STIM1 long and STIM1 gate differently TRPC1 during store-operated calcium entry

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STIM1 long and STIM1 gate differently TRPC1 during store-operated calcium entry

DYRDA, Agnieszka, KONIG, Stéphane, FRIEDEN, Maud

DYRDA, Agnieszka, KONIG, Stéphane, FRIEDEN, Maud. STIM1 long and STIM1 gate

differently TRPC1 during store-operated calcium entry. Cell Calcium , 2020, vol. 86, p. 102134

DOI : 10.1016/j.ceca.2019.102134 PMID : 31838437

Available at:

http://archive-ouverte.unige.ch/unige:140151

Disclaimer: layout of this document may differ from the published version.

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Contents lists available atScienceDirect

Cell Calcium

journal homepage:www.elsevier.com/locate/ceca

STIM1 long and STIM1 gate differently TRPC1 during store-operated calcium entry

Agnieszka Dyrda

^a

, Stéphane Koenig

^a

, Maud Frieden

^b,

*

aDepartment of Basic Neurosciences, University of Geneva, Geneva, Switzerland

bDepartment of Cellular Physiology and Metabolism, University of Geneva, Geneva, Switzerland

A R T I C L E I N F O Keywords:

Store-operated Ca²⁺entry Orai1

TRPC1 STIM1 long Patch-clamp Calcium imaging Skeletal muscle

A B S T R A C T

During myogenesis, a long splice variant of STIM1, called STIM1L is getting expressed, while the level of STIM1 remains constant. Previous work demonstrated that STIM1L is more efficient in eliciting store-operated Ca²⁺

entry (SOCE), but no current analysis of the channel(s) activated by this new STIM1L isoform was performed until now. In this study, we investigate the ionic channel(s) activated by STIM1L and whether differences exist between the two STIM1 isoforms, using HEK-293 T cells as a model system. Our data show that STIM1 and STIM1L activate Orai1 channel but also the endogenously expressed TRPC1. The channel activation occurs in two steps, with first Orai1 activation followed, in a subset of cells, by TRPC1 opening. Remarkably, STIM1L more frequently activates TRPC1 and preferentially interacts with TRPC1. In low intracellular Ca²⁺buffering condition, the frequency of TRPC1 opening increases significantly, strongly suggesting a Ca²⁺-dependent channel activation. The ability of STIM1L to open Orai1 appears decreased compared to STIM1, which might be explained by its stronger propensity towards TRPC1. Indeed, increasing the amount of STIM1L results in an enhanced Orai1 current. The role of endogenous TRPC1 in STIM1- and STIM1L-induced SOCE was confirmed by Ca²⁺imaging experiments. Overall, our findings provide a detailed analysis of the channels activated by both STIM1 isoforms, revealing that STIM1L is more prone to open TRPC1, which might explain the larger SOCE elicited by this isoform.

1. Introduction

Store-operated Ca²⁺ entry (SOCE) is an ubiquitous mechanism leading to Ca²⁺influx in response to endoplasmic/sarcoplasmic (ER/

SR) Ca²⁺depletion. The two main families of proteins supporting SOCE are STIM (stromal interaction molecule) and plasma membrane (PM) Orai channels. STIM proteins, comprising STIM1 and STIM2, are single pass transmembrane proteins localized on the ER/SR membrane that serve as ER Ca²⁺sensors. Once Ca²⁺concentration decreases in this compartment, it detaches from the luminal EF hand domain of STIM, which then oligomerizes and translocates towards the PM (reviewed in [1]). STIM oligomerization appears as punctae structures visible by fluorescent microscopy [2,3]. STIM activation is accompanied by an unfolding and exposure of key residues of the C-terminal end that bind

phospholipids of the PM, as well as residues of the CRAC activation domain (CAD) that gate the Ca²⁺-selective Orai channel (reviewed in [4]). Three members of Orai have been identified, Orai1, Orai2 and Orai3 that all are Ca²⁺-selective ion channels with very tiny unitary conductance, calculated to be in the fS range. The prototypical current flowing through Orai1 channel gated by STIM1 is called ICRAC, for Ca²⁺

release-activated Ca²⁺ current. ICRAC has several key electrophysiological characteristics that preclude confusion with other types of ionic currents. Its current-voltage (I/V) curve displays a strong inward rectification, with a reversal potential (Erev) > +60 mV reflecting the high Ca²⁺selectivity [5,6]. The current is efficiently blocked by tri- valent cations like Gd³⁺ or La³⁺. Furthermore, intracellular Ca²⁺

elevation induces both a fast and a slow Ca²⁺-dependent inactivation (FCDI and SCDI) of the current (reviewed in [7]). STIM1 does not only

https://doi.org/10.1016/j.ceca.2019.102134

Received 30 July 2019; Received in revised form 13 November 2019; Accepted 25 November 2019

Abbreviations:SOCE, store-operated Ca²⁺entry; ER/SR, endo/sarcoplasmic reticulum; Tg, thapsigargin; TRPC, transient receptor potential canonical; STIM, stromal interaction molecule; PM, plasma membrane; NFAT, nuclear factor of activated T-cells; CaMKII, calcium/calmodulin-dependent protein kinase type II; ICRAC, calcium release-activated calcium current; CAD, CRAC activation domain; I/V curve, current/voltage curve; Erev, reversal potential; MEFDKO, mouse embryonic fibroblasts double knockout; O1, Orai1; S1, STIM1; S1L, STIM1 long; C1, TRPC1; TRPC1_DN, dominant negative variant of TRPC1; HEK, human embryonic kidney;

FCDI, fast Ca²⁺-dependent inactivation; SCDI, slow Ca²⁺-dependent inactivation; co-IP, co-immunoprecipitation

⁎Corresponding author at: Department of Cell Physiology and Metabolism, University of Geneva Medical School, 1 Michel-Servet, 1211 Geneva 4, Switzerland.

E-mail address:maud.frieden@unige.ch(M. Frieden).

Cell Calcium 86 (2020) 102134

Available online 03 December 2019

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activate members of the Orai family but also non-selective cation channels from the TRPC (transient receptor potential canonical) family.

