Thermosensitive TRPV channel subunits coassemble into heteromeric channels with intermediate conductance and gating properties.
Heat-sensitive transient receptor potential (TRP) channels (TRPV1-4) form the major cellular sensors for detecting temperature increases. Homomeric channels formed by thermosensitive TRPV subunits exhibit distinct temperature thresholds. While these subunits do share significant sequence similarity, whether they can coassemble into heteromeric channels has been controversial. In the present study we investigated the coassembly of TRPV subunits using both spectroscopy-based fluorescence resonance energy transfer (FRET) and single-channel recordings. Fluorescent protein-tagged TRPV subunits were coexpressed in HEK 293 cells; FRET between different subunits was measured as an indication of the formation of heteromeric channels. We observed strong FRET when fluorescence signals were collected selectively from the plasma membrane using a "spectra FRET" approach but much weaker or no FRET from intracellular fluorescence. In addition, no FRET was detected when TRPV subunits were coexpressed with members of the TRPM subfamily or CLC-0 chloride channel subunits. These results indicate that a substantial fraction of TRP channels in the plasma membrane of cotransfected cells were heteromeric. Single-channel recordings confirmed the existence of multiple heteromeric channel forms. Interestingly, heteromeric TRPV channels exhibit intermediate conductance levels and gating kinetic properties. As these subunits coexpress both in sensory neurons and in other tissues, including heart and brain, coassembly between TRPV subunits may contribute to greater functional diversity.
Cheng
Wei
W
Department of Physiology and Membrane Biology, University of California, School of Medcine, Davis, CA 95616.
Yang
Fan
F
Takanishi
Christina L
CL
Zheng
Jie
J
D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004553:Electric Conductivity; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D021122:Protein Subunits / Q000502:physiology*; D050916:TRPV Cation Channels / Q000502:physiology*; D013696:Temperature; D014162:Transfection
D021122:Protein Subunits; D050916:TRPV Cation Channels; D049452:Green Fluorescent Proteins