Ca2+ store
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beta3-adrenergic activation causes Ca2+ release from the mitochondria and subsequent Ca2+ release from the endoplasmic reticulum (ER), evoking store-operated Ca2+ entry (SOCE) due to Ca2+ depletion from the ER in mouse brown adipocytes. In this study, we investigated how Ca2+ depletion from the ER elicits SOCE in mouse brown adipocytes using fluorometry of intracellular Ca2+ concentration ([Ca2+]i). The administration of cyclopiazonic acid (CPA), a reversible sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump blocker in the ER, caused an increase in [Ca2+]i. Moreover, CPA induced SOCE was suppressed by the administration of a Ca2+ free Krebs solution and the transient receptor potential canonical 6 (TRPC6) selective blockers 2-APB, ML-9 and GsMTx-4 but not Pico145, which blocks TRPC1/4/5. Administration of TRPC6 channel agonist 1-oleoyl-2-acetyl-sn-glycerol (OAG) and flufenamic acid elicited Ca2+ entry. Moreover, our RT-PCR analyses detected mRNAs for TRPC6 in brown adipose tissues. In addition, western blot analyses showed the expression of the TRPC6 protein. Thus, TRPC6 is one of the Ca2+ pathways involved in SOCE. These modes of Ca2+ entry provide the basis for heat production via activation of Ca2+-dependent dehydrogenase and the expression of uncoupling protein 1 (UCP1). Enhancing thermogenic metabolism in brown adipocytes may serve as broad therapeutic utility to reduce obesity and metabolic syndrome.
- MeSH
- endoplazmatické retikulum metabolismus MeSH
- hnědé tukové buňky metabolismus MeSH
- kationtové kanály TRP * metabolismus MeSH
- kationtové kanály TRPC metabolismus MeSH
- kationtový kanál TRPC6 metabolismus MeSH
- myši MeSH
- vápník metabolismus MeSH
- vápníková signalizace MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Acta Universitatis upsaliensis. Comprehensive summaries of Uppsala dissertations from the Faculty of Medicine, ISSN 0282-7476 No. 721
44 s. ; 26 sm
The Ca2+ sensor STIM1 and the Ca2+ channel Orai1 that form the store-operated Ca2+ (SOC) channel complex are key targets for drug development. Selective SOC inhibitors are currently undergoing clinical evaluation for the treatment of auto-immune and inflammatory responses and are also deemed promising anti-neoplastic agents since SOC channels are linked with enhanced cancer cell progression. Here, we describe an investigation of the site of binding of the selective inhibitor Synta66 to the SOC channel Orai1 using docking and molecular dynamics simulations, and live cell recordings. Synta66 binding was localized to the extracellular site close to the transmembrane (TM)1 and TM3 helices and the extracellular loop segments, which, importantly, are adjacent to the Orai1-selectivity filter. Synta66-sensitivity of the Orai1 pore was, in fact, diminished by both Orai1 mutations affecting Ca2+ selectivity and permeation of Na+ in the absence of Ca2+. Synta66 also efficiently blocked SOC in three glioblastoma cell lines but failed to interfere with cell viability, division and migration. These experiments provide new structural and functional insights into selective drug inhibition of the Orai1 Ca2+ channel by a high-affinity pore blocker.
- Publikační typ
- časopisecké články MeSH
The stromal interaction molecule 1 (STIM1) has two important functions, Ca2+ sensing within the endoplasmic reticulum and activation of the store-operated Ca2+ channel Orai1, enabling plasma-membrane Ca2+ influx. We combined molecular dynamics (MD) simulations with live-cell recordings and determined the sequential Ca2+-dependent conformations of the luminal STIM1 domain upon activation. Furthermore, we identified the residues within the canonical and noncanonical EF-hand domains that can bind to multiple Ca2+ ions. In MD simulations, a single Ca2+ ion was sufficient to stabilize the luminal STIM1 complex. Ca2+ store depletion destabilized the two EF hands, triggering disassembly of the hydrophobic cleft that they form together with the stable SAM domain. Point mutations associated with tubular aggregate myopathy or cancer that targeted the canonical EF hand, and the hydrophobic cleft yielded constitutively clustered STIM1, which was associated with activation of Ca2+ entry through Orai1 channels. On the basis of our results, we present a model of STIM1 Ca2+ binding and refine the currently known initial steps of STIM1 activation on a molecular level.
- MeSH
- algoritmy MeSH
- buněčná membrána metabolismus MeSH
- endoplazmatické retikulum metabolismus MeSH
- HEK293 buňky MeSH
- hydrofobní a hydrofilní interakce MeSH
- konfokální mikroskopie MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- motivy EF-ruky MeSH
- mutace MeSH
- nádorové buněčné linie MeSH
- nádorové proteiny chemie genetika metabolismus MeSH
- protein ORAI1 chemie metabolismus MeSH
- protein STIM1 chemie genetika metabolismus MeSH
- proteinové domény * MeSH
- rozbalení proteinů * MeSH
- simulace molekulární dynamiky * MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Stormorken syndrome is a multiorgan hereditary disease caused by dysfunction of the endoplasmic reticulum (ER) Ca2+ sensor protein STIM1, which forms the Ca2+ release-activated Ca2+ (CRAC) channel together with the plasma membrane channel Orai1. ER Ca2+ store depletion activates STIM1 by releasing the intramolecular "clamp" formed between the coiled coil 1 (CC1) and CC3 domains of the protein, enabling the C terminus to extend and interact with Orai1. The most frequently occurring mutation in patients with Stormorken syndrome is R304W, which destabilizes and extends the STIM1 C terminus independently of ER Ca2+ store depletion, causing constitutive binding to Orai1 and CRAC channel activation. We found that in cis deletion of one amino acid residue, Glu296 (which we called E296del) reversed the pathological effects of R304W. Homozygous Stim1 E296del+R304W mice were viable and phenotypically indistinguishable from wild-type mice. NMR spectroscopy, molecular dynamics simulations, and cellular experiments revealed that although the R304W mutation prevented CC1 from interacting with CC3, the additional deletion of Glu296 opposed this effect by enabling CC1-CC3 binding and restoring the CC domain interactions within STIM1 that are critical for proper CRAC channel function. Our results provide insight into the activation mechanism of STIM1 by clarifying the molecular basis of mutation-elicited protein dysfunction and pathophysiology.
- MeSH
- aminokyseliny metabolismus MeSH
- endoplazmatické retikulum metabolismus MeSH
- kanály aktivované uvolněním vápníku * genetika MeSH
- membránové proteiny * metabolismus MeSH
- mutace MeSH
- myši MeSH
- protein ORAI1 metabolismus MeSH
- protein STIM1 genetika MeSH
- vápník metabolismus MeSH
- vápníkové kanály metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
