Abstract: | The Ca2+ release-activated Ca2+ channel is a principal regulator of intracellular Ca2+ rise, which conducts various biological functions, including immune responses. This channel, involved in store-operated Ca2+ influx, is believed to be composed of at least two major components. Orai1 has a putative channel pore and locates in the plasma membrane, and STIM1 is a sensor for luminal Ca2+ store depletion in the endoplasmic reticulum membrane. Here we have purified the FLAG-fused Orai1 protein, determined its tetrameric stoichiometry, and reconstructed its three-dimensional structure at 21-Å resolution from 3681 automatically selected particle images, taken with an electron microscope. This first structural depiction of a member of the Orai family shows an elongated teardrop-shape 150Å in height and 95Å in width. Antibody decoration and volume estimation from the amino acid sequence indicate that the widest transmembrane domain is located between the round extracellular domain and the tapered cytoplasmic domain. The cytoplasmic length of 100Å is sufficient for direct association with STIM1. Orifices close to the extracellular and intracellular membrane surfaces of Orai1 seem to connect outside the molecule to large internal cavities.Ca2+ is an intracellular second messenger that plays important roles in various physiological functions such as immune response, muscle contraction, neurotransmitter release, and cell proliferation. Intracellular Ca2+ is mainly stored in the endoplasmic reticulum (ER).2 This ER system is distributed through the cytoplasm from around the nucleus to the cell periphery close to the plasma membrane. In non-excitable cells, the ER releases Ca2+ through the inositol 1,4,5-trisphosphate (IP3) receptor channel in response to various signals, and the Ca2+ store is depleted. Depletion of Ca2+ then induces Ca2+ influx from outside the cell to help in refilling the Ca2+ stores and to continue Ca2+ rise for several minutes in the cytoplasm (1, 2). This Ca2+ influx was first proposed by Putney (3) and was named store-operated Ca2+ influx. In the immune system, store-operated Ca2+ influx is mainly mediated by the Ca2+ release-activated Ca2+ (CRAC) current, which is a highly Ca2+-selective inwardly rectified current with low conductance (4, 5). Pathologically, the loss of CRAC current in T cells causes severe combined immunodeficiency (6) where many Ca2+ signal-dependent gene expressions, including cytokines, are interrupted (7). Therefore, CRAC current is necessary for T cell functions.Recently, Orai1 (also called CRACM1) and STIM1 have been physiologically characterized as essential components of the CRAC channel (8–12). They are separately located in the plasma membrane and in the ER membrane; co-expression of these proteins presents heterologous CRAC-like currents in various types of cells (10, 13–15). Both of them are shown to be expressed ubiquitously in various tissues (16–18). STIM1 senses Ca2+ depletion in the ER through its EF hand motif (19) and transmits a signal to Orai1 in the plasma membrane. Although Orai1 is proposed as a regulatory component for some transient receptor potential canonical channels (20, 21), it is believed from the mutation analyses to be the pore-forming subunit of the CRAC channel (8, 22–24). In the steady state, both Orai1 and STIM1 molecules are dispersed in each membrane. When store depletion occurs, STIM1 proteins gather into clusters to form puncta in the ER membrane near the plasma membrane (11, 19). These clusters then trigger the clustering of Orai1 in the plasma membrane sites opposite the puncta (25, 26), and CRAC channels are activated (27).Orai1 has two homologous genes, Orai2 and Orai3 (8). They form the Orai family and have in common the four transmembrane (TM) segments with relatively large N and C termini. These termini are demonstrated to be in the cytoplasm, because both N- and C-terminally introduced tags are immunologically detected only in the membrane-permeabilized cells (8, 9). The subunit stoichiometry of Orai1 is as yet controversial: it is believed to be an oligomer, presumably a dimer or tetramer even in the steady state (16, 28–30).Despite the accumulation of biochemical and electrophysiological data, structural information about Orai1 is limited due to difficulties in purification and crystallization. In this study, we have purified Orai1 in its tetrameric form and have reconstructed the three-dimensional structure from negatively stained electron microscopic (EM) images. |