Direct binding of STIM1 to TRPC1, 4 and 5 measured by co-immunoprecipitation (co-IP) was reported [8], and the opening of TRPC1, but also TRPC3, TRPC4, TRPC5 and TRPC6 by STIM1 was shown to occur via an electrostatic interaction between the last two lysines of STIM1 ⁶⁸⁴KK⁶⁸⁵, and two aspartate residues on the C-terminus of TRPC1 channel (⁶³⁹DD⁶⁴⁰) [9,10]. The activation of TRPC1 by STIM1 was well studied in salivary glands where upon store depletion, Ca²⁺

enters via Orai1, and the local Ca²⁺increase leads to the fusion of TRPC1 containing intracellular vesicles with the PM. The newly in- serted TRPC1 channels are then gated by STIM1 [11–13].

SOCE is considered as the main Ca²⁺entry pathway in non-excitable cells, however, in excitable tissue like skeletal muscles, it was reported to be important as well. Indeed, patients lacking functional SOCE present, beside the severe immunodeficiency, muscular hypo- tonia, a phenotype also found in animal models lacking STIM1 or Orai1 [14]. Interestingly, gain of function mutations, both in STIM1 or in Orai1 also lead to muscle pathological conditions like muscle weakness and myalgia known as tubular aggregate myopathy or the more severe Stormorken syndrome [15,16]. Our laboratory and others documented a role of SOCE in human muscle differentiation [17]. In 2011, we reported that an alternative splicing variant of STIM1 is expressed in myotubes during human myogenesis [18]. This isoform has extra 106 amino acids on the C-terminal part of the protein, and was named STIM1L (long). In human myotubes, STIM1L is bound to cortical actin filaments and thus permanently localize in the proximity to the PM [18], explaining, at least partially, the fast onset of SOCE in this tissue [18,19]. We then provided evidence that downregulation of STIM1L or TRPC1/TRPC4 leads to a slowing down of muscle differentiation with the formation of smaller myotubes as well as a delayed SOCE activation, suggesting that STIM1L functionally interacts with TRPC1 and TRPC4 in muscles [20,21]. Indeed, co-immunoprecipitation revealed that TRPC1/TRPC4 preferentially interact with STIM1L after store depletion. In addition, using mouse embryonic fibroblasts double knockout for STIM1 and STIM2 (MEFDKO) as a model system, we demonstrated that overexpression of STIM1L leads to a 20 % larger SOCE compared to STIM1-activated Ca²⁺entry. This is somehow surprising as the clusters formed by STIM1L are smaller in size compared to their STIM1 counterparts, and the cortical ER is also less abundant in cells overexpressing STIM1L compared to STIM1 [2].

Hence, the present study was designed to characterize the type of current(s) activated by STIM1L versus STIM1, using HEK293 T cells as a model system. The currents of HEK cells overexpressing STIM1 or STIM1L together with Orai1 were recorded and analyzed for potential differences which could explain the larger SOCE activated by STIM1L.

As expected, the electrophysiological recordings clearly demonstrate that both STIM1 isoforms activate Orai1. However, the STIM1L-induced ICRAC is significantly smaller, except when the amount of STIM1L is increased. More surprisingly both STIM1 isoforms also activate endogenous TRPC1, with STIM1L being more efficient in gating TRPC1 compared to STIM1. Fluorescence measurements of Ca²⁺entry confirm the involvement of TRPC1 both on STIM1 and STIM1L-activated SOCE.

2. Material and methods 2.1. Materials

Thapsigargin was purchased from Sigma-Aldrich (cat. No T9033);

Fura-2 AM from Thermo Fisher Scientific, (cat. No F1201) and Pluronic F-127 from Invitrogen (cat. No P3000MP). Orai1-His-Tag was a gift from Dr. A. Rao [22] and constructed from an Addgene plasmid

#21638 (http://n2t.net/addgene:21638; RRID:Addgene_21638), and PS-YFP-STIM1 was a kind gift from Dr. A. B. Parekh (University of Oxford, UK). PS-YFP-STIM1L was constructed as previously described [18]. PS-YFP-STIM1⁶⁸⁴EE⁶⁸⁵and PS-YFP-STIM1L⁷⁹⁰EE⁷⁹¹were created

by DNA point mutation: aag aag to gaa gaa by GeneCust (Dudelange, Luxembourg), and TRPC1 F562A was a kind gift from Muallem’s lab [23], and Flag-hTRPC1 was constructed from an Addgene plasmid

#24408 (http://n2t.net/addgene:24408; RRID:Addgene_24408). All other compounds for solution preparation were at analytical grade and were purchased from Sigma.

2.2. Cell culture and transfections

Human embryonic kidney cells, HEK293 T cell line (ATCC CRL- 11268, lot 59587035) were kept in DMEM medium (Gibco, cat. No 41966) supplemented with 1 % penicillin/streptomycin (Gibco, cat. No 15140) and 10 % FBS (Sigma, cat. No F3385), passed every 4 days and used until passage 30 to avoid changes in cell phenotype [24,25]. 24 h before the experiments, HEK239 T cells were transiently co-transfected using Lipofectamine3000 transfection reagent (Thermo Fisher Scien- tific, cat. No L3000015) according to manufacturer’s instructions with Orai1-His-Tag (0.4 μg) and PS-YFP-STIM1 or PS-YFP-STIM1L (0.5 μg).

For some experiments, Flag-hTRPC1 (0.5−1 μg) was overexpressed.

Alternatively, STIM1 mutants PS-YFP-STIM1⁶⁸⁴EE⁶⁸⁵ or PS-YFP- STIM1L⁷⁹⁰EE⁷⁹¹(0.5 μg) were co-transfected with Orai1. In some experiments, a TRPC1 mutant TRPC1 F562A (0.5 μg) was co-transfected with STIM1/L and Orai1. For the experiments were the STIM1/L to Orai1 ratio were modified, the amount of PS-YFP-STIM1 or PS-YFP- STIM1L was multiplied by 2 and 3 to achieve the 2:0.8 and 3:0.8 ratios, respectively. In the case of a ratio 6:0.8, the final amount of both plasmids was decreased by 30 %.

For experiments using siRNA against TRPC1, the cells were transfected with control siRNA (siControl) or siRNA against TRPC1 48 h before experiments, using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, cat. No 13778150) according to manufacturer’s instructions.

