11.1. Introduction

The identification of Orai and STIM proteins has opened up new avenues of research in the field of receptor-regulated calcium signaling. The ligation of phospholipase C (PLC)-coupled receptors can activate either the common store-operated calcium entry (SOCE) pathway or store-independent calcium entry (SICE) pathway. The representative conductance of the SOCE pathway is the calcium release-activated calcium (CRAC) channel encoded by Orai (CRACM) proteins. The SICE pathway biophysical manifestation is currents activated by arachidonic acid (AA) or the AA metabolite leukotriene C₄ (LTC₄) and termed arachidonate-regulated or LTC₄-regulated calcium (ARC/LRC) current encoded by channels composed of both Orai1 and Orai3 proteins.

About three decades ago, Putney first proposed the capacitative Ca²⁺ entry model (subsequently known as SOCE) [1]. Orai1 protein, the pore forming subunit of the CRAC channel, was discovered almost simultaneously by three groups in 2006 [2–4]. The Orai family of channels contains three different proteins (Orai1, 2, and 3) encoded by independent genes [5]. A large number of agonists can act on G protein-coupled receptors (GPCRs) to activate PLC. PLC hydrolyzes phosphatidylinositol-4,5-bisphopshate (PIP₂) into diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP₃) [6]. The latter binds to IP₃ receptors (IP₃R) on the membrane of the endoplasmic reticulum (ER), resulting in Ca²⁺ store emptying. The action of ER Ca²⁺ store emptying causes stromal interaction molecule 1 (STIM1), a calcium sensor to lose Ca²⁺ from its N-terminal low affinity EF hand located in the lumen of the ER [7,8]. This causes STIM1 to aggregate and to move to highly specialized areas where the ER comes close to the plasma membrane to physically trap and interact with Orai1 channels and activate Ca²⁺ entry [8]. STIM1 has one homologue, STIM2, which mediates Orai1 channel activation under resting conditions in the absence of agonist stimulation [9]. In most cells studied so far, SOCE is mediated by STIM1 and Orai1 proteins [10]. However, we reported Orai3-mediated SOCE in a subset of estrogen receptor positive breast cancer cells [11–13].

The ARC channel and its role in calcium signaling have been first reported and intensely studied by Shuttleworth and colleagues [14–17]. This group identified and characterized a conductance in HEK293 cells activated by relatively low exogenous concentrations of arachidonic acid or by low concentrations of a muscarinic agonist [18]. Polyunsaturated fatty acids were described as poor activators of these channels, and mono unsaturated or saturated fatty acids were ineffective [19]. Unlike a number of channels of the Transient Receptor Potential Canonical (TRPC3/6/7) family [20,21], ARC channels are not activated by high concentrations of DAG (100 μM) [19]. Mignen and colleagues showed that despite many similarities with CRAC currents present in the same cell type studied, ARC channels possess distinct pharmacological characteristics and biophysical properties [18]. For instance, unlike CRAC channels, ARC channels do not show the typical fast Ca²⁺-dependent inactivation (CDI), are not inhibited by a reduction in extracellular pH from 7.2 to 6.7, and are insensitive to 2-aminoethoxydiphenyl borate (2-APB) [18,22]. As is the case with CRAC channels [34,35], the absence of divalent cations in the extracellular recording medium induces the permeability of ARC channels to monovalent cations, such as Na⁺ [23]. However, this monovalent macroscopic current has different characteristics from those observed for CRAC channels especially from the perspective of their depotentiation and permeability. By blocking monovalent currents by increasing extracellular calcium concentrations as a relative measure of selectivity of calcium channels, Mignen and colleagues proposed that ARC channels have high Ca²⁺ selectivity and are 50 times more Ca²⁺-selective than CRAC channels [18,22]. These authors argued that ARC channels are the predominant calcium channels activated when cells are stimulated with low concentrations of agonists that induce repetitive calcium oscillations [24]. Using an M3 muscarinic receptor-expressing HEK293 cells and murine parotid and pancreatic acinar cells, they reported the activation of ARC channels mediating intracellular calcium oscillations by low concentrations (0.2–1 μM) of carbachol [25]. In the same cells they described the activation of the AA-producing enzyme, phospholipase A2 type IV, upon stimulation with low concentrations of carbachol.

