Genome-wide RNAi screen of Ca2?influx identifies
genes that regulate Ca2?release-activated Ca2?
Shenyuan L. Zhang*, Andriy V. Yeromin*, Xiang H.-F. Zhang†, Ying Yu*, Olga Safrina*, Aubin Penna*, Jack Roos‡,
Kenneth A. Stauderman‡, and Michael D. Cahalan*§
*Department of Physiology and Biophysics and Center for Immunology, University of California, Irvine, CA 92697;†Department of Biological Sciences,
Columbia University, New York, NY 10027; and‡TorreyPines Therapeutics, Inc., La Jolla, CA 92037
Communicated by Bertil Hille, University of Washington, Seattle, WA, April 19, 2006 (received for review April 11, 2006)
Recent studies by our group and others demonstrated a required
and conserved role of Stim in store-operated Ca2?influx and Ca2?
release-activated Ca2?(CRAC) channel activity. By using an unbi-
ased genome-wide RNA interference screen in Drosophila S2 cells,
we now identify 75 hits that strongly inhibited Ca2?influx upon
store emptying by thapsigargin. Among these hits are 11 predicted
transmembrane proteins, including Stim, and one, olf186-F, that
upon RNA interference-mediated knockdown exhibited a pro-
found reduction of thapsigargin-evoked Ca2?entry and CRAC
current, and upon overexpression a 3-fold augmentation of CRAC
control and developed more rapidly when olf186-F was cotrans-
fected with Stim. olf186-F is a member of a highly conserved family
of four-transmembrane spanning proteins with homologs from
Caenorhabditis elegans to human. The endoplasmic reticulum (ER)
Ca2?pump sarco-?ER calcium ATPase (SERCA) and the single
transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attach-
ment receptor (SNARE) protein Syntaxin5 also were required for
CRAC channel activity, consistent with a signaling pathway in
plasma membrane, and interacts with olf186-F to trigger CRAC
capacitative calcium entry (CCE) ? genome-wide screen ? CRAC channel ?
RNA interference ? store-operated calcium (SOC) influx
channels in lymphocytes and other human cell types (1, 2).
Despite the acknowledged functional importance of store-
operated Ca2?(SOC) influx in cell biology (2) and of CRAC
channels for immune cell activation (3), the intrinsic channel
components and signaling pathways that lead to channel acti-
vation remain unidentified. In previous work (4), we demon-
strated that SOC influx in S2 cells occurs through a channel that
shares biophysical properties with CRAC channels in human T
lymphocytes. In a medium-throughput RNA interference
(RNAi) screen targeting 170 candidate genes in S2 cells, we
discovered an essential conserved role of Stim and the mam-
malian homolog STIM1 in SOC influx and CRAC channel
activity (5). STIM1 and STIM2 also were identified in an
independently performed screen of HeLa cells by using the
Drosophila enzyme Dicer to generate small interfering RNA
species from dsRNA (6). Drosophila Stim and the mammalian
homolog STIM1 appear to play dual roles in the CRAC channel
activation sequence, sensing the luminal Ca2?store content
through an EF hand motif and trafficking from an endoplasmic
reticulum (ER)-like localization to the plasma membrane to
trigger CRAC channel activity (6–8). However, as single-pass
transmembrane proteins, Stim and its mammalian homolog
STIM1 are unlikely to form the CRAC channel itself. To search
systematically for additional components of the CRAC channel,
and to analyze the signaling network and other required factors
atch–clamp experiments have identified the biophysical
characteristics of Ca2?release-activated Ca2?(CRAC)
that lead to SOC channel activity, we devised and performed a
genome-wide screen on S2 cells based on a fluorescence assay of
Ca2?influx. The library at Harvard’s Drosophila RNAi Screen-
ing Center (DRSC) of 23,845 dsRNA amplicons has been used
in several functional screens (9–14).
