Our lab lab studies the molecular mechanisms of activation and regulation of TRPV1 channels. We use electrophysiology to study their functional properties, molecular biology to control their primary sequence (i.e. make mutations), and biochemistry to examine their interactions with other proteins. We are particularly interested in interactions. For ion channels, we must consider interactions between subunits within one channel, interactions with regulator proteins, interactions with the membrane, and interactions with small molecules such as second messengers. It is through understanding what interactions occur, when they occur, and how they have their effect that we make progress in addressing the function and role of ion channels within cells.
Sharona Gordon, Professor seg [at] uw.edu
Mika Munari, Research Scientist mawaya [at] uw.edu
Eric Senning, Postdoctoral Fellow endsen [at] uw.edu
I use fluorescence microscopy and Ca2+ sensitive dyes to record the time-dependent behavior of single TRPV1 ion channels opening and closing in the cell. My studies revealed that TRPV1 became slower with time that it remained open (Senning et al., eLife 2015). In the figure to the left I observe Ca2+ sparklets overlap with mobile TRPV1-GFP in plasma membrane of a HEK293T /17 cell. (A) Track of TRPV1-GFP with sparklet image as background. Clicking on image provides further examples of sparklets. (B) Fluorescence intensity of mobile TRPV1-GFP in (A). Sustained higher intensities correspond to sparklet activity. Arrowhead indicates acquisition of image in (A).
Cell unroofing is the process by which we remove the body of an adherent cell grown on a coverslip so that the only remaining cellular material on the coverslip is a plasma membrane sheet. Our recent structural studies of TRPV1 with an unnatural amino acid (UAA) inserted at different sites along its polypeptide sequence relied on sonication as the unroofing method (Zagotta et al., JGP 2016). To the left are examples of unroofed HEK293T/17 cells. (A) A plasma membrane sheet loaded with fluorescent Rhodamine B C-18. Brighter regions on periphery indicate partial unroofing where two bilayers are still present. Clicking on this image shows an unroofing movie of a cell expressing the PIP2 marker PLCδ-PH-GFP. The fluorescence intensity decreases after unroofing as the PLCδ-PH-GFP dissociates from the membrane. (B) A cell (outline in white) expressing TRPV1-GFP has a region of diffuse background fluorescence (yellow dashed line). The plasma membrane with resident TRPV1-GFP single channels remains after unroofing while intracellular TRPV1-GFP (presumably in endoplasmic reticulum and other intracellular membranes) is lost. See movie: TRPV1-GFP_unroof.m4v.
Gilbert Martinez, Postdoctoral Fellow gman [at] uw.edu
I’m interested in membrane protein structure-function with an emphasis on the 6-TM voltage gated ion channel superfamily. I use a broad array of biochemical and biophysical techniques to study the energetics of channel activation/inactivation/closing and how channel environment (cell-type, lipid composition, etc.) influence the energetics.
Mario Rosasco, Postdoctoral Fellow mrosasco [at] uw.edu
Mario received his BA in biology from the University of Chicago in 2009, where he specialized in neuroscience and endocrinology. He was awarded his Ph.D. from the department of Pharmacology at the University of Washington in 2014, and has been a postdoc in the Gordon lab since 2015.
Anastasiia Stratiievska, Graduate Student nastyna [at] uw.edu
University of Washington
HSB Room I-312
- ASBMB - The American Society for Biochemistry and Molecular Biology
- The Biophysical Society
- SGP - The Society of General Physiologists
- RStudio - A graphical interface for the R statistical programming environment
- Patcher's Power Tools - Useful electrophysiology libraries for IGOR Pro
- GIMP - A free, open-source raster drawing program
- Inkscape - A free, open-source vector drawing program
- Fiji - The ImageJ image analysis software, pre-packaged with many useful libraries