Regulation of endothelial Isoc function by calcium phosphate complexation

The endothelial I SOC 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) homologues 1 and 4 contribute to I SOC channel structure. Protein 4.1 physically and functionally links the I SOC channel to the membrane skeleton, and activation of the I SOC channel requires a proline rich region/protein 4.1 binding domain on the carboxy terminus of the TRPC4 subunit. This region contains both serine and threonine as potential phosphorylation targets. We observed that the TRPC4 subunit is phosphorylated upon thapsigargin‐induced activation of SOC entry. Calcium entry through SOC entry channels leads to high calcium concentration near the pore of the channel. We hypothesized that free calcium coming through the channel pore complexes with the phosphate group(s) to regulate the channel open/closed state. As a proof of concept, calcium (Ca 2+ ) and lanthanum (La 3+ ) complex formation of phosphoserine was evaluated using nuclear magnetic resonance (NMR) spectroscopy. The change in 2 J C‐P and 3 J C‐P coupling constants in carbon‐13 and phosphorus‐31 NMR following Ca 2+ (or La 3+ ) addition to phosphoserine indicated that Ca 2+ (or La 3+ ) had readily complexed with the phosphate group. This observation is compatible with the idea that the formation of a calcium phosphate complex is important in I SOC channel function. Supported by HL60024 and HL66299.