severe combined immune deficiency (SCID) (15). The screen in
this study made use of the ability of thapsigargin (TG) to send
GFP-tagged nuclear factor of activated T cells (NFAT) to the
nucleus in S2 cells, providing an assay for disruption of signaling
anywhere in the cascade from elevated [Ca2?]i to calcineurin
activation and nuclear relocalization of NFAT. The fly gene
olf186-F (named Orai) was identified in the screen, and a human
homolog on chromosome 12 was shown to be mutated in SCID
patients, resulting in the loss of CRAC channel activity. Heter-
ologous expression of the wild-type human homolog, which was
named Orai1, restored CRAC channel activity in SCID T cell
Here, based on direct Ca2?influx measurements in a genome-
wide screen, we identify several genes that are required for
CRAC channel function in S2 cells. Our results confirm the
functional requirement of olf186-F (Orai) for Ca2?signaling and
extend these results to investigate effects of knockdown and
sarco-?ER calcium ATPase (SERCA) pump and the trafficking
protein Syntaxin 5 are required for CRAC channel activity.
Genome-Wide Screen for SOC Influx. Each well of 63 separate
384-well plates contained an individual dsRNA amplicon. Ca2?-
indicator fluorescence measurements were made in each well to
monitor cytosolic Ca2?([Ca2?]i) before (basal) and after [ca-
pacitive calcium entry (CCE)] addition of TG. TG inhibits
SERCA pump-mediated reuptake of Ca2?into cellular stores,
depleting them and triggering CCE in S2 cells (4, 16), as well as
in mammalian cells (2). Hits in the screen were defined by
significantly reduced CCE?basal values, as described in Methods
and illustrated by a tail in the histogram shown in Fig. 1A. The
‘‘top 10 hits,’’ with strong suppressive effects comparable with
were selected for further evaluation (Fig. 1B; see also Table 1,
which is published as supporting information on the PNAS web
site). Among the 75 filtered hits with z-scores of CCE?basal ?
?3 (see Table 2, which is published as supporting information on
the PNAS web site), only 11 contained transmembrane seg-
Conflict of interest statement: No conflicts declared.
Abbreviations: CCE, capacitive calcium entry; CRAC, Ca2?release-activated Ca2?; ER, en-
doplasmic reticulum; RNAi, RNA interference; SERCA, sarco-?ER calcium ATPase; SNARE,
single transmembrane-soluble N-ethylmaleimide-sensitive attachment receptor; SOC,
store-operated Ca2?; TG, thapsigargin.
§To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
June 13, 2006 ?
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ments, as shown in Fig. 1C. Among these hits, the five strongest
are annotated in Flybase (www.flybase.org) as Ca-P60A, Stim,
olf186-F, sec61alpha, and Syx5.
The consistent suppressive effect of Stim dsRNA validates the
present screen. However, Stim is unlikely to constitute the
CRAC channel, because multiple transmembrane segments are
found in all known ion-channel pore-forming subunits. The
protein product of sec61alpha is a subunit of the translocon
complex, which recognizes and delivers newly synthesized mem-
synthesis or localization of other essential components. Ca-P60A
is the SERCA pump gene in fly, whose products are located in
the ER for filling?refilling the Ca2?store. Syx5 generates a single
transmembrane-soluble N-ethylmaleimide-sensitive (NSF) at-
cells. RT-PCR analysis on olf186-F, Stim, CG11059, and a control gene, Presenilin (Psn). (B) [Ca2?]iin eight representative S2 cells treated with CG11059 dsRNA.
Solution exchanges are indicated. (C) [Ca2?]iin eight cells treated with olf186-F dsRNA. (D) Averaged [Ca2?]ivalues ? SEM for control cells (n ? 195 cells in three
Ca2?before TG treatment (Ca0 3 Ca2), peak [Ca2?]iduring TG-evoked release transient (Ca0 ? TG), and maximal and sustained (3 min) [Ca2?]iafter readdition
of 2 mM external Ca2?. (E) Representative time course of whole-cell currents recorded in control cells treated with CG11059 dsRNA and in cells treated with
olf186-F dsRNA. (F) Suppression of CRAC current by olf186-F dsRNA pretreatment. Each point represents the maximal inward CRAC current density (pA?pF) in
a single cell, plotted as absolute values in consecutive order from left to right within three groups of cells: untreated, cells treated with dsRNA to suppress
CG11059, or olf186-F (P ? 5 ? 10?6compared with either control group). The untreated cell group includes two cells each with current density ?12 pA?pF.
