Reconstitution of calcium channel protein Orai3 into liposomes for functional studies

Abstract

Store-operated calcium entry (SOCE) is the main mechanism for the Ca2+ influx in non-excitable cells. The two major components of SOCE are stromal interaction molecule 1 (STIM1) in the endoplasmic reticulum and Ca2+ release-activated Ca2+ channel (CRAC) Orai on the plasma membrane. SOCE requires interaction between STIM1 and Orai. Mammals have three Orai homologs: Orai1, Orai2, and Orai3. Although Orai1 has been widely studied and proven to be essential for numerous cellular processes, Orai3 has also attracted a significant attention recently. The gating and activation mechanisms of Orai3 have yet to be fully elucidated. Here, we expressed, purified, and reconstituted Orai3 protein into liposomes and investigated its orientation and oligomeric state in the resulting proteoliposomes. STIM1 interacted with the Orai3-containing proteoliposomes and mediated calcium release from them, suggesting that the Orai3 channel was functional and that recombinant STIM1 could directly open the Orai3 channel in vitro. The developed in vitro calcium release system could be used to study the structure, function, and pharmacology of Orai3 channel.