Journal Article

A subset of Orai1alpha and Orai1beta subunits heteromerizes to form CRAC channels

Jardín, Isaac, Nieto-Felipe, Joel, Macías-Díaz, Alvaro, Lopez, Jose J, Alvarado, Sandra, Rosado, Juan A, Smani, Tarik, Jiménez-Velarde, Vanesa, Martín-Romero, Francisco J

2025

Overview

Ca.sup.2+ release-activated Ca.sup.2+ (CRAC) channels are highly Ca.sup.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, Orai1[alpha], which comprises 301 amino acids, and a short variant, Orai1[beta], 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 Orai1[alpha] and Orai1[beta] exhibit different lipid raft distributions in resting cells when expressed individually, likely due to the presence of a caveolin-binding domain exclusively in Orai1[alpha]. 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. 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.sup.2+ mobilization was determined using fura-2 and G-GECO1.2 fused to Orai1[alpha] 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. Our results indicate that Orai1[alpha] and Orai1[beta] exhibit different lipid raft partitioning in resting cells when expressed individually, likely attributed to the presence of a caveolin-binding domain in Orai1[alpha]. 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 Orai1[beta] mutant has been found to interfere with Orai1[alpha]-mediated Ca.sup.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 Orai1[alpha] and Orai1[beta] although both variants form mostly independent channels. Our results indicate that while Orai1[alpha] and Orai1[beta] 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.

• Publisher: BioMed Central Ltd
• Subject: Amino acids / Calcium channels / Ethylenediaminetetraacetic acid / Gene expression / Genetic aspects / Genetic variation / Health aspects / Medical research / Medicine, Experimental / Physiological aspects