sites, and costly sample preparation. Here, we introduce an approach combining cell-derived nanovesicles, microfluidics, and single-molecule fluorescence colocalization microscopy to track individual binding events at a cyclic nucleotide-gated TAX-4 ion channel critical for sensory transduction. Our observations reveal dynamics of both nucleotide binding and a subsequent conformational change likely preceding pore opening. Kinetic modeling suggests that binding of the second ligand is either independent of the first ligand or exhibits up to ~10-fold positive binding cooperativity. This approach is broadly applicable to studies of binding dynamics for proteins with extracellular or intracellular domains in native cell membrane.

Date: 2021
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DOI: 10.1038/s41467-021-26816-5

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