The sense strands of siRNA were:

siControl (Microsynth): 5’ GCU AUU GCA UGU CGA AAU AdTdT 3′

siTRPC1 (Qiagen): 5’ CGG ACU UCU AAA UAU GCU AdTdT 3′

2.3. Electrophysiology

Patch-clamp recordings were performed in whole-cell mode using electrodes of resistance between 2.5–3 MΩ, which were pulled from 1.5 mm thin-wall glass capillaries (World Precision Instruments, cat. No TW150F-4) using a vertical PC-10 Narishige puller. Pipette solution contained (in mM): 130 cesium methansulfonate, 8 MgCl2, 10 Hepes, 10 BAPTA and 0.001 thapsigargin, pH 7.2 (CsOH). In pipette solution containing 1 mM BAPTA, cesium methansulfonate was increased to 134 mM. Extracellular solution contained (in mM): 135 NaCl, 10 CsCl, 4.4 MgCl2, 2.8 KCl, 10 Hepes, 0.5 EDTA, 0.5 EGTA, 10 glucose and 11 CaCl2(free Ca²⁺ 10 mM, calculated with maxchelator maxchelator.- stanford.edu), pH 7.4 (NaOH). In 0 mM CaCl2, no calcium was added and NaCl was increased to 145 mM. After establishing the whole-cell mode, the cells were let for minimum 3 min in the absence of extracellular Ca²⁺before adding 10 mM CaCl₂. 10 μM Gd³⁺was added at the end of the recording to block the current and estimate the leak.

Currents were recorded during 1 s ramp of potentials ranging from

−130 mV to +85 mV applied every 5 s with Axon™pCLAMP™ 10.7 Electrophysiology Data Acquisition & Analysis Software (Molecular Devices), using an Axopatch 200B Amplifier (Axon; Molecular Devices), low-pass filtered at 1 kHz and digitized with the Digidata 1322 A. For each time point the currents at −100 mV and +80 mV were plotted over time.

2.4. Ca²⁺measurements

For cytosolic Ca²⁺measurements, cells were loaded with 2 μM of Fura-2 AM and 0.1 % pluronic acid (Pluronic F-127) in the dark at room temperature for 25 min in Ca²⁺-containing solution, washed twice and

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allowed de-esterification of the dye for 20 min. Fluorescence was recorded using Zeiss Axio Observer A1 microscope (Carl Zeiss AG, Zurich, Switzerland) equipped with a Lambda XL illumination system (Sutter Instrument, Novato, CA, USA) which rapidly changed the excitation wavelengths between 340 nm (ET340x; Chroma) and 380 nm (ET380x;

Chroma). Emission was collected through a 415 DRLP dichroic mirror, and a 510WB40 filter (Omega Optical) by a cooled 16-bit CMOS camera (pco.Edge sCMOS, Visitron Systems, Puchheim, Germany). Image acquisition were performed with the VisiWiew software (Visitron Systems, Puchheim, Germany), and the analysis was done with ImageJ Software [26]. Cells were stimulated in Ca²⁺-free solution with 1 μM of thapsigargin (Tg) for 8 min and then 2 mM Ca²⁺was added to reveal SOCE. The slope and amplitude of Ca²⁺entry were analyzed on iso- lated cells to better correspond to patch-clamp recordings. The bath contained (in mM): 140 NaCl, 5 KCl, 1 MgCl2, 20 Hepes, 10 glucose, 2 CaCl₂ in Ca²⁺-containing solution or 1 mM EGTA without Ca²⁺ in Ca²⁺-free solution, pH 7.4 (NaOH).

2.5. RTqPCR

The RNA was extracted using TRIzol reagent (Invitrogen, cat. No AM97738) according to the manufacturer’s instructions (phenol- chloroform RNA isolation). All following steps were performed by the iGE3 Genomics Platform of the University of Geneva (http://www.ige3.

unige.ch/genomics-platform.php) and are described in more details in [27]. In brief: 0.5 μg of total RNA was reverse-transcribed with the PrimeScript RT reagent kit (TAKARA, Bio Company, Japan) according to manufacturer’s protocol. The impact of siRNA against TRPC1 was tested for four genes: TRPC1, TRPC4, Orai1 and STIM1. Additionally 2 housekeeping genes were assessed β2-microglobulin and EE-EF1. PCR was performed on an SDS 7900 H T instrument (Applied Biosystems).

Raw threshold-cycle (Ct) values obtained with SDS 2.2 (Applied Bio- systems) were imported into Excel. Normalization factor and fold changes were calculated using the GeNorm method [28]. Fold changes obtained for each condition were normalized to the siControl.

The following primers were used:

TRPC1: F: 5’ GGT TTC GTC TTG ATA TCT ATA G 3′; R: 5’ TCG TTT GTC AAG AGG CTC ATC 3′.

Orai1: F: 5’ GCT CAT GAT CAG CAC CTG CAC 3′; R: 5’ GGG ACT CCT TGA CCG AGT TG 3′.

STIM1: F: 5’ CTG ACG GAG CCA CAG CAT G 3′; R: 5’GGA ATG GGT CAA ATC CCT CTG 3′.

TRPC4: F: 5’ TCA GCA CAT CGA CAG GTC AGA C 3′; R: 5’CCA CGG TAA TAT CAT CCA CTC GAC 3′.

β2-microglobulin: F: 5’ TGC TCG CGC TAC TCT CTC TTT 3′; R: 5’

TCT GCT GGA TGA CGT GAG TAA AC 3′.

EE-EF1: F, 5’ AGC AAA AAT GAC CCA CCA ATG 3′; R 5’ GGC CTG GAT GGT TCA GGT A3′.

2.6. Western blot

The proteins were extracted using CHAPS lysis buffer (1 % CHAPS, 50 mM Tris−HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 10 % glycerol).

