Modification of Store-operated Channel Coupling and Inositol Trisphosphate Receptor Function by 2-Aminoethoxydiphenyl Borate in DT40 Lymphocytes

Abstract

Store-operated channels (SOCs) provide an important means for mediating longer-term Ca2؉ signals and replenishment of Ca 2؉ stores in a multitude of cell types. However, the coupling mechanism between endoplasmic reticulum stores to activate plasma membrane SOCs remains unknown. In DT40 chicken B lymphocytes, the permeant inositol trisphosphate receptor (InsP 3 R) modifier, 2-aminoethoxydiphenyl borate (2-APB), was a powerful activator of store-operated Ca 2؉ entry between 1-10 M. Ca 2ϩ signals control a vast array of cellular functions ranging from short-term responses such as contraction and secretion to longer-term regulation of cell growth and proliferation (1, 2). The cytosolic Ca 2ϩ signals generated in response to receptors are complex involving two closely coupled components: rapid, transient release of Ca 2ϩ stored in the endoplasmic reticulum (ER) 1 followed by slowly developing extracellular Ca 2ϩ entry (1, 3-7). G protein-coupled receptors and tyrosine kinase receptors, through activation of phospholipase C, generate the second messenger, InsP 3 , which diffuses rapidly within the cytosol to interact with InsP 3 Rs on the ER; the InsP 3 Rs serve as Ca 2ϩ channels to release luminally stored Ca 2ϩ and generate the initial Ca 2ϩ signal phase (1, 4). The resulting depletion of Ca 2ϩ stored within the ER lumen serves as the primary trigger for a message that is returned to the plasma membrane resulting in the slow activation of "storeoperated channels" (SOCs), which mediate capacitative Ca Coupling to activate SOCs has been hypothesized to involve direct coupling between the ER and plasma membrane (9, 10), and evidence indicates that physical docking of the ER with the plasma membrane may be involved in SOC activation (11)(12)(13)(14). Recent evidence has indicated that the InsP 3 R may be a component in mediating the coupling process between stores and Ca 2ϩ entry channels. In particular, a role for the InsP 3 R has been implicated in the activation of the TRP family of receptoractivated channels, which share a number of functional parameters with SOCs including reports that they are store-operated (15-21). Evidence from reconstitution studies has indicated a direct functional communication between TRP channels and InsP 3 Rs (17,(22)(23)(24), and a number of reports have revealed a physical interaction between TRP channels and the InsP 3 R (24 -29).Recently, we have probed the role played by InsP 3 Rs in Ca 2ϩ entry mechanisms utilizing cells from the DT40 chicken B lymphocyte line (DT40InsP 3 R-ko) in which the InsP 3 R genes have been disrupted (30). It was observed that these cells have store-operated Ca 2ϩ entry that is functionally the same as that occurring in the wild type DT40 cells (30 -32). Moreover, we observed that TRPC3 channels transiently expressed in * This work was supported by National Institutes of Health Grant HL55426 (to D. L. G.), a fellowship from the American Heart Association, Maryland Affiliate (to H.-T. M.), and Grant 99/08 of the Concerted Actions from K.U. Leuven (to J. B. ...

STIM protein coupling in the activation of Orai channels

“…2-APB is a powerful modifier of SOC function (16,(19)(20)(21)(22)(23)(24)(25)(26), and we reveal here that 2-APB induces rapid, profound, and either direct or very close interactions between the STIM and Orai proteins, locking the C terminus of STIM proteins together with Orai1 to facilitate complete functional channel coupling. We show that just the C-terminal coiled-coil domain of STIM proteins is sufficient for Orai1 activation.…”

“…Our approach was to examine the STIM-Orai interactions by using the reactive borate 2-aminoethoxydiphenyl borate (2-APB), a powerful modifier of SOC function (11,(19)(20)(21). Its actions are complex, enhancing SOC activation at 5-10 M, yet blocking it at 50 M (20, 21).…”

“…Our approach was to examine the STIM-Orai interactions by using the reactive borate 2-aminoethoxydiphenyl borate (2-APB), a powerful modifier of SOC function (11,(19)(20)(21). Its actions are complex, enhancing SOC activation at 5-10 M, yet blocking it at 50 M (20,21). Recent evidence emphasizes the remarkable effectiveness and the complexities of 2-APB in modifying STIM protein function and Orai channel gating (22)(23)(24)(25).…”

Cyclosporin A-insensitive Permeability Transition in Brain Mitochondria

“…2-APB may block the activation machinery of the channels participating in SOCE and not the channels themselves (80), (81). Therefore, 2-APB-sensitive channel-activating proteins similar to those involved in SOCE may be present in mitochondria in the absence of TRP channels.…”

Human Golgi Antiapoptotic Protein Modulates Intracellular Calcium Fluxes

“…Rabbit polyclonal antibodies against Bax and Bak were from Cell Signaling Technology (Beverly, MA). Rabbit polyclonal antibody (Rbt 476) against IP3R (all isoforms) was described previously (Ma et al, 2002). Coelenterazine-W, coelenterazine-N, fura-2 acetoxymethyl ester (fura-2/AM), and Rhod-2/AM were from Molecular Probes (Eugene, OR), histamine and ionomycin from Sigma-Aldrich (Poole, Dorset, United Kingdom), 2-APB from Calbiochem (La Jolla, CA), and STS from Roche (East Sussex, United Kingdom).…”