Murine ORAI2 Splice Variants Form Functional Ca2+ Release-activated Ca2+ (CRAC) Channels

Abstract

The stimulation of membrane receptors coupled to the phopholipase C pathway leads to activation of the Ca 2؉ release-activated Ca 2؉ (CRAC) channels. Recent evidence indicates that ORAI1 is an essential pore subunit of CRAC channels. STIM1 is additionally required for CRAC channel activation. The present study focuses on the genomic organization, tissue expression pattern, and functional properties of the murine ORAI2. Additionally, we report the cloning of the murine ORAI1, ORAI3, and STIM1. Two chromosomal loci were identified for the murine orai2 gene, one containing an intronless gene and a second locus that gives rise to the splice variants ORAI2 long (ORAI2L) and ORAI2 short (ORAI2S). Northern blots revealed a prominent expression of the ORAI2 variants in the brain, lung, spleen, and intestine, while ORAI1, ORAI3, and STIM1 appeared to be near ubiquitously expressed in mice tissues. Specific antibodies detected ORAI2 in RBL 2H3 but not in HEK 293 cells, whereas both cell lines appeared to express ORAI1 and STIM1 proteins. Co-expression experiments with STIM1 and either ORAI1 or ORAI2 variants showed that ORAI2L and ORAI2S enhanced substantially CRAC current densities in HEK 293 but were ineffective in RBL 2H3 cells, whereas ORAI1 strongly amplified CRAC currents in both cell lines. Thus, the capability of ORAI2 variants to form CRAC channels depends strongly on the cell background. Additionally, CRAC channels formed by ORAI2S were strongly sensitive to inactivation by internal Ca 2؉ . When co-expressed with STIM1 and ORAI1, ORAI2S apparently plays a negative dominant role in the formation of CRAC channels.

Bombyx mori Homologs of STIM1 and Orai1 Are Essential Components of the Signal Transduction Cascade That Regulates Sex Pheromone Production

“…In contrast, the BmOrai1B transcript is generated from exons 1, 2, 3, and 4 but with exons 1 and 2 giving rise to the 5Ј UTR with the start methionine in exon 3. Two mouse Orai2 variants that differ only in the length of their N termini are generated from a similar alternative splicing mechanism (43).…”

The STIM/Orai coupling machinery

“…The three highly homologous Orai1, Orai2 and Orai3 proteins are widely expressed at the mRNA level and display similar plasma-membrane expression. 28,73 All three channels activate in a store-dependent manner when co-expressed with STIM1, while the extent of their current amplitudes is 2-3 fold smaller for Orai2 and Orai3 compared to Orai1. These reduced currents may reflect differences in their expression levels, efficiency in coupling to STIM1 and distinct single channel properties.…”

“…37 Hence, the positively charged 2-APB 74 might exert part of its stimulatory action via interaction with the Orai3 selectivity filter together with further structural requirements located within transmembrane 2 and 3 that finally result in stimulated currents with altered permeation properties. For a more detailed view on other drugs that affect STIM as well as Orai proteins, we refer to Derler et al 14 Besides these three Orai channels, further Orai2 splice variants have been identified on two chromosomal loci, 73 one containing an intronless gene and a second locus that gives rise to the splice variants Orai2 long (Orai2L) and Orai2 short (Orai2S). When co-expressed with STIM1 both Orai2S and Orai2L represent Ca 2+ selective channels.…”

Plasticity in Ca 2+ selectivity of Orai1/Orai3 heteromeric channel

“…Orai1 is strongly expressed in lymphoid organs, skeletal muscle, liver, and skin (6,7). While Orai3 is detected in similar tissues as Orai1, Orai2 is predominantly found in murine brain (8) or human kidney, lung, and spleen (9). T-cells of patients with a severe combined immunodeficiency lacked CRAC currents due to non-functional Orai1 channels, caused by a single point mutation (6,(10)(11)(12).…”