The Ca
2+ release-activated Ca
2+ channel is a
principal regulator of intracellular Ca
2+ rise, which conducts
various biological functions, including immune responses. This channel,
involved in store-operated Ca
2+ 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
Ca
2+ 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.Ca
2+ 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
Ca
2+ 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 Ca
2+ through the inositol 1,4,5-trisphosphate
(IP
3) receptor channel in response to various signals, and the
Ca
2+ store is depleted. Depletion of Ca
2+ then induces
Ca
2+ influx from outside the cell to help in refilling the
Ca
2+ stores and to continue Ca
2+ rise for several
minutes in the cytoplasm (
1,
2). This Ca
2+ influx
was first proposed by Putney
(
3) and was named
store-operated Ca
2+ influx. In the immune system, store-operated
Ca
2+ influx is mainly mediated by the Ca
2+
release-activated Ca
2+ (CRAC) current, which is a highly
Ca
2+-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 Ca
2+
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 Ca
2+ 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.
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