Earlier work by the Shuttleworth group suggested that the pharmacological inhibition of PLA2 with isotetrandrine blocks the activation of ARC channels, while the pharmacological inhibition of the lipoxygenase and cyclooxygenase pathways had no effect on ARC activation [14,26], indicating that AA is produced by receptor-mediated activation of PLA2 and that AA processing into downstream metabolites is not required for ARC channel activation. After identification of STIM and Orai proteins, Shuttleworth and colleagues showed that both Orai1 and Orai3 are required for ARC channel activation [27], in addition to the minor pool of STIM1 located in the plasma membrane [28]. More recent work from our laboratory identified a SICE channel in primary aortic vascular smooth muscle cells (VSMC). We found that this conductance is activated by AA, but AA metabolism into LTC₄ by the enzymatic activity of LTC₄ synthase (LTC₄S) provided a more robust activation of these channels; LTC₄ acts intracellularly when applied through the patch pipette but not extracellularly when added to the bath solution. We named this channel LTC₄-regulated calcium (LRC) channel [23,29–31]. Collectively, our data in VSMC showed that receptor activation causes production of AA through sequential activation of PLC and DAG lipase and that AA metabolism by 5-lipooxygenase and LTC₄S into LTC₄ is required for LRC channel activation [29,31]. A molecular knockdown on LTC₄ synthase (LTC₄S) abrogated receptor-mediated LRC channel activation (using the PAR1 agonist thrombin), while direct application of LTC₄ through the patch pipette robustly activated LRC currents. The biophysical properties of LRC channels were identical to those of ARC channels, prompting us to undertake a side by side comparison in VSMC and HEK293 cells to determine whether these two conductances are mediated by the same or by different cellular pools of STIM and Orai proteins [23]. Briefly, using protein knockdown, pharmacological inhibitors, and a nonmetabolizable form of AA, we found that regardless of the cell type considered (HEK293 cells or VSMC), ARC and LRC currents are the manifestation of the same channel that can be activated by AA but is more robustly activated by LTC₄ [23]. We also found that in both cell types, ARC/LRC currents depended on Orai1, Orai3, and STIM1 [23,29], but unlike findings from the Shuttleworth group, we were able to rescue ARC/LRC activity in HEK293 cells and VSMC with expressed STIM1 constructs that do not traffic to the plasma membrane when using Fura-2 calcium imaging and perforated patch recording in intact cells but not in whole-cell recordings. These results suggest a facilitatory role for PM-STIM1 in ARC/LRC channel activation [23]. Orai1 exists in two variants generated through alternative translation-initiation of the Orai1 mRNA: a longer Orai1α form contains an additional N-terminal (NT) 63 amino acids upstream of the conserved start site of a shorter Orai1β [32]. A study from our group showed that while Orai1α and Orai1β are interchangeable for forming CRAC channels, only Orai1α can support ARC/LRC channels by forming a unique heteromeric channel with Orai3. Studies by the Shuttleworth group were performed before Orai1α variant was discovered; it is therefore unclear which Orai1 subtype was used [33]. We also showed that a specific interaction of STIM1 second C-terminal (CT) coiled-coil (CC2) with Orai3 CT region is required for LRC channel activation by LTC₄ [31].

In summary, the SICE pathway appears to be mediated by one channel entity. In succeeding text, we will refer to this channel as either ARC or LRC, depending on whether we are referring to experiments that used either AA or LTC₄ to activate this conductance. ARC/LRC channels are encoded by Orai1 and Orai3 and regulated by STIM1. There are two major points of contention between our findings and those of the Shuttleworth group: (1) the requirement for AA metabolic conversion into LTC₄ and (2) the cellular pool of STIM1 required for ARC/LRC activation, that is, ER-resident versus PM-resident STIM1.

