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J. Biol. Chem., Vol. 283, Issue 29, 20261-20267, July 18, 2008

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2-Aminoethoxydiphenyl Borate Alters Selectivity of Orai3 Channels by Increasing Their Pore Size^*

Rainer Schindl¹², Judith Bergsmann¹, Irene Frischauf, Isabella Derler³, Marc Fahrner, Martin Muik⁴, Reinhard Fritsch, Klaus Groschner, and Christoph Romanin⁵

From the Institute of Biophysics, University of Linz, A-4040 Linz and the Department of Pharmacy, University of Graz, A-8010 Graz, Austria

Stim1 in the endoplasmic reticulum and the three Orai (also termed CRACM) channels in the plasma-membrane are main components of native Ca²⁺ release-activated Ca²⁺ channels. A pharmacological hallmark of these channels is their distinct sensitivity to 2-aminoethoxydiphenyl borate (2-APB). Here we report that Orai3 currents can be robustly stimulated by 75 µM 2-APB independent of Stim1, whereas 2-APB at similar concentrations inhibited store-operated Orai1 currents. 2-APB did not only promote currents through Orai3 channels but also dramatically altered ion selectivity of Orai3 channels. This allowed for permeation of monovalent cations both in the inward as well as outward direction, which is in sharp contrast to the high Ca²⁺ selectivity of store-operated Orai3 currents. An Orai3-R66W mutant, which lacked in analogy to the severe combined immune deficiency mutant Orai1-R91W store-operated activation, was also found to be resistant to 2-APB stimulation. The change in selectivity by 2-APB was associated with an increase in Orai3 minimum pore size from about 3.8Å to more than 5.34Å_. In line with a potential interaction of 2-APB with the Orai3 pore, among three pore mutants tested, the Orai3 E165Q mutant particularly resembled in its permeation properties those of 2-APB stimulated Orai3 and additionally exhibited a reduced response to 2-APB. In aggregate, stimulation of Orai3 currents by 2-APB occurred along with an alteration of the permeation pathway that represents a unique mechanism for regulating ion channel selectivity by chemical compounds.

Received for publication, April 23, 2008 , and in revised form, May 19, 2008.

^* This work was supported by Austrian Science Foundation (FWF) projects P18280 [GenBank] (to K. G.) and P18169 [GenBank] as well as subproject 11 within W1201 (to C. R.). 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 Both authors contributed equally.

³ Graduate student with a scholarship from the Austrian Academy of Sciences.

⁴ Graduate student within the PhD-Program W1201 "Molecular Bioanalytics" from the Austrian Science Foundation (FWF).

² To whom correspondence may be addressed. E-mail: rainer.schindl{at}jku.at. ⁵ To whom correspondence may be addressed. Tel.: 43-732-2468-9272; Fax: 43-732-2468-9280; E-mail: christoph.romanin{at}jku.at.

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