Oligomeric state of the endothelial ISOC channel

Activation of store operated Ca 2+ (SOC) entry promotes inter‐endothelial cell gap formation. Mammalian transient receptor potential proteins 1 and 4 (TRPC‐1, 4) are subunits of SOC entry channels and contribute to an endothelial cell Ca 2+ ‐selective SOC entry channel ( I SOC ). Based on homology with voltage‐gated K + channels it is currently believed that TRPC proteins form channels by coalescence of four subunits. The idea of a tetrameric structure was supported in a study by Schaefer et al using heterologously expressed TRPC6 in a FRET based approach. While heterologously expressed TRPC6 forms a homotetramer, it is presently unclear whether endogenous TRPC proteins also form tetramers. We therefore sought to determine the oligomeric state of the endogenous I SOC channel. Our approach was to cross‐link lysine residues residing in the extracellular loops of TRPC1 and TRPC4. Rat pulmonary artery endothelial cells (PAEC) were treated with cross‐linking agents of spacer arm‐lengths 7.7 Å, 11.4 Å and 16.1 Å. Cells were then lysed and homogenized, and proteins analyzed via SDS‐PAGE. Immunoblotting for TRPC4 revealed a band representative of a tetramer configuration in samples cross‐linked by the 7.7 Å spacer arm. Thus TRPC4 contributes to an endogenous tetrameric channel configuration in PAECs. Supported by HL60024 and AHA0625311B.