Horizontal lines indicate the mean value of current density in each group.
Suppression of TG-dependent Ca2?influx and CRAC current by olf186-F dsRNA. (A) Reduction of olf186-F mRNA expression in olf186-F dsRNA-treated
basal Ca2?, displayed as a histogram. (Inset) The distribution of averaged CCE?basal values for each well. Low values of CCE?basal are enlarged to show the tail
of the distribution, representing amplicons that dramatically suppressed TG-evoked calcium entry. (B) The top 10 hits with strongest effect on TG-evoked Ca2?
influx. Averaged values of CCE?basal are shown for all 48,384 wells tested in the assay (‘‘mean’’), for the top 10 hits from the screen, and for the positive control
well that contained Stim dsRNA in each assay plate (‘‘Stim Ave’’). Striped bars represent hits with transmembrane regions. (C) Transmembrane (TM) protein hits.
Identification of genes involved in store-operated calcium entry. (A) The effect of individual gene silencing on TG-evoked Ca2?entry (CCE) relative to
www.pnas.org?cgi?doi?10.1073?pnas.0603161103 Zhang et al.
tachment receptor (SNARE) protein (Syntaxin 5), which is
essential for vesicle fusion and may modulate CCE by altered
protein trafficking rather than serving as the channel pore. Thus,
among the top 10 hits, olf186-F is the only gene of unknown
structure and function that is predicted to contain multiple
Effects of olf186-F Knockdown and Overexpression on Ca2?Influx and
CRAC Currents in Single Cells. To clarify effects of suppressing
olf186-F at the level of single cells, we examined Ca2?signaling
and CRAC currents in cells treated with dsRNA for olf186-F, in
comparison with untreated cells or with cells treated with
dsRNA for CG11059, an irrelevant cell adhesion molecule (5),
as controls. RT-PCR showed ?50% decrease of olf186-F mRNA
expression, compared with controls (Fig. 2A). Fig. 2B illustrates
ratiometric fura-2 [Ca2?]imeasurements before and after TG-
evoked store depletion in eight individual control cells. Addition
of TG in zero-Ca2?solution to deplete the store elicited a Ca2?
release transient caused by net leak of Ca2?from the store when
a robust Ca2?signal was observed in every cell. In cells pre-
treated with olf186-F dsRNA, neither the resting [Ca2?]ilevel
nor the release transient were significantly altered, but the rise
in [Ca2?]i upon readdition of external Ca2?was strongly
suppressed in the vast majority of the individual cells (Fig. 2C).
Fig. 2D clearly demonstrates that suppression of olf186-F effec-
tively inhibits both the early and sustained components of Ca2?
entry evoked by TG at the single-cell level. Comparable inhibi-
tion was obtained in cells pretreated with Stim dsRNA as a
positive control (data not shown), consistent with our previous
Patch–clamp experiments confirmed a dramatic suppression
of CRAC currents after knockdown of olf186-F (Fig. 2 E and F).
CRAC current normally develops after establishing the whole-
cell recording configuration as the cytoplasm is dialyzed by a
pipette solution containing a strong Ca2?chelator to reduce
cytosolic [Ca2?]iand deplete internal stores. With this method of
to a maximum value before declining slowly. However, in the
majority of cells pretreated with olf186-F dsRNA, CRAC cur-
rent was completely suppressed, as illustrated by the represen-
tative traces in Fig. 2E and by a chart of CRAC current densities
(Fig. 2F). As we showed previously for Stim (5), olf186-F
expression is required for normal CRAC channel activity.
