Controlling Engineered P2X Receptors with Light

Methods Mol Biol. 2020:2041:301-309. doi: 10.1007/978-1-4939-9717-6_22.

Abstract

This chapter details methods to express and modify ATP-gated P2X receptor channels so that they can be controlled using light. Following expression in cells, a photoswitchable tool compound can be used to covalently modify mutant P2X receptors, as previously demonstrated for homomeric P2X2 and P2X3 receptors, and heteromeric P2X2/3 receptors. Engineered P2X receptors can be rapidly and reversibly opened and closed by different wavelengths of light. Light-activated P2X receptors can be mutated further to impart ATP-insensitivity if required. This method offers control of specific P2X receptor channels with high spatiotemporal precision to study their roles in physiology and pathophysiology.

Keywords: ATP; Azobenzene; Ion channel; Optochemicals; P2X; Photoswitchable; Synthetic optogenetics.

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Electrophysiology
  • Genetic Engineering / methods*
  • HEK293 Cells
  • Humans
  • Ion Channel Gating / physiology*
  • Ion Channel Gating / radiation effects
  • Light*
  • Mutation
  • Optogenetics / methods*
  • Receptors, Purinergic P2X2 / chemistry
  • Receptors, Purinergic P2X2 / physiology*
  • Receptors, Purinergic P2X2 / radiation effects
  • Receptors, Purinergic P2X3 / chemistry
  • Receptors, Purinergic P2X3 / physiology*
  • Receptors, Purinergic P2X3 / radiation effects

Substances

  • Receptors, Purinergic P2X2
  • Receptors, Purinergic P2X3
  • Adenosine Triphosphate