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Originally published In Press as doi:10.1074/jbc.M100944200 on March 19, 2001

J. Biol. Chem., Vol. 276, Issue 22, 18888-18896, June 1, 2001
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Assessment of the Role of the Inositol 1,4,5-Trisphosphate Receptor in the Activation of Transient Receptor Potential Channels and Store-operated Ca2+ Entry Channels*

Hong-Tao MaDagger , Kartik VenkatachalamDagger , Hong-Sheng Li§, Craig Montell§, Tomohiro Kurosaki, Randen L. Patterson, and Donald L. Gill||

From the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, § Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and  Department of Molecular Genetics, Kansai Medical University, Moriguchi 570-8506, Japan

The mechanism for coupling between Ca2+ stores and store-operated channels (SOCs) is an important but unresolved question. Although SOCs have not been molecularly identified, transient receptor potential (TRP) channels share a number of operational parameters with SOCs. The question of whether activation of SOCs and TRP channels is mediated by the inositol 1,4,5-trisphosphate receptor (InsP3R) was examined using the permeant InsP3R antagonist, 2-aminoethoxydiphenyl borate (2-APB) in both mammalian and invertebrate systems. In HEK293 cells stably transfected with human TRPC3 channels, the actions of 2-APB to block carbachol-induced InsP3R-mediated store release and carbachol-induced Sr2+ entry through TRPC3 channels were both reversed at high agonist levels, suggesting InsP3Rs mediate TRPC3 activation. However, electroretinogram recordings of the light-induced current in Drosophila revealed that the TRP channel-mediated responses in wild-type as well as trp and trpl mutant flies were all inhibited by 2-APB. This action of 2-APB is likely InsP3R-independent since InsP3Rs are dispensable for the light response. We used triple InsP3R knockout DT40 chicken B-cells to further assess the role of InsP3Rs in SOC activation. 45Ca2+ flux analysis revealed that although DT40 wild-type cells retained normal InsP3Rs mediating 2-APB-sensitive Ca2+ release, the DT40InsP3R-k/o cells were devoid of functional InsP3Rs. Using intact cells, all parameters of Ca2+ store function and SOC activation were identical in DT40wt and DT40InsP3R-k/o cells. Moreover, in both cell lines SOC activation was completely blocked by 2-APB, and the kinetics of action of 2-APB on SOCs (time dependence and IC50) were identical. The results indicate that (a) the action of 2-APB on Ca2+ entry is not mediated by the InsP3R and (b) the effects of 2-APB provide evidence for an important similarity in the function of invertebrate TRP channels, mammalian TRP channels, and mammalian store-operated channels.

* This work was supported by National Institutes of Health (NIH) Grant HL55426 (to D. L. G.), a fellowship from the American Heart Association, Maryland Affiliate (to H-T. M.), a fellowship from the Interdisciplinary Muscle Training Program (to R. L. P.), and NIH Grant EY10852 (to C. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger The contribution of these two authors was equal.

|| To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene St., Baltimore, MD 21201. Tel.: 410-706-2593 (office) and 410-706-7247 (laboratory); Fax: 410-706-6676; E-mail: dgill@umaryland.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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