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Functional requirement for Orai1 in store-operated TRPC1-STIM1 channels.
J Biol Chem. 2008 May 09; 283(19):12935-40.JB

Abstract

Orai1 and TRPC1 have been proposed as core components of store-operated calcium release-activated calcium (CRAC) and store-operated calcium (SOC) channels, respectively. STIM1, a Ca(2+) sensor protein in the endoplasmic reticulum, interacts with and mediates store-dependent regulation of both channels. We have previously reported that dynamic association of Orai1, TRPC1, and STIM1 is involved in activation of store-operated Ca(2+) entry (SOCE) in salivary gland cells. In this study, we have assessed the molecular basis of TRPC1-SOC channels in HEK293 cells. We report that TRPC1+STIM1-dependent SOCE requires functional Orai1. Thapsigargin stimulation of cells expressing Orai1+STIM1 increased Ca(2+) entry and activated typical I(CRAC) current. STIM1 alone did not affect SOCE, whereas expression of Orai1 induced a decrease. Expression of TRPC1 induced a small increase in SOCE, which was greatly enhanced by co-expression of STIM1. Thapsigargin stimulation of cells expressing TRPC1+STIM1 activated a non-selective cation current, I(SOC), that was blocked by 1 microm Gd(3+) and 2-APB. Knockdown of Orai1 decreased endogenous SOCE as well as SOCE with TRPC1 alone. siOrai1 also significantly reduced SOCE and I(SOC) in cells expressing TRPC1+STIM1. Expression of R91WOrai1 or E106QOrai1 induced similar attenuation of TRPC1+STIM1-dependent SOCE and I(SOC), whereas expression of Orai1 with TRPC1+STIM1 resulted in SOCE that was larger than that with Orai1+STIM1 or TRPC1+STIM1 but not additive. Additionally, Orai1, E106QOrai1, and R91WOrai1 co-immunoprecipitated with similar levels of TRPC1 and STIM1 from HEK293 cells, and endogenous TRPC1, STIM1, and Orai1 were co-immunoprecipitated from salivary glands. Together, these data demonstrate a functional requirement for Orai1 in TRPC1+STIM1-dependent SOCE.

Authors+Show Affiliations

Secretory Physiology Section, Molecular Physiology and Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

18326500

Citation

Cheng, Kwong Tai, et al. "Functional Requirement for Orai1 in Store-operated TRPC1-STIM1 Channels." The Journal of Biological Chemistry, vol. 283, no. 19, 2008, pp. 12935-40.
Cheng KT, Liu X, Ong HL, et al. Functional requirement for Orai1 in store-operated TRPC1-STIM1 channels. J Biol Chem. 2008;283(19):12935-40.
Cheng, K. T., Liu, X., Ong, H. L., & Ambudkar, I. S. (2008). Functional requirement for Orai1 in store-operated TRPC1-STIM1 channels. The Journal of Biological Chemistry, 283(19), 12935-40. https://doi.org/10.1074/jbc.C800008200
Cheng KT, et al. Functional Requirement for Orai1 in Store-operated TRPC1-STIM1 Channels. J Biol Chem. 2008 May 9;283(19):12935-40. PubMed PMID: 18326500.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Functional requirement for Orai1 in store-operated TRPC1-STIM1 channels. AU - Cheng,Kwong Tai, AU - Liu,Xibao, AU - Ong,Hwei Ling, AU - Ambudkar,Indu S, Y1 - 2008/03/07/ PY - 2008/3/11/pubmed PY - 2008/6/18/medline PY - 2008/3/11/entrez SP - 12935 EP - 40 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 283 IS - 19 N2 - Orai1 and TRPC1 have been proposed as core components of store-operated calcium release-activated calcium (CRAC) and store-operated calcium (SOC) channels, respectively. STIM1, a Ca(2+) sensor protein in the endoplasmic reticulum, interacts with and mediates store-dependent regulation of both channels. We have previously reported that dynamic association of Orai1, TRPC1, and STIM1 is involved in activation of store-operated Ca(2+) entry (SOCE) in salivary gland cells. In this study, we have assessed the molecular basis of TRPC1-SOC channels in HEK293 cells. We report that TRPC1+STIM1-dependent SOCE requires functional Orai1. Thapsigargin stimulation of cells expressing Orai1+STIM1 increased Ca(2+) entry and activated typical I(CRAC) current. STIM1 alone did not affect SOCE, whereas expression of Orai1 induced a decrease. Expression of TRPC1 induced a small increase in SOCE, which was greatly enhanced by co-expression of STIM1. Thapsigargin stimulation of cells expressing TRPC1+STIM1 activated a non-selective cation current, I(SOC), that was blocked by 1 microm Gd(3+) and 2-APB. Knockdown of Orai1 decreased endogenous SOCE as well as SOCE with TRPC1 alone. siOrai1 also significantly reduced SOCE and I(SOC) in cells expressing TRPC1+STIM1. Expression of R91WOrai1 or E106QOrai1 induced similar attenuation of TRPC1+STIM1-dependent SOCE and I(SOC), whereas expression of Orai1 with TRPC1+STIM1 resulted in SOCE that was larger than that with Orai1+STIM1 or TRPC1+STIM1 but not additive. Additionally, Orai1, E106QOrai1, and R91WOrai1 co-immunoprecipitated with similar levels of TRPC1 and STIM1 from HEK293 cells, and endogenous TRPC1, STIM1, and Orai1 were co-immunoprecipitated from salivary glands. Together, these data demonstrate a functional requirement for Orai1 in TRPC1+STIM1-dependent SOCE. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/18326500/Functional_requirement_for_Orai1_in_store_operated_TRPC1_STIM1_channels_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=18326500 DB - PRIME DP - Unbound Medicine ER -