To examine further the function of olf186-F, we cloned its
full-length cDNA from S2 cells and inserted it into a Drosophila
expression vector. The olf186-F clone was overexpressed with or
without a cotransfected Stim clone in S2 cells, by using a
cotransfected GFP construct for identification of transfected
cells. Increased expression levels of olf186-F and Stim after
separate transfections or cotransfection were verified by RT-
PCR (see Fig. 6A, which is published as supporting information
on the PNAS web site). Fig. 3A illustrates the time course of
current development after break-in to achieve whole-cell re-
cording in four representative cells. Expression of Stim by itself
had no significant effect on current amplitude compared with
control, untransfected cells. However, when olf186-F was over-
expressed, CRAC current increased significantly, and when
Stim, olf186-F, and olf186-F plus Stim. (B) Ca2?current in olf186-F ? Stim cotransfected cell. Arrows a and b indicate the time corresponding to current–voltage
curves in C. (C) Current–voltage relationship of CRAC current in the same cell. (D) CRAC current density in transfected S2 cells, plotted as in Fig. 2F, within four
groups of cells: GFP-transfected control; Stim and GFP cotransfected (not significantly different from controls); olf186-F and GFP cotransfected (P ? 10?3); and
(E) Method to analyze kinetics of CRAC current development. (F) Effect of cotransfected Stim on delay kinetics. Delay times are significantly reduced (P ? 5 ?
10?6), but time1/2values are not altered when Stim is expressed together with olf186-F, compared with olf186-F alone.
Overexpression of olf186-F leads to increased CRAC currents in S2 cells. (A) Representative CRAC currents in S2 cells transfected with GFP only (control),
Zhang et al.
June 13, 2006 ?
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olf186-F was coexpressed with Stim, CRAC current was further
enhanced. The induced current after cotransfection of olf186-F
with Stim exhibited Ca2?selectivity and current–voltage shapes
indistinguishable from native CRAC current (Fig. 3 B and C).
When external Ca2?was elevated 10-fold, the current magni-
tudes approximately doubled, as is the case for native CRAC
current in S2 cells (4), and current–voltage curves had the same
inwardly rectifying characteristic. Fig. 3D illustrates CRAC
current densities for individual cells in each group of transfected
cells. Overexpression of olf186-F increased the average current
density 3-fold, and although Stim by itself did not alter current
density, cotransfection with olf186-F produced a remarkable
8-fold enhancement. Interestingly, cotransfection with Stim also
decreased the initial delay to the onset of current development
(Fig. 3 A, E, and F). Together, these results show that overex-
pression of olf186-F is sufficient to increase CRAC current
density, that coexpression with Stim produces a further enhance-
for channel activation.
Apart from much larger current amplitudes, the Ca2?-
selective current in cells cotransfected with olf186-F and Stim
native CRAC currents. Monovalent ion selectivity upon removal
of external Ca2?(divalent-free), Na?current inactivation, and
potentiation of Ca2?current upon readdition of external Ca2?
were similar to that described for native CRAC current in
lymphocytes and S2 cells (see Fig. 7A, which is published as
supporting information on the PNAS web site) (4, 17–19).
Current–voltage relations for the monovalent Na?current also
showed inward rectification and a reversal potential of ?45 mV
(Fig. 7B), the same as native monovalent CRAC current and
consistent with low permeability to Cs?(4). The response to
voltage steps was also the same, with currents that increase
slightly at very negative potentials (Fig. 7 C and D), as seen
previously in S2 cells (4). Furthermore, the Ca2?current in
olf186-F ? Stim transfectants was sensitive to pharmacological
agents that act on native CRAC currents (Fig. 7 E and F). Gd3?
(50 nM) and 2-aminoethyldiphenyl borate (2-APB; 20 ?M)
blocked the enhanced Ca2?currents, and at lower concentration
(5 ?M) 2-APB exhibited a characteristic potentiation of current
before blocking. In summary, the ion selectivity, development
and inactivation kinetics, and pharmacological profile of the
large induced Ca2?current after overexpression of olf186-F plus
Stim match native CRAC currents. Because the current is not
the possibility that olf186-F itself is part of the channel.