Protein concentrations were assessed using Bradford method (Coomassie Protein Assay Reagent, Thermo Scientific, cat. No 1856209). For endogenous TRPC1 detection maximal amount of proteins were loaded (min 50 μg per lane) whereas to estimate STIM1 to Orai1 ratio – 5 to 10 μg of proteins were loaded per lane. All the following steps are described in more details in [27]. In brief: proteins were separated on SDS-PAGE and transferred to nitrocellulose mem- branes which then were blocked with a PVA solution (Polyvinyl Alcohol 146–186k MW, 0.01 %). The blots were incubated with the primary antibodies diluted in T-TBS (0.1 % Tween 20, 20 mM Tris−HCl pH 7.5, and 137 mMNaCl) and 5 % BSA (Sigma, cat. No 05482, lot STBG0968 V) overnight at +4 °C. The horseradish peroxidase (HRP)- conjugated secondary antibodies were prepared in T-TBS and 5 % non-

fat milk. The Western blots were revealed using ECL reagents chemi- luminescence (western-lightning plus ECL, Perkin Elmer; Hyperfilm MP, Amersham Biosciences). ImageJ Software was used to quantify the level of protein expression. The total amount of the protein loaded per lane was used as a loading control [29] and for band intensity normalization (stained with the Ponceau S, Sigma, cat. No P7170, and according to the manufacturer’s instructions). The protein size was followed using pre-stained Protein Marker VI (10–245, Apli Chem cat.

No A8889, lot 114029310703). The following primary antibodies were used: TRPC1 (Santa Cruz, cat. No sc133076, lot E2615, mouse monoclonal, 1:300), STIM1 (Millipore, cat. No AB9870; lot 3015362, rabbit polyclonal, 1:2000), Orai1 (Sigma, cat. No O8264, lot 055M4783 V, rabbit polyclonal, 1:5000) and Anti-6X His tag® antibody [HIS.H8]

(Abcam, cat. No 18184, lot GR3257990-2, mouse monoclonal, 1:3000).

The following secondary antibodies were used: goat anti-mouse-IgG (H + L) HRP conjugate (Bio-Rad, cat. No 170-65161721011, 1:5000), goat anti-rabbit-IgG (H + L) HRP conjugate (Cell Signaling, cat. No 09/

2017, lot 27, 1:5000).

2.7. Co-Immunoprecipitation experiments

Briefly, HEK cells (2 × 35 mm dishes) were scraped 24 h after transfection with YFP-STIM1 or YFP-STIM1L together with Flag- hTRPC1, resuspended in 500 μl IP buffer (30 mM Tris−HCl pH7.5, 250 mM NaCl, 0.5 % NP40 and 4 % glycerol) and maintained for 1 h under agitation at +4 °C. After mild centrifugation (800gfor 5 min), 25 μl of the supernatant were kept frozen as “input”, and 5 μg of anti- GFP antibody (Abcam, cat. No Ab290, lot GR3251545-1 rabbit polyclonal) were added to the rest of the supernatant and incubated at +4 °C overnight with constant mixing. The protein-antibody complex was then incubated for 3 h at +4 °C with constant mixing with 30 μl of SureBeads™ Protein G Magnetic Beads (Bio-Rad, cat. No 1614023). The immune complexes were washed four times in IP buffer. After dena- turing, samples were subjected to SDS-PAGE. The negative control was performed using lysates from cells overexpressing GFP and Flag- hTRPC1. Immunoblots were performed with anti-GFP (Abcam, cat. No Ab290, lot GR3251545-1, rabbit polyclonal, 1:2000) or anti-TRPC1 (UC Davis/NIH NeuroMab Facility, clone 1F1, Cat. No73-278, lot 437 5VA 45, mouse monoclonal supernatant, 1:300) antibodies.

2.8. Statistical analysis

The density distributions of Ca²⁺ amplitudes were estimated by kernel based methods. The difference between conditions was tested using a Kolmogorov-Smirnov test. Statistical significance was assessed at a two-sided alpha level of 5 % for all tests. All analyses were done with the R software, version 3.3.1 (R Foundation for Statistical Computing, Vienna, Austria) by the biostatisticians from the Clinical Research Support Units of the University-Hospital Clinical Research Center’s, Geneva, Switzerland (https://www.hug-ge.ch/en/clinical- research-center). For all other data, a Student t-test was used to de- termine the statistically significant differences: * p < 0.05, **

p < 0.01, *** p < 0.001, ****p < 0.0001. Error bars and I/V curves are mean ± SEM.

3. Results

3.1. STIM1L and STIM1 activate two types of currents

As a first approach, we overexpressed Orai1 together with either STIM1 or STIM1L in HEK cells, to determine the characteristics of the current activated by each STIM1 isoforms. In order to activate the current, the cells were kept in external Ca²⁺- free solution and 1 μM of thapsigargin in the patch pipette. After minimum 3 min, 10 mM Ca²⁺

was added to the external solution and current development was recorded. As expected under these conditions, cells displayed ICRAC

A. Dyrda, et al. Cell Calcium 86 (2020) 102134

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current when transfected with STIM1 and Orai1. As well, I_CRACdevel- oped in cells transfected with STIM1L and Orai1, however, the maximal current amplitude (at −100 mV) was around 40 % smaller than with STIM1 (Fig. 1A). The E_rev(> +80 mV) and the inhibition by Gd³⁺

were very similar between STIM1- and STIM1L-activated ICRAC. Un- expectedly however, in more than 50 % of cells displaying ICRAC, a second current developed with a delay between 1–3 min after the onset of ICRAC. The current-voltage relationship (I/V curve) of this second current was almost linear with a left shift of Erevto around +20 mV (Fig. 1B). A careful analysis of the second current revealed that it was present either as a small or a large amplitude, that was associated with a moderate or large shift of the Erev, respectively (Supplementary Fig. 1). The electrophysiological characteristics of the second current suggest the activation of a non-selective channel, such as TRPC channels that are endogenously present in HEK cells [30–33].

3.2. TRPC1 is activated by STIM1 and STIM1L

It is well described that TRPC1 can be activated by STIM1 [34,35].

