In 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. / Biochemistry
| Identifer | oai:union.ndltd.org:TEMPLE/oai:scholarshare.temple.edu:20.500.12613/1420 |
| Date | January 2013 |
| Creators | HENDRON, EUNAN |
| Contributors | Gill, Donald L., Rothberg, Brad S., Soboloff, Jonathan, Chong, Parkson Lee-Gau |
| Publisher | Temple University. Libraries |
| Source Sets | Temple University |
| Language | English |
| Detected Language | English |
| Type | Thesis/Dissertation, Text |
| Format | 123 pages |
| Rights | IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available., http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | http://dx.doi.org/10.34944/dspace/1402, Theses and Dissertations |
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