Effects of Ca-P60A, Syx5, and tsr dsRNA Treatment on Ca2?Dynamics
and CRAC Current. The SERCA pump also emerged from the
RNAi screen as a putative regulator of SOC influx. However,
because the screen was based on Ca2?influx induced by TG
(which blocks the SERCA pump), we were concerned about the
potential for a false-positive hit. We therefore performed single-
cell Ca2?imaging and patch–clamp experiments using alterna-
tive stimuli (ionomycin, passive stores depletion) to deplete the
Ca2?store. Selective lowering of Ca-P60A mRNA was first
verified by RT-PCR (Fig. 6B). Knockdown of Ca-P60A signifi-
cantly increased resting [Ca2?]i,reduced the store release tran-
sient upon addition of TG and strongly suppressed Ca2?influx
upon readdition of external Ca2?(Fig. 4 A and B). In addition,
ionomycin in zero-Ca2?solution applied to control cells evoked
a sharp Ca2?release transient with a peak that averaged ?200
nM, but a greatly reduced release transient in Ca-P60A dsRNA-
treated cells (Fig. 4 C and D), indicating reduced Ca2?store
content as a consequence of reduced SERCA pump activity. As
shown by the summary of Ca2?imaging experiments (Fig. 4E),
knockdown of SERCA has a strong Ca2?phenotype, raising
resting [Ca2?]i, reducing release transients, and suppressing
influx evoked by TG. Furthermore, patch–clamp experiments
demonstrated that CRAC currents also were suppressed when
stores were depleted passively by dialysis of a Ca2?chelator (Fig.
4F), confirming a requirement of Ca-P60A for activation of
functional CRAC channels.
Several trafficking proteins also were identified as putative
regulators of SOC activity (Table 2). Syx5 is a syntaxin, several
of which have been implicated in SNARE complexes that
regulate vesicle trafficking; and tsr is referred to as an actin-
binding protein that regulates cytoskeleton remodeling. A pu-
tative role of its human homolog, cofilin, has been reported in
activation of store-operated calcium entry in platelets (20). Both
Syx5 and tsr dsRNA preincubation caused significant and selec-
tive lowering of mRNA levels (Fig. 6 C and D) and a corre-
sponding inhibition of TG-dependent Ca2?influx in S2 cells,
without altering the resting [Ca2?]ior store release (compare
Fig. 5 A–C). Fig. 5D summarizes the inhibition of TG-evoked
[Ca2?]iinflux when Syx5 or tsr expression was knocked down.
Patch–clamp experiments confirmed that CRAC currents were
indeed suppressed during passive stores depletion when Syx5 was
knocked down, but effects of tsr knockdown on CRAC currents
did not achieve statistical significance (Fig. 5E).
Our genome-wide screen, based on direct Ca2?influx measure-
are required for CRAC channel activity. We independently
identified olf186-F (Orai) as essential for Ca2?signaling and
Averaged [Ca2?]iin cells treated with control CG11059 dsRNA. (B) Averaged
[Ca2?]iin cells treated with Ca-P60A dsRNA. (C and D) Ca2?release evoked by
1 ?M ionomycin in control cells and in cells treated with Ca-P60A dsRNA to
knock down SERCA expression. (E) Averaged [Ca2?]ivalues ? SEM for control
2D and including peak [Ca2?]i during ionomycin-evoked release transient
(Ca0 ? Iono). (F) Summary of inward CRAC current densities in control
CG11059- and Ca-P60A dsRNA-treated cells (P ? 0.002), using the same plot-
ting format as in Fig. 2F.