Hence, we tested whether TRPC1 could be responsible for the linear current recorded on HEK cells. In order to prevent TRPC1 activity we applied three strategies: transfection of the cells with a siRNA against TRPC1 channel (siC1), overexpression of a not conductive TRPC1 mutant (F562A) that behaves as a dominant negative TRPC1 (C1_DN [23];), or overexpression of a STIM1 mutant (STIM1⁶⁸⁴EE⁶⁸⁵ and STIM1L⁷⁹⁰EE⁷⁹¹, called then S1_EE and S1L_EE for simplicity), in which the last two lysine residues are replaced by glutamate. Indeed, it was shown that TRPC1 gating by STIM1 occurs through electrostatic interaction between the two last lysines of STIM1 and two aspartate residues on TRPC1 [9]. Such STIM1⁶⁸⁴EE⁶⁸⁵and STIM1L⁷⁹⁰EE⁷⁹¹mutants would be unable to gate TRPC1 channel. The efficiency of siTRPC1 at the mRNA and protein level is shown in Supplementary Fig. 2. Each of the three strategies tested led to the activation of ICRAC-like current only, without the development of a second linear current, both for STIM1- and STIM1L-overexpressing cells (Fig. 2). Hence, these data are in line with the hypothesis that although we overexpress only STIM1/L and Orai1 on HEK cells, endogenous TRPC1 activation (ITRPC1) is taking

place, at least in a subset of cells. We call this mix current between Orai1 and TRPC1, ICRAC+TRPC1. Importantly, ICRAC+TRPC1 was more frequently observed in cells expressing STIM1L (70 %) compared to STIM1 (50 %;Fig. 3A). Additionally, we never recorded I_TRPC1alone, without ICRAC being first activated, which is consistent with a Ca²⁺- dependent process leading to ITRPC1activation. To assess the role of intracellular Ca²⁺elevation for I_TRPC1activation, we repeated the experiments, lowering the intracellular BAPTA concentration to 1 mM.

Indeed, the pipette solution contains a high amount of BAPTA (10 mM), which is not in favor of Ca²⁺-dependent process. In low Ca²⁺-buffering condition, the frequency of ICRAC+TRPC1 development drastically increased to around 80 % and 90 % for STIM1 and STIM1L overexpressing cells, respectively (Fig. 3B). The shape and the Erevof ICRAC

and ICRAC+TRPC1were very similar in low BAPTA condition (Fig. 3C-D) compared to 10 mM BAPTA. Finally, the delay between the onset of ICRAC development and the activation of ICRAC+TRPC1 shortened significantly in low intracellular BAPTA concentration compared to 10 mM, which further strength the Ca²⁺-dependency of TRPC1 activation (Fig. 3E). Thus, altogether, our data strongly point to an activation of endogenous TRPC1 channel occurring after an initial Orai1- induced Ca²⁺ entry, an event that takes place more frequently in STIM1L-expressing cells.

To determine whether STIM1 or STIM1L physically interact with TRPC1, and if yes, whether this interaction is different between both STIM1 isoforms, we performed co-immunoprecipitation experiments on HEK cells overexpressing STIM1 or STIM1L together with TRPC1. The data showed that the interaction between STIM1L and TRPC1 is stronger than the one between STIM1 and TRPC1 (Fig. 4).

3.3. Preventing endogenous TRPC1 activation reduces SOCE

In the next set of experiments, we wanted to establish how electrophysiological data correlate with fluorescent Ca²⁺measurements in HEK cells overexpressing STIM1/L and Orai1. In line with what we previously reported in MEFDKO cells [2], the SOCE induced by STIM1L was around 30 % bigger than the one activated by STIM1 (Fig. 5A-B). Western blot analysis revealed that the STIM1/L proteins are overexpressed at similar Fig. 1.Both STIM1 splice variants activate two types of current: ICRACand a linear current.

Cells were transfected with Orai1 and either STIM1 (O1S1) or STIM1L (O1S1L) plasmids 24 h before electrophysiological recordings. Representative time course of ICRAC(Ai) and ICRACplus the linear current (Bi) development, from STIM1L overexpressing cells. Outward currents were monitored at +80 mV (open circles) and inward currents at −100 mV (filled circles). Black points were recorded in the absence of external Ca²⁺, green points in the presence of 10 mM of Ca²⁺, and gray points with 10 μM Gd³⁺. Only the last minute in the external Ca²⁺-free solution are shown. The black and red I/V curves (mean ± SEM) are shown for cells displaying only ICRAC (Aii) and cells displaying both ICRACand the linear current (Bii). “n” represents the number of recorded cells from > 5 transfections.

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Fig. 2.Only ICRACwas observed after suppression of endogenous TRPC1.

In both STIM1 and STIM1L overexpressing cells only ICRAC-like current was recorded after (A) TRPC1 protein downregulation (siRNA against TRPC1), (B) overexpression of a dominant negative variant of TRPC1 (TRPC1_DN), or (C) overexpression of mutated STIM1 protein unable to activate TRPC1 (mutant EE). The black and red I/V curves (mean ± SEM) correspond to cells overexpressing STIM1 (O1S1) or STIM1L (O1S1L). “n” represents the number of recorded cells from > 5 transfections.

Fig. 3.ICRAC+TRPC1is activated more frequently in cells overexpressing STIM1L, and in condition of low intracellular BAPTA concentration.

(A) Percentage of cells in which either only ICRACor ITRPC1in addition to ICRACdeveloped in conditions where 10 mM BAPTA was present in the patch pipette. A total of 41 and 30 cells were recorded for STIM1 (O1S1) and STIM1L (O1S1L) overexpressing cells, respectively. (B) Percentage of cells in which either only ICRACor ITRPC1

in addition to ICRACdeveloped in conditions where the concentration of BAPTA was reduced to 1 mM in the patch pipette. A total of 9 and 8 cells were recorded for STIM1 (O1S1) and STIM1L (O1S1L) overexpressing cells, respectively. I/V curves (mean ± SEM) recorded in cells displaying (C) ICRACalone or (D) ITRPC1in addition to ICRACwhen either STIM1 (O1S1) or STIM1L (O1S1L) was overexpressed with Orai1, in 1 mM intracellular BAPTA. (E) Measurements of the delay between the onset of ICRACdevelopment and the activation of ICRAC+TRPC1, in conditions of 10 mM or 1 mM BAPTA inside the patch pipette. Numbers within the bars represent the number of recordings and the bars are mean ± SEM.

Fig. 4.Interaction between STIM1L and TRPC1.

(A) Representative co-immunoprecipitation experiments on HEK cells that overexpressed YFP-STIM1 and Flag-hTRPC1 or YFP-STIM1L and Flag-hTRPC1. Inputs show the level of overexpressed proteins in cell lysates treated with 1 μM Tg. Lysates of cells were incubated with antibodies against GFP (IP), and Western blots (IB, immunoblot) of the immunoprecipitated proteins were probed with antibodies against GFP and hTRPC1 (B) Quantification of the ratio between TRPC1 and immunoprecipitated GFP. Bars are mean ± SEM, n = 3 independent transfections.

