Small Fluorescein Arsenical Hairpin-Based Förster Resonance Energy Transfer Analysis Reveals Changes in Amino- to Carboxyl-Terminal Interactions upon OAG Activation of Classical Transient Receptor Potential 6

Mol Pharmacol. 2019 Jul;96(1):90-98. doi: 10.1124/mol.119.115949.


Although the overall structure of many classical transient receptor potential proteins (TRPC), including human and murine TRPC6, were recently resolved by cryoelectron microscopy analysis, structural changes during channel activation by 1-oleoyl-1-acetyl-sn-glycerol (OAG), the membrane-permeable analog of diacylglycerol, were not defined. Moreover, data on carboxyl- and amino-terminal interactions were not provided, as the amino-terminal regions of murine and human TRPC6 were not resolved. Therefore, we employed a Förster resonance energy transfer (FRET) approach using a small fluorescein arsenical hairpin (FlAsH) targeted to a short tetracysteine sequence at the unresolved amino-terminus and cerulean, a cyan fluorescent protein, as a tag at the carboxyl-terminus of the murine TRPC6 protein. After OAG as well as GSK-1702934A activation, FRET efficiency was simultaneously and significantly reduced, indicating a decreased interaction between the amino to carboxyl termini in the functional tagged murine TRPC6 tetramer (TRPC6 WT) heterologously expressed in human embryonic kidney 293T cells. There was a significant reduction in the FRET signal obtained from analysis of murine TRPC6 FRET constructs with homologous amino-terminal mutations (M131T, G108S) that had been identified in human patients with inherited focal segmental glomerulosclerosis, a condition that can lead to end-stage renal disease. A novel, designed loss-of-function TRPC6 mutation (N109A) in the amino-terminus in close proximity to the carboxyl-terminus produced similar FRET ratios. SIGNIFICANCE STATEMENT: Our data show for the first time that FlAsH-tagging of ion channels is a promising tool to study conformational changes after channel opening and may significantly advance the analysis of ion channel activation as well as their mutants involved in channelopathies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Diglycerides / chemistry
  • Diglycerides / pharmacology*
  • Fluorescence Resonance Energy Transfer
  • Green Fluorescent Proteins / chemistry*
  • HEK293 Cells
  • Humans
  • Mice
  • Mutation
  • Patch-Clamp Techniques
  • TRPC6 Cation Channel / chemistry*
  • TRPC6 Cation Channel / genetics
  • TRPC6 Cation Channel / metabolism*


  • 1-oleoyl-2-acetoyl-sn-glycerol
  • Cyan Fluorescent Protein
  • Diglycerides
  • TRPC6 Cation Channel
  • Green Fluorescent Proteins