Abstract
We describe two methods to study CRAC channel function in human lung mast cells. Both methods involve suppression of endogenous channel function. In the first we use Orai-targeting shRNAs to knock down Orai channel mRNA transcripts. In the second we overexpress dominant-negative mutants of the three members of the Orai channel family. To overcome the poor transfection efficiency of mast cells, we employ an adenoviral delivery system for cell transduction. Knockdown of CRAC channel transcripts is assessed initially using quantitative RT-PCR. We describe an assay for β-hexosaminidase release as a measure of mast cell degranulation to assess the effect of overexpression of dominant-negative mutants.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Vig M, DeHaven WI, Bird GS et al (2008) Defective mast cell effector functions in mice lacking the CRACM1 pore subunit of store-operated calcium release-activated calcium channels. Nat Immunol 9:89–96
Ashmole I, Duffy SM, Leyland ML et al (2012) CRACM/Orai ion channel expression and function in human lung mast cells. J Allergy Clin Immunol 129:1628–1635
Bradding P, Walls AF, Holgate ST (2006) The role of the mast cell in the pathophysiology of asthma. J Allergy Clin Immunol 117:1277–1284
Russell WC (2000) Update on adenoviruses and its vectors. J Gen Virol 81:2573–2604
Bergelson JM, Cunningham JA, Droguet G et al (1997) Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 275:1320–1323
Fire A, Xu S, Montgomery MK et al (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811
Fellmann C, Lowe SW (2014) Stable RNA interference rules for silencing. Nat Cell Biol 16:10–18
Hou X, Pedi L, Diver MM et al (2012) Crystal structure of the calcium release-activated calcium channel Orai. Science 338:1308–1313
Vig M, Beck A, Billingsley JM et al (2006) CRACM1 multimers form the ion-selective pore of the CRAC channel. Curr Biol 16:2073–2079
Lis A, Peinelt C, Beck A et al (2007) CRACM1, CRACM2, and CRACM3 are store-operated Ca2+ channels with distinct functional properties. Curr Biol 17:794–800
DeHaven WI, Smyth JT, Boyles RR et al (2007) Calcium inhibition and calcium potentiation of Orai1, Orai2, and Orai3 calcium release-activated calcium channels. J Biol Chem 282:17548–17556
Ashmole I, Duffy SM, Leyland ML et al (2013) The contribution of Orai (CRACM)1 and Orai (CRACM)2 channels in store-operated Ca2+ entry and mediator release in human lung mast cells. PLoS One 8:e74895. https://doi.org/10.1371/journal.pone.0074895
Sanmugalingam D, Wardlaw AJ, Bradding P (2000) Adhesion of human lung mast cells to bronchial epithelium: evidence for a novel carbohydrate-mediated mechanism. J Leukoc Biol 68:38–46
Kaelin WG Jr (2012) Molecular biology. Use and abuse of RNAi to study mammalian gene function. Science 337:421–422
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Ashmole, I., Bradding, P. (2018). Study of Endogenous CRAC Channels in Human Mast Cells Using an Adenoviral Delivery System to Transduce Cells with Orai-Targeting shRNAs or with cDNAs Expressing Dominant-Negative Orai Channel Mutations. In: Penna, A., Constantin, B. (eds) The CRAC Channel. Methods in Molecular Biology, vol 1843. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8704-7_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8704-7_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8702-3
Online ISBN: 978-1-4939-8704-7
eBook Packages: Springer Protocols