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levels (Supplementary Fig. 3), and thus the uneven SOCE amplitudes are not related to different levels of STIM1 and STIM1L expression (Fig. 5). To evaluate the involvement of TRPC1 in SOCE, we applied the same strategies as for patch-clamp experiments, i.e. transfection of the cells with siTRPC1, DN TRPC1 or STIM1 mutants (S1_EE and S1L_EE). The siRNA against TRPC1 decreased by about 40 % and 20 % the SOCE amplitude in cells overexpressing STIM1 or STIM1L, respectively, and the slope of Ca²⁺

entry was reduced by 70 and 30 % for STIM1 and STIM1L, respectively (Fig. 5A). Overexpression of DN TRPC1 together with STIM1 or STIM1L also diminished the slopes and amplitudes of SOCE (Fig. 5B). These data

thus confirmed the involvement of TRPC1 both in STIM1- and STIM1L- induced SOCE. Surprisingly however, while overexpression of the STIM1L_EE mutant decreased Ca²⁺ entry elicited by STIM1L, overexpression of STIM1_EE mutant increased SOCE induced by STIM1 (Sup- plementary Fig. 4A, B). This increase remains unexplained at the moment.

We also performed experiments where we overexpressed TRPC1 together with STIM1/Orai1 or STIM1L/Orai1. As shown on Supplemental Fig. 4C, D, the amplitudes and the slopes of SOCE slightly increased with the overexpression of TRPC1 while not significantly. The lack of obvious effect of TRPC1 overexpression was already reported, and is likely due to an Fig. 5.Preventing endogenous TRPC1 activation reduces STIM1- and STIM1L- induced SOCE.

HEK cells were loaded with Fura-2 AM to monitor changes in cytosolic Ca²⁺concentration, and stimulated with 1 μM Tg to elicit SOCE. HEK cells were transiently transfected with Orai1 and either STIM1 (O1S1) or STIM1L (O1S1L), and (A) siRNA control (siCtr), or siRNA against TRPC1 (siC1), or (B) a mutated TRPC1 (dominant negative TRPC1, C1_DN). Representative measurements (the average of signals collected from one coverslip) of the Ca²⁺-re-addition step of the protocol are presented for cells with downregulation of endogenous TRPC1 (Ai) and for cells which overexpress the mutated TRPC1 (Bi). SOCE amplitudes (Aii and Bii) and slopes (Aiii and Biii) were calculated upon Ca²⁺re-addition in Tg-treated cells after either STIM1 (black border bars) or STIM1L (red border bars) overexpression.

SOCE was assessed (Aii-Aiii) after TRPC1 downregulation (siC1, green filled bars; siRNA control, gray filled bars) and (Bii-Biii) after overexpression of a dominant negative TRPC1 channel (C1_DN, blue filled bars). Numbers within the bars indicate the total number of the coverslips analyzed (between 3 and 62 cells were analyzed per coverslip). Bars represent mean ± SEM.

Fig. 6.Frequency distributions of SOCE amplitudes significantly differ between STIM1 and STIM1L overexpressing cells.

Distribution of SOCE amplitudes in STIM1 (A) and STIM1L (B) overexpressing cells with corresponding smoothing splines. (C) Results from STIM1 and STIM1L are shown as an overlay for comparison. Statistical significance was assessed at a two-sided alpha level of 5 %.

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inappropriate stoichiometry between STIM, Orai and TRPC [36].

Our electrophysiological data clearly demonstrated activation of two channels following ER Ca²⁺depletion: Orai1 (CRAC current) and, in a sub-population of the cells, TRPC1. Additionally, we found that TRPC1 channels are activated more frequently in the presence of STIM1L. If we assume that the same phenomenon is taking place in intact cells (fluorescence Ca²⁺imaging), it should give rise to distinct distributions of SOCE amplitudes between STIM1 and STIM1L overexpressing cells. Indeed, analysis of the Ca²⁺amplitudes in terms of cell populations revealed a different density distributions between STIM1- and STIM1L-activated SOCE (Fig. 6). For STIM1, the distribution is best fitted by a curve (smoothing spline) displaying two peaks centered at around 0.5 and 1.7 of SOCE delta ratio amplitude (Fig. 6A), and for STIM1L, a major peak is centered at 1.7 of SOCE delta ratio amplitude and two others, one at a smaller and the other at a larger SOCE delta ratio amplitude (0.9 and 2.3, respectively; Fig. 6B). The overall distributions of STIM1 and STIM1L SOCE amplitudes are significantly different and can be appreciated by the overlay of the two curves shown in Fig. 6C. It is tempting to speculate that the smaller amplitudes of SOCE (recorded for STIM1 and STIM1L) correspond to Orai1 activation, while the larger ones are due to activation of both Orai1 and TRPC1.

This assumption was, at least partially, confirmed by analyzing the changes of the SOCE amplitude distribution upon reduction of TRPC1 activation. Downregulation of TRPC1 (siC1) or overexpression of dominant negative TRPC1 (C1_DN) together with STIM1 resulted in a loss of the higher SOCE amplitudes and increase in the lower SOCE

amplitudes, leading to a leftward shift of SOCE density distributions (Fig. 7A, C). This shift was also noticeable for STIM1L albeit the effect was less pronounced (Fig. 7B, D).

3.4. The STIM1/L:Orai1 ratio impacts the ICRACamplitude differently In the last set of experiments, we addressed the issue related to the smaller ICRACinduced by STIM1L compared to STIM1. We questioned whether it might be linked to the relative amount of STIM1/L and Orai1 proteins, which for all above described experiments was 1:0.8. To investigate this point, we increased the amount of plasmid encoding STIM1/L, keeping the Orai1 plasmid level constant, a strategy which was shown to alter the protein expression level [37–39]. Western blots confirmed the changes in STIM1/L:Orai1 protein ratio (1:0.8, 2:0.8, 3:0.8 and 6:0.8; SupplementaryFig. 5). Functionally, for STIM1, increasing by 2 or 3 the amount of STIM1 (2:0.8, 3:0.8) did not change the amplitude of I_CRACcompared to the initial amount (1:0.8). On the other hand, 6 times more STIM1 decreased ICRAC(Fig. 8A). For STIM1L, interestingly, increasing by 3 the amount of STIM1L (3:0.8) induced a larger I_CRAC, comparable to the one activated by STIM1. At the highest ratio tested (6:0.8) ICRAC also strongly diminished, as for STIM1 (Fig. 8B). Changing the STIM1/L:Orai1 ratio only affected the current amplitude but neither the shape of the I/V curve nor the E_revwere modified (Supplementary Fig. 6). These data suggest that the optimal STIM1:Orai1 ratio might be different for the two splice variants of STIM1.

