Subunit stoichiometry of the endothelial ISOC channel

Abstract

Activation of store operated Ca2+ (SOC) entry promotes inter-endothelial cell gap formation. The endothelial cell ISOC channel is a Ca2+-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 ISOC channel, the subunit stoichiometry is unknown. We sought to determine the subunit stoichiometry of the endothelial ISOC 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 ISOC channel. Isolated ISOC channel was treated with Cy3 or Cy 5 labeled TRPC4 antibody and fluorescence saturation curves generated. At the 50% saturation concentration, ISOC 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 ISOC channel. Supported by HL60024 and AHA0625311B.