Browsing Theses and Dissertations by Subject "2-apb"
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Dissecting the mechanism of STIM coupling to OraiStore-operated Ca2+ entry (SOCE) triggered by the depletion of endoplasmic reticulum (ER) luminal Ca2+ stores is a major Ca2+ entry pathway in non-excitable cells and is essential in physiological Ca2+ signaling and homeostasis. STIM proteins are sensors of ER luminal Ca2+, which translocate to ER-plasma membrane (PM) junctional regions to activate the family of Orai channels mediating Ca2+ entry. This study is focused on dissecting the mechanism of STIM interacting with Orai. A powerful modifier of SOCE, 2-aminoethoxydiphenyl borate (2-APB) is utilized. First, the action of 2-APB on the mammalian Orai homologues are characterized using the DT40 STIM knockout cells. 50 ìM 2-APB directly activates Orai3 but not Orai1 or Orai2. Second, while it stimulates the STIM2-mediated constitutive Ca2+ entry through Orai, 2-APB also induces the cytosolic STIM C-terminus fragments to translocate to the PM and activate Orai1. These data reveal 50 ìM 2-APB enhances STIM-Orai coupling. Further, this enhanced binding of STIM and Orai leads to a conformational change within the STIM-Orai complex, which is possibly the underlying mechanism for the 50 ìM 2-APB inhibitory effect on SOCE. Finally, six residues (344-349) at the N-terminus of the STIM-Orai activation region (SOAR) prove to be critical for this inhibitory action. These same six amino acid region also constitutes an ancillary Orai binding site within SOAR, in addition to the main polybasic region. The deletion of this ancillary site abolishes the ability of SOAR to bind to and activate Orai1, but can be compensated for by the STIM-Orai binding enhancing effect of 50 ìM 2-APB. The majority of STIM1 is located on the ER membrane, while a small proportion of STIM1 is on the PM. Using an extracellularly applied STIM1 antibody, the PM STIM1 can be aggregated to exert an influence on the ER STIM1. Although the PM STIM1 is not obligatory for STIM1-mediated Orai activation, it nevertheless may have a functional presence in the PM. Lastly, a regulatory link between voltage-gated Ca2+ channels (Cav channels) and the STIM proteins is established. After activation by store depletion, STIM strongly suppresses the Cav1.2 channels. There is a biochemical interaction between STIM1 and the Cav1.2 pore subunit á1C. This inhibitory effect is independent of Orai1 activation. Hence, STIM1 interacts with and reciprocally controls two major Ca2+ channels.
Potent and specific actions of 2-Aminoethoxydiphenyl borate (2-APB) derivatives on Orai channel functionIn an effort to dissect the mechanism of SOCe activation, I used two novel 2-APB analogs (DPB162-AE and DPB163-AE) which are ~50-100 times more potent at modifying SOCe than 2-APB. In the presence of STIM1, both compounds (2 µM) differentially affected Orai subtypes, fully blocking endogenous Orai1, but not Orai2 or Orai3 mediated SOCe in DT40 Orai-specific knockout cells. Neither analog directly activated Orai3 over-expressed alone in HEK293 cells. Analysis of constitutively active Orai1 mutant, Orai1V102C, showed an increase in Ca2+ entry after application of DPB162-AE independent of STIM1. When STIM1 was co-expressed with Orai1V102C, there was no inhibitory effect of the analog on the mutant channel complex. DPB162-AE appeared to have a long term effect on the channel complex revealed a lack of SOCe 10 minutes after washout of the analog. STIM1ct-Orai1 Ca2+ entry was moderately increased by DPB162-AE yet constitutively active Stim1ct4EA-Orai1 Ca2+ entry was robustly inhibited. The activation of mutant Orai1V102C indicated the analogs are capable of interacting with Orai1, perhaps to widen the pore, and pointing to a putative mechanism of action for inhibition. FRET analysis indicated no effect on STIM1-Orai1, STIM1ct-Orai1 or SOAR-Orai1 coupling. Thus, the inhibitory effect on STIM1-Orai may be through physical alteration of Orai1 gating. Previously reported as having biphasic effect on SOCe proteins, DPB163-AE appeared to effect its potentiation exclusively via STIM2 with no evident inhibition of STIM2 SOCe. Inhibition by both analogs was mediated by STIM1. DPB162-AE and DPB163-AE had remarkable specificity on Orai1 as opposed to other Ca2+ permeant channels. Neither compound affected Ca2+ entry through TRPC3, TRPC6, or strontium entry through Cav1.2 channels at concentrations (2 µM) that completely inhibited Orai1-mediated SOCe. In summary, DPB162-AE and DPB163-AE are highly specific inhibitors of Orai1 SOCe, with little effect on Orai2 and Orai3, and no effect on other Ca2+ channels. They do not disrupt STIM-Orai coupling but may modify functional Orai1 channel structure to effect their inhibitory action on SOCe.