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Resolution of the subunit stoichiometry of the endogenous endothelial ISOC channel
Author(s) -
Cioffi Donna L.,
St. Croix Claudette M.,
Pitt Bruce R.,
Stevens Troy
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1178.16
Subject(s) - trpc1 , transient receptor potential channel , protein subunit , biophysics , photobleaching , förster resonance energy transfer , immunoprecipitation , trpc , chemistry , biology , biochemistry , receptor , fluorescence , gene , physics , quantum mechanics
The endothelial ISOC channel is a calcium selective store‐operated calcium (SOC) entry channel whose activation is an important step leading to intercellular gap formation and increased permeability. The transient receptor potential canonical (TRPC) proteins 1 and 4 contribute to ISOC channel structure. SOC entry channels are comprised of four subunits which coalesce to form the channel pore. To date, the number of each type of subunit, i.e. the subunit stoichiometry, remains unknown. We sought to determine the subunit stoichiometry of the endogenous endothelial ISOC channel using Förster Resonance Energy Transfer (FRET). Prior studies demonstrated that TRPC4 interacts with protein 4.1. Thus, protein 4.1 immunoprecipitation was performed to isolate an enriched population of endothelial ISOC channels. TRPC1 and TRPC4 subunits were tagged with fluorophore (Cy3 and Cy5) labeled antibodies. FRET experiments were performed using the sensitized emission approach and confirmed via acceptor photobleaching. Positive FRET transitions occurred between TRPC1‐Cy3 and TRPC4‐Cy5, TRPC1‐Cy3 and TRPC1‐Cy5 as well as between TRPC4‐Cy3 and TRPC4‐Cy5, indicating that the tetrameric ISOC channel is comprised of at least two TRPC1 and at least two TRPC4 subunits. Thus the subunit stoichiometry of the endogenous endothelial ISOC channel is two TRPC1 subunits plus two TRPC4 subunits. Supported by HL60024 and 1F32HL086124.