Disruption of the proline rich region/protein 4.1 binding domain on the endothelial Isoc channel inhibits intercellular gap formation

Abstract

The endothelial ISOC channel is a calcium selective store-operated calcium entry channel. We have previously shown that activation of the ISOC channel is an important step leading to intercellular gap formation and increased permeability. The transient receptor potential canonical (TRPC) homologues 1 and 4 contribute to ISOC channel structure. Protein 4.1 physically and functionally links the ISOC channel to the membrane skeleton, and activation of the ISOC channel requires a proline rich region/protein 4.1 binding domain on the carboxy terminus of the TRPC4 subunit. However, it is not clear whether disruption of the proline rich region/protein 4.1 binding domain on TRPC4 inhibits the formation of intercellular gaps. A competitive peptide to the proline rich region/protein 4.1 binding domain on TRPC4 (prr/4.1 peptide) and its scrambled counterpart were transfected into monolayers of rat pulmonary artery endothelial cells. Thapsigargin (1 μM) was applied and intercellular gap formation monitored by videomicroscopy. Intercellular gap formation was observed in the transfection control and with the scrambled peptide. Gaps formed within 3 to 5 minutes of thapsigargin application and resealed over the time course of the experiment. In cells transfected with the prr/4.1 peptide, the formation of gaps was significantly reduced; the gaps were smaller and resealed faster compared to control. Thus, disruption of the proline rich region/protein 4.1 binding domain on TRPC4 inhibits intercellular gap formation. Supported by HL60024 and HL66299.