A subset of Orai1α and Orai1β subunits heteromerizes to form CRAC channels.

Publisher: BioMed Central Country of Publication: England NLM ID: 101170464 Publication Model: Electronic Cited Medium: Internet ISSN: 1478-811X (Electronic) Linking ISSN: 1478811X NLM ISO Abbreviation: Cell Commun Signal Subsets: MEDLINE

Original Publication: [London] : BioMed Central, c2003-

ORAI1 Protein*/metabolism ; ORAI1 Protein*/chemistry ; ORAI1 Protein*/genetics ; Protein Multimerization* ; Protein Subunits*/metabolism ; Protein Subunits*/chemistry ; Calcium Release Activated Calcium Channels*/metabolism; Humans ; Membrane Microdomains/metabolism ; HEK293 Cells ; Calcium/metabolism

Competing Interests: Declarations. Ethical approval: Experimental procedures were approved by the local ethical committee (University of Extremadura and Extremadura Health Service). Competing interests: The authors declare no competing interests.
Background: Ca ²⁺ release-activated Ca ²⁺ (CRAC) channels are highly Ca ²⁺ selective plasma membrane channels formed by the hexameric assembly of Orai subunits, with a predominant role for Orai1. Two Orai1 variants have been identified, Orai1α, which comprises 301 amino acids, and a short variant, Orai1β, lacking the first N-terminal 63 or 71 amino acids; however, little is known about their possible heteromerization to form CRAC channels. Here we show that Orai1α and Orai1β exhibit different lipid raft distributions in resting cells when expressed individually, likely due to the presence of a caveolin-binding domain exclusively in Orai1α. However, when both variants are co-expressed, they show a similar distribution predominantly in the lipid raft domains, indicating potential interaction between the two Orai1 forms.
Methods: A lipid raft isolation protocol in combination with Western blotting assay was conducted to detect the expression of each Orai1 variants in the isolated membrane fractions. Ca ²⁺ mobilization was determined using fura-2 and G-GECO1.2 fused to Orai1α fluorescence. Evidence of physical interaction between both Orai1 variants was provided using co-immunoprecipitation, APEX2 peroxidase-catalyzed proximity labeling, Förster resonance energy transfer (FRET) and super-resolution microscopy.
Results: Our results indicate that Orai1α and Orai1β exhibit different lipid raft partitioning in resting cells when expressed individually, likely attributed to the presence of a caveolin-binding domain in Orai1α. However, when both variants are co-expressed, they show a similar distribution predominantly in the lipid raft domains, indicating potential interaction between the two Orai1 forms. Expression of a dominant-negative Orai1β mutant has been found to interfere with Orai1α-mediated Ca ²⁺ entry. Using co-immunoprecipitation, APEX2 peroxidase-catalyzed proximity labeling, Förster resonance energy transfer (FRET) and super-resolution microscopy our results indicate that there is certain interaction between Orai1α and Orai1β although both variants form mostly independent channels.
Conclusions: Our results indicate that while Orai1α and Orai1β mostly form separate CRAC channels, a small subset of both Orai1 variants combine to form heteromeric channels. These findings provide new insights on the nature of CRAC channels.
(© 2025. The Author(s).)

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PID2022-136279NB-C21 Agencia Estatal de Investigación; PID2022-136279NB-C21 Agencia Estatal de Investigación; PID2022-136279NB-C21 Agencia Estatal de Investigación; PID2022-136279NB-C21 Agencia Estatal de Investigación; PID2022-136279NB-C21 Agencia Estatal de Investigación; PID2022-136279NB-C21 Agencia Estatal de Investigación; PID2022-136279NB-C22 Agencia Estatal de Investigación; PID2022-136279NB-C21 Agencia Estatal de Investigación; IB20007 Junta de Extremadura; IB20007 Junta de Extremadura; IB20007 Junta de Extremadura; IB20007 Junta de Extremadura

Keywords: Lipid rafts; Orai1α; Orai1β; Store-operated Ca2+ entry

0 (ORAI1 Protein)
SY7Q814VUP (Calcium)
0 (Protein Subunits)
0 (ORAI1 protein, human)
0 (Calcium Release Activated Calcium Channels)

Date Created: 20250602 Date Completed: 20250603 Latest Revision: 20250605

20250605

PMC12131441

10.1186/s12964-025-02271-3

40457251