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Canonical Transient Receptor Potential 5 Channel in Conjunction with Orai1 and STIM1 Allows Sr²⁺ Entry, Optimal Influx of Ca²⁺, and Degranulation in a Rat Mast Cell Line¹

Hong-Tao Ma^2,*, Ze Peng^3,*, Takaaki Hiragun^4,*, Shoko Iwaki, Alasdair M. Gilfillan and Michael A. Beaven^2,*

^* Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute and Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

Degranulation of mast cells in response to Ag or the calcium mobilizing agent, thapsigargin, is dependent on emptying of intracellular stores of Ca²⁺ and the ensuing influx of external Ca²⁺, also referred to as store-operated calcium entry. However, it is unlikely that the calcium release-activated calcium channel is the sole mechanism for the entry of Ca²⁺ because Sr²⁺ and other divalent cations also permeate and support degranulation in stimulated mast cells. In this study we show that influx of Ca²⁺ and Sr²⁺ as well as degranulation are dependent on the presence of the canonical transient receptor potential (TRPC) channel protein TRPC5, in addition to STIM1 and Orai1, as demonstrated by knock down of each of these proteins by inhibitory RNAs in a rat mast cell (RBL-2H3) line. Overexpression of STIM1 and Orai1, which are known to be essential components of calcium release-activated calcium channel, allows entry of Ca²⁺ but not Sr²⁺, whereas overexpression of STIM1 and TRPC5 allows entry of both Ca²⁺ and Sr²⁺. These and other observations suggest that the Sr²⁺-permeable TRPC5 associates with STIM1 and Orai1 in a stoichiometric manner to enhance entry of Ca²⁺ to generate a signal for degranulation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Intramural Programs of the National Heart, Lung, and Blood Institute and the National Institute of Allergy and Infectious Diseases.

² Address correspondence and reprint requests to Dr. Michael A. Beaven or Dr. Hong-Tao Ma, Laboratory of Molecular Immunology, Room 8N109, Building 10, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda MD 20892-1760. E-mail addresses: beavenm{at}nhlbi.nih.gov or mah{at}nhlbi.nih.gov

³ Current address: Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892.

⁴ Current address: Department of Dermatology, Hiroshima University Faculty of Medicine, 1-2-3, Kasumi, Minami-ku 734-8551, Hiroshima, Japan.

⁵ Abbreviations used in this paper: SOCE, store-operated calcium entry; TRP, transient receptor potential; TRPC, canonical TRP; CRAC, calcium release-activated calcium; I_CRAC, CRAC current; eYFP, enhanced yellow fluorescent protein; PLC, phospholipase C; siRNA, small inhibitory RNA; shRNA, short hairpin RNA.