Fig. 7.Suppression of TRPC1 led to a leftward shift of SOCE amplitude distributions.

Distribution of SOCE amplitudes in STIM1/Orai1 (A) and STIM1L/Orai1 (B) overexpressing cells in control conditions (gray lines, O1S1+siCtr and O1S1L + siCtr) and after TRPC1 downregulation (green lines, O1S1+siC1 and O1S1L + siC1). Distribution of SOCE amplitudes in STIM1/Orai1 (C) and STIM1L/Orai1 (D) overexpressing cells in control conditions (STIM1: black line, O1S1; STIM1L: red line, O1S1L) and after overexpression of a dominant negative TRPC1 channel (STIM1:

O1S1+C1_DN; STIM1L: O1S1L + C1_DN; blue lines). Statistical significance was assessed at a two-sided alpha level of 5 % for all tests.

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4. Discussion

In the present work, we studied the ionic channels activated by STIM1 and STIM1L, a splice variant of STIM1 highly expressed in skeletal muscles, using HEK293 T cells as a model system. The patch- clamp data revealed that both STIM1 isoforms activate two types of channels: the highly Ca²⁺selective Orai1 channel and in a large pro- portion of cells the non-selective TRPC1 channel. Two main differences were observed between STIM1- and STIM1L-activated currents. The most obvious difference was the amplitude of STIM1L-activated ICRAC, which was around 40 % smaller compared to STIM1-induced ICRAC. The second important difference was the frequency of TRPC1 activation, that was significantly higher in cells overexpressing STIM1L compared to STIM1. In agreement, the interaction between STIM1L and TRPC1 was stronger that between STIM1 and TRPC1, as shown by co-immunoprecipitation experiments. Remarkably, Orai1-mediated Ca²⁺

influx was a prerequisite for TRPC1 activation both by STIM1 and STIM1L. Ca²⁺imaging recordings confirmed that endogenous TRPC1 participate in SOCE activated by both STIM1 isoforms.

We provide several lines of evidence to support endogenous TRPC1 channel activation by STIM1/L. Activation of TRPC1 was revealed by a change of the I/V curve that became more linear compared to ICRACand by a left shift of the Erev, highlighting the decrease of the current Ca²⁺

selectivity. To confirm the involvement of TRPC1, we applied three strategies to prevent the TRPC1 activation and all three resulted in the generation of only one type of the I/V curve very similar to ICRAC. This outcome was obtained with both STIM1 isoforms. It is important to highlight that we never recorded TRPC1 current only, it was always preceded by the activation of Orai1, which indicates that Orai1 opening is absolutely necessary for TRPC1 to be activated upon store depletion, an observation that was already made in several cell types [23,40–44].

The sequential development of ICRAC followed by ICRAC+TRPC1 upon store depletion is consistent with the sequence of events taking place in salivary glands following cell stimulation and reported by the group of I. Ambudkar [11,13]. According their model, STIM1 first activates Orai1 and the consequent Ca²⁺ entry triggers the fusion of TRPC1- containing vesicles with the PM. The TRPC1 activation results, even- tually, from STIM1 gating. Our findings can be explained by a very similar scenario, where consistently Orai1 first activates followed by TRPC1. The increase percentage of cells displaying TRPC1 activation under low intracellular Ca²⁺ buffer (i.e. 1 mM BAPTA in the patch pipette), supports a Ca²⁺-dependency of TRPC1 activation. Under this experimental condition, the (local) cytosolic Ca²⁺elevation after I_CRAC activation is expected to be higher, and thus would favor TRPC1 opening. Hence, overall, our data are in line with an initial local Ca²⁺

elevation promoting a subsequent TRPC1 activation, such as described in salivary glands, and involving the TRPC1-containing vesicle fusion.

However, we cannot rule out another mechanism of TRPC1 activation recently proposed in vascular smooth muscle, whereby the initial cytosolic Ca²⁺ elevation promotes PKC-dependent TRPC1

phosphorylation and activation [45,46]. As well, we cannot rule out the involvement of other TRPC channels that could form heteromultimers with TRPC1 such as TRPC4. Indeed, TRPC1 and TRPC4 were reported to be part of SOCE in skeletal muscle [21,47], cardiac muscle [48], but also other cell types like mesangial cells [49]. Regarding the kinetic of current activation, it is well-known that in skeletal muscle the activation of SOCE is much faster than in other cellular systems, a property due in part to the present of STIM1L. In HEK cells, we did not observe a fast ICRACdevelopment linked to STIM1L (data not shown, performed in conditions where ICRACwas activated in 10 mM extracellular Ca²⁺).

This is actually in line with our recent report on MEFDKO cells, where no kinetic difference was measured between Ca²⁺entry activated by STIM1 or STIM1L [2].

I_CRACamplitude was shown to be dependent on the relative amount of STIM1 and Orai1 proteins [37–39,50]. Here we investigated whether the same applies to STIM1L and could explain the reduced amplitude of the STIM1L-activated I_CRAC. As already reported for STIM1 [37], a large excess of STIM1 or STIM1L compared to Orai1 led to an important decrease of ICRACamplitude. Interestingly however, transfecting three times more STIM1L plasmid significantly increased ICRAC amplitude, while no change was observed with STIM1. Importantly, Erevwas not affected, showing that the ionic selectivity was not modified by changing STIM1/L:Orai1 ratio. These experiments suggest that the optimal ratio between STIM1:Orai1 and STIM1L:Orai1 is different. We speculate that STIM1L being more frequently associated to TRPC1, a higher amount of the protein is necessary to fully open Orai1. This hypothesis is in line with our result using siRNA against TRPC1. This condition was the only one where the amount of endogenous TRPC1 was decreased, and was also the only one where the amplitude of ICRACwas very similar between STIM1- and STIM1L-expressing cells (Fig. 2A). Hence, we propose that the relative amount of STIM1/L-Orai1-TRPC1 and the preference of STIM1L towards TRPC1 might explain the reduced ICRAC

activated by STIM1L compared to STIM1. Physiologically, the potential discrepancy in the optimal ratio between STIM1 and STIM1L and the gated channels could be relevant in the context of myogenesis, as with time of muscle differentiation, the expression level of STIM1L increases while STIM1 expression remains stable [18,21]. Thus, the relative amount of each STIM1 isoform varies during the process of muscle differentiation, and could lead to variation in Orai1 and/or TRPC1 activation which might have physiological consequences.