Effects of Ca-P60A dsRNA on Ca2?dynamics in individual S2 cells. (A)
www.pnas.org?cgi?doi?10.1073?pnas.0603161103Zhang et al.
activation of CRAC current in S2 cells, confirming two recent
reports (15, 21). In addition, we provide evidence based on
overexpression that it may form an essential part of the CRAC
channel. In mammalian cells overexpression of STIM1 increases
Ca2?influx rates and CRAC currents by ?2-fold (7, 8), but in
S2 cells we show that overexpression of Stim alone does not
increase CRAC current, consistent with Stim serving as a
channel activator rather than the channel itself. In contrast,
transfection of olf186-F by itself increased CRAC current den-
sities 3-fold, and cotransfection of olf186-F with Stim resulted in
an 8-fold enhancement and the largest CRAC currents ever
recorded. These results support the hypothesis that olf186-F
constitutes part of the CRAC channel and that Stim serves as the
messenger for its activation. Consistent with this hypothesis, the
CRAC channel activation kinetics during passive Ca2?store
depletion were significantly faster with cotransfected Stim. Many
fundamental aspects of the mechanism of CRAC channel acti-
vation remain to be clarified, including the protein–protein
interactions that underlie trafficking and channel activation.
Site-directed mutagenesis in a heterologous expression system
may help to define the putative pore-forming region.
Similar to Stim, knockdown of olf186-F did not produce a
severe cell growth phenotype (data not shown). It was neither a
hit in a previous screen of cell survival (9) nor in any other
published Drosophila whole-genome RNAi screen (10–14). The
olf186-F gene is a member of a highly conserved gene family that
contains three homologs in mammals, two in chicken, three in
zebrafish, and one member only in fly and worm (see Fig. 8A,
which is published as supporting information on the PNAS web
site). C09F5.2, the only homolog in Caenorhabditis elegans, is
expressed in intestine, hypodermis, and reproductive system as
well as some neuron-like cells in the head and tail regions
(www.wormbase.org). Worms under RNAi treatment against
C09F5.2 are sterile (22). Analysis of hydrophobic regions of the
predicted protein from the fly gene and the three mammalian
homologs (Fig. 8B) suggested the presence of four conserved
transmembrane segments. Cytoplasmic C termini are suggested
by the presence of coiled-coil motifs in each sequence. A
predicted transmembrane topology and the sequence for the fly
gene are shown in Fig. 8C. Sequence alignment between mem-
bers from human, chicken, and fly revealed strong sequence
conservation in putative transmembrane regions and conserved
negatively charged residues in loops between transmembrane
segments. All three human members are expressed in the
immune system (GNF Symatlas; http:??symatlas.gnf.org?
SymAtlas). Mutation of a human homolog of Drosophila
olf186-F, ORAI1 on chromosome 12, appears to be the cause of
defective CRAC channel activity in severe combined immune
deficiency patient T cells (15), consistent with a requirement for
functional CRAC channels in the immune response. Interest-
ingly, microarray data from public databases (GEO profiles;
www.ncbi.nlm.nih.gov) combined with tissue-specific EST
counts show that all three human members are expressed in a
variety of nonexcitable tissues including thymus, lymph node,
intestine, dermis, and many other tissues including the brain,
although expression patterns and levels are different among the
Ca-P60A has been proposed to be the only Drosophila SERCA
gene (23). We validated its ER pump function by showing that
ionomycin did not induce significant store release from S2 cells
pretreated with dsRNA against Ca-P60A, consistent with a
previous report (23). The elevation in resting [Ca2?]iand rapidly
changing Ca2?transients during changes in external Ca2?before
addition of TG may indicate a low level of constitutive CRAC
channel activity induced by store depletion. In addition, SERCA
knockdown inhibited CRAC channel activity after passive store
labeled as in Fig. 2D. (E) Summary of inward CRAC current densities in Syx5 and tsr dsRNA-treated cells, using the same plotting format as in Fig. 2F. Mean values
for CG11059 and Syx5 are significantly different (P ? 0.004). The mean values for CG11059 and tsr are not significantly different (P ? 0.65).
Zhang et al.
June 13, 2006 ?
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depletion in whole-cell patch recordings. These results are Download full-text
consistent with the SERCA pump being required for normal
activity of CRAC channels but do not rule out indirect inhibition
of CRAC current as a consequence of residual high resting
[Ca2?]ior store depletion. The role of SERCA in CRAC channel
function merits further study.
