The Isoc Channel is a Critical Determinant of Interendothelial Gap Formation

Abstract

Inflammatory agonists deplete Ca2+ from the endoplasmic reticulum and activate Ca2+ entry in a process referred to as store‐operated Ca2+ entry (SOC). Activation of a Ca2+‐selective SOC current, Isoc, increases permeability and induces intercellular gap formation in pulmonary artery endothelial cells. However, the mechanism by which Ca2+ entry through the ISOC leads to barrier disruption is unclear. Endothelial cells express the transient receptor potential canonical (TRPC) homologues 1 and 4 that contribute to the molecular basis of ISOC. TRPC4 specifically links the Isoc channel to the membrane skeleton via interaction with protein 4.1. We therefore sought to determine whether inhibition of the TRPC4‐4.1 interaction is sufficient to prevent thapsigargin from inducing gaps. Thapsigargin activates SOC entry and induces a large rise in cytosolic Ca2+. Introduction of a competitive peptide that disrupts the TRPC4‐4.1 interaction reduces the global Ca2+ response to thapsigargin and abolishes Isoc. Moreover, peptide inhibition of the TRPC4‐4.1 interaction nearly abolished thapsigargin‐induced gap formation, and increased the rate of gap resealing. Thus, Ca2+ entry through the Isoc channel is critical to intercellular gap formation. Supported by HL60024.