1. Department of Physiology & Biophysics and Center for Immunology, University of California, Irvine, California 92697, USA
2. *These authors contributed equally to this work

Correspondence to: Michael D. Cahalan¹ Correspondence and requests for materials should be addressed to M.D.C. (Email: mcahalan@uci.edu).

Abstract

Recent RNA interference screens have identified several proteins that are essential for store-operated Ca²⁺ influx and Ca²⁺ release-activated Ca²⁺ (CRAC) channel activity in Drosophila and in mammals, including the transmembrane proteins Stim (stromal interaction molecule)^{1, 2} and Orai^{3, 4, 5}. Stim probably functions as a sensor of luminal Ca²⁺ content and triggers activation of CRAC channels in the surface membrane after Ca²⁺ store depletion^{1, 6}. Among three human homologues of Orai (also known as olf186-F), ORAI1 on chromosome 12 was found to be mutated in patients with severe combined immunodeficiency disease, and expression of wild-type Orai1 restored Ca²⁺ influx and CRAC channel activity in patient T cells³. The overexpression of Stim and Orai together markedly increases CRAC current^{5, 7, 8, 9}. However, it is not yet clear whether Stim or Orai actually forms the CRAC channel, or whether their expression simply limits CRAC channel activity mediated by a different channel-forming subunit. Here we show that interaction between wild-type Stim and Orai, assessed by co-immunoprecipitation, is greatly enhanced after treatment with thapsigargin to induce Ca²⁺ store depletion. By site-directed mutagenesis, we show that a point mutation from glutamate to aspartate at position 180 in the conserved S1–S2 loop of Orai transforms the ion selectivity properties of CRAC current from being Ca²⁺-selective with inward rectification to being selective for monovalent cations and outwardly rectifying. A charge-neutralizing mutation at the same position (glutamate to alanine) acts as a dominant-negative non-conducting subunit. Other charge-neutralizing mutants in the same loop express large inwardly rectifying CRAC current, and two of these exhibit reduced sensitivity to the channel blocker Gd³⁺. These results indicate that Orai itself forms the Ca²⁺-selectivity filter of the CRAC channel.

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