Among the hits, several are known to be involved in protein
trafficking. The gene products of both Syx5 and Syx1A are
t-SNARE proteins involved in vesicle fusion in many cell types.
We verified the RNAi effects of Syx5 at the single-cell level and
demonstrated strong suppression of CRAC channel activity as
well as the SOC influx. tsr may regulate SOC influx indirectly by
controlling cell metabolism because RNAi of tsr did not signif-
icantly influence CRAC current density in whole-cell patch–
clamp experiments. Membrane trafficking previously was sug-
gested to be important for SOC channel activity in Xenopus
oocytes, based on inhibition by botulinum toxin or by a domi-
nant-negative SNAP-25 construct (24), and our results further
suggest a requirement for syntaxins and SNARE-complex for-
mation, possibly to mediate translocation of Stim to the plasma
membrane (6, 7). The screen also revealed three other groups of
hits that may influence calcium dynamics. These results set the
stage for experiments targeting specific genes to understand the
fine tuning of Ca2?homeostasis and signaling.
Drosophila S2 cells were cultured in 384-well plates containing
?0.25 ?g of dsRNA (?104cells per well). Each plate included
a well with dsRNA targeting Stim as a positive control. After 5
days, cells were loaded with a [Ca2?]iindicator fluo-4?AM (10
?M; Molecular Probes); free dye then was washed by Ringer
solution containing 2 mM Ca2?(see Table 3, which is published
as supporting information on the PNAS web site, for all solution
recipes). Three fluorescence measurements were systematically
performed: basal (resting intracellular free Ca2?), CCE (TG-
dependent Ca2?influx assessed 4 min after addition of TG), and
Fmax (maximal fluorescence 15 min after addition of Triton
X-100 to a final concentration of ?2% to detect changes in cell
number). A schematic diagram is shown in Fig. 9A, which is
published as supporting information on the PNAS web site.
Values of ‘‘basal?Fmax’’ were calculated for each well to indicate
the normalized resting [Ca2?]ilevel, and values of ‘‘CCE?basal’’
were computed to represent the relative CCE levels. The screen
was carried out in duplicate. To correct for variation in dye
loading or cell number, we computed ratios of fluorescence
values (CCE?basal) as an index for Ca2?influx evoked by TG.
A scatter plot showed reasonable agreement for the replicate
assays for most amplicons (Fig. 9B), particularly for hits with
reduced Ca2?influx reflected in lower CCE?basal values. Be-
cause most amplicons did not influence the dynamics of Ca2?
signaling, the average for a given plate was very close to that of
nontreated wells. Therefore, z-scores of basal?Fmaxand CCE?
basal equal to the value of the well minus the average of the plate
divided by the standard deviation for the plate were calculated
for each well. The averaged z-scores (Fig. 9C) represent varia-
tions in the distribution of CCE?basal measurements for each
amplicon. Hits in the screen, defined by values of ?3 standard
deviations from the mean (z-score ? ?3 or ?3) fell into four
categories: (i) decreased resting [Ca2?]i; (ii) increased resting
[Ca2?]i; (iii) decreased CCE (Table 2); and (iv) increased CCE.
To eliminate false-positive outcomes, putative hits with a z-score
of Fmax? ?2, or with more than five off-targets, were generally
filtered out from the lists. Overlapping hits between groups i and
iv and groups ii and iii were removed from group iv and iii,
The remaining methods can be found in Supporting Materials
and Methods and Table 4, which are published as supporting
information on the PNAS web site.
We thank Sindy Wei for help with [Ca2?]iimaging; J. Ashot Kozak for
helpful discussion; Karinne Cahalan for assistance with illustrations; Dr.
Weihua Jiang for data processing; Dr. Luette Forrest for help with cell
culture; and B. Mathey-Prevot, N. Perrimon, and staff at the Drosophila
RNAi Screening Center at Harvard. This work was supported by
National Institutes of Health Grant NS14609 (to M.D.C.), a George E.
Hewitt Foundation fellowship (to S.L.Z.), and American Heart Associ-
ation Scientist Development Grant 0630117N (to Y.Y.).
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