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

Author

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  • Vishal R. Patel

    (The University of Texas at Austin
    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)

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.

Suggested Citation

  • Vishal R. Patel & Arturo M. Salinas & Darong Qi & Shipra Gupta & David J. Sidote & Marcel P. Goldschen-Ohm, 2021. "Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel," Nature Communications, Nature, vol. 12(1), pages 1-13, December.
  • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-26816-5
    DOI: 10.1038/s41467-021-26816-5
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    References listed on IDEAS

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