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Inositol 1,4,5‐trisphosphate receptors (IP3Rs) are a family of intracellular Ca2+ channels that exist as homo‐ or heterotetramers. In order to determine whether the N‐terminal ligand‐binding domain is in close physical proximity to the C‐terminal pore domain, we prepared microsomal membranes from COS‐7 cells expressing recombinant type I and type III IP3R isoforms. Trypsin digestion followed by cross‐linking and co‐immunoprecipitation of peptide fragments suggested an inter‐subunit N‐ and C‐terminal interaction in both homo‐ and heterotetramers. This observation was further supported by the ability of in vitro translated C‐terminal peptides to interact specifically with an N‐terminal fusion protein. Using a 45Ca2+ flux assay, we provide functional evidence that the ligand‐binding domain of one subunit can gate the pore domain of an adjacent subunit. We conclude that common structural motifs are shared between the type I and type III IP3Rs and propose that the gating mechanism of IP3R Ca2+ channels involves the association of the N‐terminus of one subunit with the C‐terminus of an adjacent subunit in both homo‐ and heterotetrameric complexes. [ABSTRACT FROM AUTHOR]

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are a family of intracellular Ca2+ channels that exist as homo- or heterotetramers. In order to determine whether the N-terminal ligand-binding domain is in close physical proximity to the C-terminal pore domain, we prepared microsomal membranes from COS-7 cells expressing recombinant type I and type III IP3R isoforms. Trypsin digestion followed by cross-linking and co-immunoprecipitation of peptide fragments suggested an inter-subunit N- and C-terminal interaction in both homo- and heteratetramers. This observation was further supported by the ability of in vitro translated C-terminal peptides to interact specifically with an N-terminal fusion protein. Using a 45Ca2+ flux assay, we provide functional evidence that the ligand-binding domain of one subunit can gate the pore domain of an adjacent subunit. We conclude that common structural motifs are shared between the type I and type III IP3Rs and propose that the gating mechanism of IP3R Ca2+ channels involves the association of the N-terminus of one subunit with the C-terminus of an adjacent subunit in both homo- and heterotetrameric complexes. [ABSTRACT FROM AUTHOR]

Store-operated Ca2+ entry is a central component of intracellular Ca2+ signaling pathways. The Ca2+ release-activated channel (CRAC) mediates store-operated Ca2+ entry in many different cell types. The CRAC channel is composed of the plasma membrane (PM)-localized Orai1 channel and endoplasmic reticulum (ER)-localized STIM1 Ca2+ sensor. Upon ER Ca2+ store depletion, Orai1 and STIM1 form complexes at ER-PM junctions, leading to the formation of activated CRAC channels. Although the importance of CRAC channels is well described, the underlying mechanisms that regulate the recruitment of Orai1 to ER-PM junctions are not fully understood. Here, we describe the rapid and transient S-acylation of Orai1. Using biochemical approaches, we show that Orai1 is rapidly S-acylated at cysteine 143 upon ER Ca2+ store depletion. Importantly, S-acylation of cysteine 143 is required for Orai1-mediated Ca2+ entry and recruitment to STIM1 puncta. We conclude that store depletion-induced S-acylation of Orai1 is necessary for recruitment to ER-PM junctions, subsequent binding to STIM1 and channel activation. [ABSTRACT FROM AUTHOR]