Reconstitution of Calcium Channel Protein Orai3 into Liposomes for Functional Studies

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Chuangxuan Liang^1,a and Fuyun Wu^1,2,b*

¹School of Basic Medical Sciences, Hubei University of Medicine, 442000 Shiyan, China

²Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, 300071 Tianjin, China

^* To whom correspondence should be addressed.

Received March 29, 2023; Revised July 14, 2023; Accepted August 2, 2023

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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 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 the 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.

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KEY WORDS: store-operated Ca²⁺ entry, Orai3, STIM1, reconstitution, liposomes

DOI: 10.1134/S0006297923090092

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