Fluorescent toxins as ion channel activity sensors

Robert Stewart, Bruce E. Cohen, Jon T. Sack

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Voltage gated ion channels (VGICs) shape the electrical character of cells by undergoing structural changes in response to membrane depolarization. High-resolution techniques have provided a wealth of data on individual VGIC structures, but the conformational changes of endogenous channels in live cell membranes have remained unexplored. Here, we describe methods for imaging structural changes of voltage-gated K+ channels in living cells, using peptidyl toxins labeled with fluorophores that report specific protein conformations. These Endogenous Voltage-sensor Activity Probes (EVAPs) enable study of both VGIC allostery and function in the context of endogenous live-cell membranes under different physiological states. In this chapter, we describe methods for the synthesis, imaging, and analysis of dynamic EVAPs, which can report K+ channel activity in complex tissue preparations via 2-photon excitation microscopy, and environment-sensitive EVAPs, which report voltage-dependent conformational changes at the VGIC-toxin interface. The methods here present the utility of current EVAPs and lay the groundwork for the development of other probes that act by similar mechanisms. EVAPs can be correlated with electrophysiology, offering insight into the molecular details of endogenous channel function and allostery in live cells. This enables investigation of conformational changes of channels in their native, functional states, putting structures and models into a context of live-cell membranes. The expansive array of state-dependent ligands and optical probes should enable probes more generally for investigating the molecular motions of endogenous proteins.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
DOIs
StateAccepted/In press - 2021

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Fluoroscent probe
  • Gating modifier toxin
  • Ion channel
  • Potassium channel
  • Voltage gated ion channel
  • Voltage sensor
  • Voltage sensor toxin

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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