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Subunit stoichiometry of the endothelial ISOC channel
Author(s) -
Cioffi Donna L.,
St. Croix Claudette M.,
Pitt Bruce R.,
Stevens Troy
Publication year - 2007
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.21.6.a1432-c
Subject(s) - trpc1 , förster resonance energy transfer , biophysics , protein subunit , immunoprecipitation , transient receptor potential channel , chemistry , trpc , microbiology and biotechnology , fluorescence , biology , biochemistry , receptor , gene , physics , quantum mechanics
Activation of store operated Ca 2+ (SOC) entry promotes inter‐endothelial cell gap formation. The endothelial cell I SOC channel is a Ca 2+ ‐selective SOC entry channel which is comprised of transient receptor potential canonical (TRPC) proteins. While it is known that TRPC1 and TRPC4 contribute subunits to the I SOC channel, the subunit stoichiometry is unknown. We sought to determine the subunit stoichiometry of the endothelial I SOC channel using a Förster Resonance Energy Transfer (FRET) approach. Previously we determined that the TRPC4 subunit interacts with protein 4.1. Thus, protein 4.1 immunoprecipitation was performed to isolate the endothelial I SOC channel. Isolated I SOC channel was treated with Cy3 or Cy 5 labeled TRPC4 antibody and fluorescence saturation curves generated. At the 50% saturation concentration, I SOC channel immunocomplexes were treated with Cy3 labeled TRPC4 antibody, Cy5 labeled TRPC4 antibody or a combination of the two. Samples were irradiated at Cy3 excitation wavelength and fluorescence measured at the Cy5 emission wavelength. Sensitized emission was observed in samples labeled with both Cy3 and Cy5, indicating positive FRET. These data indicate that at least two TRPC4 subunits are present in the endothelial I SOC channel. Supported by HL60024 and AHA0625311B.