11.2. Biophysical Properties and Molecular Composition of SICE Channels

In 1996, it was first demonstrated that AA-activated noncapacitative Ca²⁺ entry through an unknown channel in avian nasal gland cells [14]. Four years later, using the whole-cell patch clamp technique, Ca²⁺ currents activated by exogenous application of AA were recorded under conditions where cytosolic Ca²⁺ was buffered to ∼100 nM in the pipette solution [18]. This novel non-store-operated channel was named the arachidonate-regulated calcium (ARC) channel [18]. A recording of ARC channel current (I_ARC) in M3 muscarinic receptor-expressing HEK293 cells has revealed that, like CRAC, ARC channels possess a small, highly calcium-selective conductance [18] (see Chapter 1). When external solution contained 10 mM Ca²⁺ and the pipette solution was buffered to 100 nM Ca²⁺, application of 8 μM AA to the bath solution activated I_ARC that averaged 0.56 ± 0.05 pA/pF at -80 mV. I_ARC displays marked inward rectification, reversal potentials greater than +30 mV, and inhibition by La³⁺. Unlike CRAC channels, however, ARC channels are insensitive to 2-APB, are unaffected by reduction of extracellular pH, and do not show Ca²⁺-dependent fast inactivation [18]. Moreover, like most voltage-gated Ca²⁺ channels and CRAC channels [34,35], in external divalent cation-free (DVF) solutions, ARC channels start conducting large monovalent currents [22,23]. However, unlike CRAC channels [36], ARC channels do not show the typical inactivation during a short pulse of DVF bath solution, called depotentiation [23] (see Figure 11.1). However, there is an example where overexpressed STIM1 and Orai1 generate CRAC currents that did not depotentiate in DVF solutions (e.g., see [37]). This could be due to different STIM1/Orai1 stoichiometries and/or limiting endogenous regulatory proteins during overexpression. The Shuttleworth group used arachidonyl coenzyme-A (ACoA), a membrane-impermeant analog of AA to show that activation of the ARC channels reflects an action of the fatty acid specifically at the inner surface of the plasma membrane [19]. A more recent study proposed that the N-terminus of Orai3 is required for AA action on ARC channels [38], but how AA interacts with ARC channels to gate them is entirely unknown.

Figure 11.1

Whole-cell patch clamp electrophysiological recording from HEK293 cells shows that CRAC current activated by 20 mM BAPTA in the patch pipette exhibits depotentiation during a short pulse of divalent cation-free (DVF) bath solution, whereas ARC currents (more...)

Recordings performed in our lab revealed similar LRC channel biophysical properties, such as small conductance, inward rectification, >+30 mV reversal potentials, no depotentiation during the pulse of divalent-free bath (DVF) solution, and inhibition by Gd³⁺. Comparisons of the properties of ARC, LRC, and CRAC are presented in Table 11.1. We also showed that N-methyl LTC₄ (NMLTC₄), a nonmetabolizable form of LTC₄ delivered through the patch pipette was fully capable of activating LRC currents, suggesting the metabolism of LTC₄ into downstream metabolites is not required. However, NMLTC₄ did not activate LRC channels when it was applied extracellularly, indicating that LTC₄ acts through the inner side of the cell plasma membrane [23]. Exactly how LTC₄ interacts and gates the channel remains unclear.

Table 11.1

Biophysical Properties of ARC, LRC, and CRAC Channels.

The crystal structure of the founding member of Orai channels, Drosophila Orai (dOrai), was recently resolved at a resolution of 3.35 Å [39]. This dOrai structure shows a drastically different molecular organization from other ion channels and reveals dOrai as a homohexameric channel arranged around a central pore with a ring of six extracellular glutamate residues (E106 in human Orai1) representing the selectivity filter (see Chapters 2, 3, 14). While this structure strongly suggests that human Orai most likely also form hexameric channels, how many subunits of each of Orai1 and Orai3 are required to form functional ARC/LRC channels that fully recapitulate the properties of native channels remains a contentious issue. The Shuttleworth group suggested that CRAC channels are homotetramers of four Orai1 [40]. Subsequent studies from the same group proposed that native ARC channels are heteropentamers of Orai1 and Orai3 (either organized as 31113 or 31311) [41] (discussed in Chapter 2). They also published a report questioning the hexameric assembly of dOrai as a possible artifact of crystallization [42]. It is worth noting that studies challenging the crystal structure were performed with concatemers ectopically expressed in HEK293 cells expressing endogenous Orai isoforms. In this case, native Orai channels could potentially assemble with concatenated tetramers/pentamers to form hexamers.

11.3. Methods for Measuring SICE Channel Function

11.3.1. Whole-Cell Patch Clamp Recording

The patch clamp technique is the best approach to study ion channel function. Due to the tiny unitary conductance of Orai channels, for instance, single CRAC channel conductance was estimated using stationary noise analysis around 24 fS [43] (Chapter 1). Therefore, for CRAC as well as ARC channel current recordings, the whole-cell configuration of the patch clamp technique is the best choice.

