Contribution of protein 4.1 to endothelial store-operated calcium entry

Store-operated calcium (SOC) entry represents the principal Ca 2+ entry pathway into non-excitable cells and triggers interendothelial cell gap formation leading to increased permeability. Importantly, SOC entry-mediated interendothelial cell gap formation occurs only in the absence of a rise in cAMP. The type 6 adenylyl cyclase (AC6) is inhibited by Ca2+ from SOC entry channels while the type 8 adenylyl cyclase (AC8) is stimulated by Ca2+ from SOC entry channels. Inflammatory mediators such as thrombin activate SOC entry, in addition to other second messenger-mediated events, and cause interendothelial cell gap formation. It is uncertain however whether thrombin-induced gap formation requires Ca2+ entry through SOC entry channels. In the present studies, we heterologously expressed AC8 in pulmonary microvascular endothelial cells to convert normal Ca 2+ inhibition to Ca2+ stimulation of cAMP. In AC8 expressing cells, thrombin-induced gap formation was nearly abolished. These data indicate that SOC entry contributes to both adenylyl cyclase regulation and thrombin-induced gap formation.; Despite intensive investigation, mechanisms underlying activation of SOC entry are still unclear. The endothelial ISOC channel is a Ca2+-selective SOC entry channel which is comprised, at least in part, of the transient receptor potential (TRP) proteins TRPC1 and TRPC4. While it is known that activation of ISOC is regulated by the spectrin-actin membrane skeleton, it is unclear how the ISOC channel is coupled to the membrane skeleton. In these studies we show that protein 4.1 interacts with both the TRPC4 subunit of the ISOC channel and the membrane skeleton. Deletion of the protein 4.1 binding domain and adjacent proline-rich region on TRPC4 prevents ISOC activation. Similarly, peptide competition to the protein 4.1 binding domain and adjacent proline-rich region on TRPC4 abolishes ISOC. Together, these findings reveal that protein 4.1 functionally links the TRPC4 subunit of the ISOC channel to the underlying membrane skeleton.