Calcium entry through the store-operated calcium current, ISOC, disrupts the spectrin-F-actin interaction inducing interendothelial gaps

Activation of a calcium-selective store-operated calcium (SOC) current, Isoc, disrupts the endothelial barrier and increases permeability. However, the mechanism in which calcium entry through the Isoc leads to barrier disruption is unclear. The transient receptor potential canonical (TRPC) homologues 1, 3, and 4 may contribute to the molecular basis of Isoc. TRPC4 binds to protein 4.1 and links the Isoc channel to the spectrin-actin membrane skeleton. The protein 4.1-spectrin-F-actin interaction contributes to membrane stability and increases in cytosolic calcium disrupt this interaction inducing membrane instability in erythrocytes. We therefore sought to determine: (i) whether calcium entry through Isoc disrupts the spectrin-F-actin interaction, and (ii) whether disruption of the spectrin-F-actin interaction induces interendothelial gaps.;First, we confirmed that SOC entry induces gaps in pulmonary artery endothelial cells (PAECs). However, introduction of a competitive peptide inhibiting Isoc activation reduced gap formation suggesting calcium entry through Isoc is sufficient to disrupt the endothelial barrier. Secondly, thapsigargin-induced SOC entry increased cytosolic and membrane calcium to ≈ 1 muM. Furthermore, SOC entry reduced the co-immunoprecipitation of actin with spectrin in PAECs. Pulmonary microvascular endothelial cells (PMVECs) also activate SOC channels and increase cytosolic calcium, but, thapsigargin is not sufficient to activate Isoc. In PMVECs, activation of SOC channels increased cytosolic calcium concentrations but did not reduce the co-immunoprecipitation of actin with spectrin, suggesting a global rise in calcium is not sufficient to disrupt the spectrin-F-actin interaction. Finally, an antibody disrupting the spectrin-F-actin interaction was introduced by microinjection or with the Chariot Reagent SystemRTM. Antibody introduction produced gaps in both PMVECs and PAECs indicating the spectrin-F-actin interaction helps stabilize the membrane skeleton in both cell types. Moreover, introducing an antibody that binds to spectrin inhibiting the spectrin-protein 4.1 interaction has no effect on membrane stability. Collectively, our findings suggest that calcium entry through Isoc disrupts the spectrin-F-actin interaction inducing interendothelial gaps.