STIM1 and calmodulin interact with Orai1 to induce Ca2+-dependent inactivation of CRAC channels.
Ca(2+)-dependent inactivation (CDI) is a key regulator and hallmark of the Ca(2+) release-activated Ca(2+) (CRAC) channel, a prototypic store-operated Ca(2+) channel. Although the roles of the endoplasmic reticulum Ca(2+) sensor STIM1 and the channel subunit Orai1 in CRAC channel activation are becoming well understood, the molecular basis of CDI remains unclear. Recently, we defined a minimal CRAC activation domain (CAD; residues 342-448) that binds directly to Orai1 to activate the channel. Surprisingly, CAD-induced CRAC currents lack fast inactivation, revealing a critical role for STIM1 in this gating process. Through truncations of full-length STIM1, we identified a short domain (residues 470-491) C-terminal to CAD that is required for CDI. This domain contains a cluster of 7 acidic amino acids between residues 475 and 483. Neutralization of aspartate or glutamate pairs in this region either reduced or enhanced CDI, whereas the combined neutralization of six acidic residues eliminated inactivation entirely. Based on bioinformatics predictions of a calmodulin (CaM) binding site on Orai1, we also investigated a role for CaM in CDI. We identified a membrane-proximal N-terminal domain of Orai1 (residues 68-91) that binds CaM in a Ca(2+)-dependent manner and mutations that eliminate CaM binding abrogate CDI. These studies identify novel structural elements of STIM1 and Orai1 that are required for CDI and support a model in which CaM acts in concert with STIM1 and the N terminus of Orai1 to evoke rapid CRAC channel inactivation.
Mullins
Franklin M
FM
Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Park
Chan Young
CY
Dolmetsch
Ricardo E
RE
Lewis
Richard S
RS
D015220:Calcium Channels / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004594:Electrophysiology; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D015640:Ion Channel Gating / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism*; D008954:Models, Biological; D016296:Mutagenesis; D009363:Neoplasm Proteins / Q000378:metabolism*; D000071740:ORAI1 Protein; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D000071737:Stromal Interaction Molecule 1; D014162:Transfection
D015220:Calcium Channels; D002147:Calmodulin; D017931:DNA Primers; D018076:DNA, Complementary; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1
J Physiol. 2009 Jun 15;587(Pt 12):2903-18
19403622
Nature. 2006 Sep 14;443(7108):226-9
16921385
Channels (Austin). 2008 Jul-Aug;2(4):261-8
18769136
Neuron. 1999 Apr;22(4):645-8
10230783
Neuron. 2001 Sep 27;31(6):973-85
11580897
Biochim Biophys Acta. 1999 Aug 17;1433(1-2):56-67
10446359
J Cell Biol. 2006 Sep 11;174(6):815-25
16966423
Nature. 2008 Jul 24;454(7203):538-42
18596693
Annu Rev Physiol. 2002;64:289-311
11826271
Curr Biol. 2005 Jul 12;15(13):1235-41
16005298
J Physiol. 1993 Jun;465:359-86
8229840
Trends Pharmacol Sci. 2009 Mar;30(3):118-28
19187978
Cell. 2008 Oct 3;135(1):110-22
18854159
Physiol Rev. 2005 Apr;85(2):757-810
15788710
Curr Biol. 2006 Oct 24;16(20):2073-9
16978865
J Cell Biol. 2006 Sep 11;174(6):803-13
16966422
J Gen Physiol. 2003 Aug;122(2):207-23
12860924
Biochemistry. 2008 Jun 10;47(23):6089-91
18484746
J Biol Chem. 2001 Aug 17;276(33):30794-802
11408490
J Gen Physiol. 2004 Feb;123(2):167-82
14744989
Trends Biochem Sci. 1990 Feb;15(2):59-64
2186516
J Gen Physiol. 2007 Nov;130(5):525-40
17968026
J Gen Physiol. 1995 Feb;105(2):209-26
7760017
Nat Immunol. 2008 Apr;9(4):432-43
18327260
Annu Rev Immunol. 2001;19:497-521
11244045
Cell. 2009 Mar 6;136(5):876-90
19249086
J Physiol. 2004 Jul 1;558(Pt 1):85-97
15226409
J Biol Chem. 2009 Mar 27;284(13):8421-6
19189966
Nat Cell Biol. 2009 Mar;11(3):337-43
19182790
Nature. 2005 Oct 6;437(7060):902-5
16208375
J Struct Funct Genomics. 2000;1(1):8-14
12836676
J Physiol. 2004 Jan 15;554(Pt 2):255-61
14500775
Cell Calcium. 2007 Aug;42(2):163-72
17499354
Proc Natl Acad Sci U S A. 2007 May 29;104(22):9301-6
17517596
Nature. 2006 Sep 14;443(7108):230-3
16921383