11.3.1.1. Equipment Setup for Patch Clamp Recording

11.3.1.1.1. Amplifier, Low-Noise Digitizer, and Software

The Axopatch 200B amplifier (Molecular Devices) works together with either the Digidata 1550B or 1440A low-noise digitizer (Molecular Devices). For connecting the amplifier and digitizer, the manuals provide step-by-step instructions. For acquiring and analyzing data, the software of Clampex 10 and Clampfit 10 are used, respectively. Similar equipment used by other investigators is also available from HEKA Elektronik, Germany. The HEKA EPC 10 USB Single is a good choice for a patch clamp amplifier. A fully equipped setup includes the EPC 10 amplifier combined with a computer and PATCHMASTER software, a digital storage oscilloscope, a variable analog filter, and a sophisticated pulse generator.

11.3.1.1.2. Microscope

A Nikon ECLIPSE Ti quantitative phase contrast microscope equipped with 20x fluorescence objective is a good choice for the patch clamp setup. Other suitable microscopes are available from various vendors, including Olympus, Leica, and Zeiss.

11.3.1.1.3. Micromanipulator

The MP-225 micromanipulator (Sutter Instrument) is designed primarily for positioning patch and intracellular recording pipettes. Its speed and resolution of movement are easily selected with a multiple position thumbwheel, allowing fast/coarse movement and slow/ultrafine movement in 10 increments. Two commonly used robotic movements have been incorporated for user convenience. A single button press can initiate a move to a home position for pipette exchange or to a user-defined work position for the quick positioning of the pipette near the recording location. Other manufacturers provide similar micromanipulators.

11.3.1.1.4. Vibration Isolation Table

Both Kinetic and TMC brand products are suitable. The minimum size for patch clamp setup is 30 x 36 in. to give enough space for the microscope, perfusion system, and micromanipulator controller with a gas cylinder connected to the air table near the setup.

11.3.1.1.5. Faraday Cage

Proper grounding is essential for obtaining low-noise patch clamp recording from small conductance ARC/LRC channels. Therefore, careful grounding of all instruments including microscope, perfusion system, amplifier, digitizer, micromanipulator, and computer through low-resistance ground cables and use of Faraday cage will minimize noise. The Faraday cage (Kinetic systems) mounted on the top of the vibration isolation table should also be grounded.

11.3.1.1.6. Micropipette Puller

A well-designed micropipette puller can help you deliver a successful whole-cell patch clamp experiment. For the micropipette puller, the Sutter micropipette pullers MP-97 or MP-1000 (Sutter instrument) are a good choice to use with borosilicate glass capillaries (World Precision Instruments) with 1.5 mm OD and 0.86 mm ID to obtain patch pipettes with 0.5–1 μm diameter tips.

11.3.1.1.7. Microforge

For obtaining superior gigaohm (GΩ) seals between the patch pipette and the plasma membrane of cells, tips of patch pipettes should be polished using a microforge controller, for example, DMF1000 (World Precision Instruments) under a microscope, like Revelation III (LW Scientific). After polishing, the seal resistance obtained can be improved by up to 5–10-fold, compared to unpolished pipettes. Therefore, this step is necessary for low noise recordings of small currents such as ARC/LRC.

11.3.1.1.8. Computer

The computer system requirements of patch clamp setup are similar to the calcium imaging system described in the following text. For example, an Intel Pentium-4 processor or faster, Microsoft Windows XP or later, CD-ROM drive, 1 GB or more system memory (RAM), 128 GB or more disk space, and 24-bit graphics display can essentially meet the required needs.

11.3.1.2. Solutions for Electrophysiological Recordings

DVF solution composition: 155 mM Na-methanesulfonate, 10 mM HEDTA, 1 mM EDTA, and 10 mM HEPES (pH 7.4, adjusted with NaOH). We and other researchers have used DVF external solutions to amplify the ARC and CRAC channel currents [18,22,23,29,31,44].

11.3.1.2.1. For Activation of Currents Using Exogenous AA Delivered in the Bath

• Ca ²⁺ -containing bath solution: 115 mM Na-methanesulfonate, 10 mM CsCl, 1.2 mM MgSO₄, 10 mM HEPES, 20 mM CaCl₂, and 10 mM glucose (pH adjusted to 7.4 with NaOH).
• Ca ²⁺ -containing pipette solution: 115 mM Cs-methanesulfonate, 10 mM Cs-BAPTA, 5 mM CaCl₂, 8 mM MgCl₂, and 10 mM HEPES (pH adjusted to 7.2 with CsOH). Calculated free Ca²⁺ was 150 nM using Maxchelator software (http://maxchelator.stanford.edu/).

