Channelpedia

PubMed 26755770


Referenced in Channelpedia wiki pages of: none

Automatically associated channels: TRP , TRPV , TRPV1



Title: Measuring distances between TRPV1 and the plasma membrane using a noncanonical amino acid and transition metal ion FRET.

Authors: William N Zagotta, Moshe T Gordon, Eric N Senning, Mika A Munari, Sharona E Gordon

Journal, date & volume: J. Gen. Physiol., 2016 Feb , 147, 201-16

PubMed link: http://www.ncbi.nlm.nih.gov/pubmed/26755770


Abstract
Despite recent advances, the structure and dynamics of membrane proteins in cell membranes remain elusive. We implemented transition metal ion fluorescence resonance energy transfer (tmFRET) to measure distances between sites on the N-terminal ankyrin repeat domains (ARDs) of the pain-transducing ion channel TRPV1 and the intracellular surface of the plasma membrane. To preserve the native context, we used unroofed cells, and to specifically label sites in TRPV1, we incorporated a fluorescent, noncanonical amino acid, L-ANAP. A metal chelating lipid was used to decorate the plasma membrane with high-density/high-affinity metal-binding sites. The fluorescence resonance energy transfer (FRET) efficiencies between L-ANAP in TRPV1 and Co(2+) bound to the plasma membrane were consistent with the arrangement of the ARDs in recent cryoelectron microscopy structures of TRPV1. No change in tmFRET was observed with the TRPV1 agonist capsaicin. These results demonstrate the power of tmFRET for measuring structure and rearrangements of membrane proteins relative to the cell membrane.