Our findings reporting the activation of endogenous TRPC1 channels on HEK that overexpress STIM1 and Orai1 might appears surprising as this expression system was and is still widely used to study ICRACcurrent properties, and such endogenous TRPC1 activation was not reported/studied, even if it was mentioned to take place [44]. It should be also mentioned that TRPC1 was shown to be part of the native SOCE in HEK cells [23]. Two important remarks have to be provided at this point: first, the protocol we were using in this study, addition of 10 mM Ca²⁺after a time in Ca²⁺-free medium, is not that frequently used in electrophysiological recordings, with the notable Fig. 8.STIM1/Orai1 and STIM1L/

Orai1 ratios modulate ICRACamplitude.

Cells were transiently transfected with increasing amounts of either STIM1 (A) or STIM1L (B) plasmid (Orai1 plasmid level was kept constant) and ICRAC

amplitude was assessed at −100 mV.

Bars represent mean ± SEM and P > 0.05 values are in brackets. For STIM1L the X6 condition the one sample Wilcoxon test was applied (median theoretical value -1.112).

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exception of studies on SCDI of ICRAC[51,52]. This protocol certainly favored the occurrence of Ca²⁺-dependent channel activation, as a sudden addition of high Ca²⁺concentration would lead to a significant cytosolic Ca²⁺elevation around Orai1, despite the presence of Ca²⁺

chelator. Hence, we hypothesize that the more commonly used protocol where the cells are bathed in high Ca²⁺from the beginning of the recording is less prone to reveal Ca²⁺-dependent channel activation like TRPC1. The second aspect is that the TRPC1 activation taking place with some delay following ICRACdevelopment looks very much like an increase of the leak current, i.e. a degradation of the seal between the pipette and the cell. Hence, the appearance of a linear current might have been overlooked in many recordings.

Our electrophysiological recordings showed that STIM1L is less efficient than STIM1 to open Orai1, that both splice variants of STIM1 gate endogenous TRPC1 and that STIM1L is more prone to activate TRPC1. But how these findings translate to Ca²⁺entry as measured with cytosolic Ca²⁺indicator was an open question. Our results showed that STIM1L-induced SOCE was larger by about 30 % compared to STIM1-induced SOCE, an observation similar to the one we found in MEFDKO cells overexpressing STIM1 or STIM1L [2]. In addition, Ca²⁺

imaging experiments confirmed that both splice variants of STIM1 activate TRPC1. In case of STIM1L, all three strategies to eliminate TRPC1 (the same as applied in electrophysiological studies) diminished SOCE, whereas for STIM1, two strategies reduced (TRPC1_DN and siTRPC1) while the third one (mutated STIM1_EE) unexpectedly enhanced SOCE.

It is important to remember that Ca²⁺imaging results were based on analysis done on the average Ca²⁺responses from many cells, while patch-clamp examined the channel activation in one cell. Therefore, to get a better insight into the distribution of the SOCE amplitudes activated by both splice variants of STIM1, we constructed the histograms of the SOCE amplitudes. This graphical way of data presentation clearly shows that: 1) the distributions of the SOCE amplitudes are different between STIM1 and STIM1L and 2) STIM1L-activated SOCE resulted more frequently in large Ca²⁺elevations. In addition, the large SOCE amplitudes became less frequent in cells treated with siTRPC1, while the small SOCE amplitudes became more frequent. The same tendency was observed in cells overexpressing DN TRPC1, even if the effect was less pronounced. The hypothesis that the small and large SOCE amplitudes correspond to Orai1 and Orai1+TRPC1 activation, respectively, was only partially confirmed by the experiments. And unexpectedly, the involvement of TRPC1 in SOCE was more obvious with STIM1 compared to STIM1L, which is not in line with the electrophysiological recordings. However, one should consider that the two techniques are very different, with intact versus dialyzed cells, and without intracellular Ca²⁺chelator versus high cytosolic BAPTA concentration, for Ca²⁺imaging and patch-clamp recording, respectively, and could explain the discrepancy we observed. The physiological impact of the different channel activation by STIM1 and STIM1L remains now to be determined. Ca²⁺ signaling are crucial for proper myotube differentiation, and several Ca²⁺-dependent signaling pathways processes are activated during differentiation, like NFAT and CaMKII [53–55]. It would thus be of interest to determine if specific Ca²⁺entry routes (Orai1 vs. TRPC1) are linked to certain downstream pathways like it was reported in many cellular systems [12,56,57].

5. Conclusion

In conclusion, this report provide evidences that both STIM1 and STIM1L activate Orai1, but also TRPC1, that TRPC1 activation is preceded by Orai1 opening, and that STIM1L is more prone to activate TRPC1. This finding might explain why Orai1 activation by STIM1L is less efficient than STIM1-activated Orai1. The ability of STIM1L to better/more often gate TRPC1, might also well explain the larger Ca²⁺

entry activated by this isoform compared to STIM1.

Author contributions

M. F. designed and coordinated the study. M. F. and A. D. wrote the manuscript. A. D. performed and analyzed the Ca²⁺ imaging and electrophysiology experiments. A.D. performed the Q-PCR and Western blot experiments under the supervision of S.K, who performed the co- immunoprecipitation experiments and constructed YFP-STIM1L plasmid. All authors designated are qualified for authorship.

Funding sources

This work was supported by Swiss National Foundation (SNF) grant 310030_166313 (M. F.), Foundation Marcel Levaillant (M. F.).

Declaration of Competing Interest

None of the author reports conflicts of interest.

Acknowledgements

We are grateful to Prof. Laurent Bernheim for valuable discussions and suggestions during project's evolution as well as for critical reading of the manuscript. We are thankful to Prof. Nicolas Demaurex for a critical discussions and suggestions. We would like to thanks to Olivier Dupont and Cyril Castelbou for excellent technical assistance. We would also like to give our thanks to Christopher Henry who helped with the analysis of Ca²⁺data.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.ceca.2019.102134.

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