title: Measuring distances between TRPV1 and the plasma membrane using a noncanonical amino acid and transition metal ion FRET.
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.
authors.affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195 seg@uw.edu zagotta@uw.edu.
authors.forename: William N
authors.initials: WN
authors.lastname: Zagotta
authors.affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195.
authors.forename: Moshe T
authors.initials: MT
authors.lastname: Gordon
authors.affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195.
authors.forename: Eric N
authors.initials: EN
authors.lastname: Senning
authors.affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195.
authors.forename: Mika A
authors.initials: MA
authors.lastname: Munari
authors.affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195 seg@uw.edu zagotta@uw.edu.
authors.forename: Sharona E
authors.initials: SE
authors.lastname: Gordon
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D002211:Capsaicin / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D050916:TRPV Cation Channels / Q000819:agonists / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D008670:Metals; D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; D002211:Capsaicin