11.3.1.2.2. For Activation of Currents Using Intracellular LTC₄ Delivered through the Patch Pipette

• Ca ²⁺ -containing bath solution: 115 mM Na-methanesulfonate, 10 mM CsCl, 1.2 mM MgSO₄, 10 mM HEPES, 20 mM CaCl₂, and 10 mM glucose (pH was adjusted to 7.4 with NaOH).
• Ca ²⁺ -containing pipette solution: 115 mM Cs-methanesulfonate, 10 mM Cs-BAPTA, 5 mM CaCl₂, 8 mM MgCl₂, and 10 mM HEPES (pH adjusted to 7.2 with CsOH). 50–100 nM LTC₄ is added.

11.3.1.2.3. For Activation of Store Depletion-Activated CRAC Currents

• Bath solution (same as the previous): 115 mM Na-methanesulfonate, 10 mM CsCl, 1.2 mM MgSO₄, 10 mM HEPES, 20 mM CaCl₂, and 10 mM glucose (pH was adjusted to 7.4 with NaOH).
• Ca ²⁺ -free pipette solution: 115 mM Cs-methanesulfonate, 20 mM Cs-BAPTA, 8 mM MgCl₂, and 10 mM HEPES (pH adjusted to 7.2 with CsOH).

Note: CRAC current recordings are used as controls to highlight the biophysical, pharmacological, and molecular distinction between I_CRAC and I_ARC.

11.3.1.3. Experimental Procedures

11.3.1.3.1. Seeding Cells

Twelve to twenty-four hours before patch clamp experiments, cells are seeded onto 30 mm round glass coverslips (Thermo Scientific) in 6-well tissue culture plates (VWR) at a low density to allow easy identification of single cells for recordings.

11.3.1.3.2. Preparing Patch Pipettes

Pipettes should be pulled on the day of recordings and every pipette should be inspected under the microforge microscope for imperfections before fire-polishing. Polished patch pipettes are stored in a vacuum container for the rest of the day.

11.3.1.3.3. Performing Patch Clamp Electrophysiology Experiments

Before starting the recording, coverslips with attached cells are mounted in recording chambers, each containing a 1 mL bath solution, and transferred to the microscope stage. After identifying a single cell under the microscope, the patch pipette is filled with filtered pipette solution and mounted into the pipette holder. Resistances of filled glass pipettes are 1–3 MΩ. The liquid-junction potential offsets are corrected before each recording. Since ARC/LRC current amplitudes are usually small (several picoamperes) [31], only cells forming tight seals (>16 GΩ) are selected for whole-cell configuration. Immediately after establishing the whole-cell patch clamp configuration, the recording is initiated by applying voltage ramps (typically from 100 to - 140 mV) lasting 250 ms at 0.5 Hz (Figure 11.2a). An initial DVF application is performed before the current has been activated (by addition of AA) or before the current has developed on inclusion of LTC₄ in the patch pipette. The first DVF pulse allows the determination of basal currents or “leaks” that should be subsequently subtracted from total currents obtained after full activation. Specifically, the initial I-voltage (V) relations obtained in Ca²⁺-containing bath solutions (position 1 in Figure 11.2b) and DVF bath solutions (position 2) represent background currents that are subtracted from AA- or LTC₄-activated Ca²⁺ currents (position 3) and Na⁺ currents (obtained in DVF bath solutions; position 4), respectively. After currents are fully activated by AA or LTC₄, I-V curves are obtained for Ca²⁺ currents (in Ca²⁺-containing bath solutions) and Na⁺ currents (in DVF bath solutions). Using Origin software (OriginLab), I-V curves corresponding to background currents obtained in Ca²⁺ and Na⁺ are subtracted from the I-V curves obtained in Ca²⁺ and Na⁺ after AA/LTC₄ stimulation and maximal current activation. Namely, for Ca²⁺ currents (curve 3-curve 1) and Na⁺ currents (curve 4–curve 2), respectively. For recording ARC/LRC, cells are maintained at a 0 or +30 mV holding potential [28]. Reverse ramps from positive to negative voltages are recommended in certain cell types in order to inhibit voltage-gated Na⁺ channels expressed in these cells. Inclusion of 8 mM MgCl₂ in the pipette solution is designed to inhibit TRPM7 currents that are expressed in most cell lines, including HEK293 cells [35]. Experiments are typically performed at room temperature.

Figure 11.2

Example of ramp protocol (a) and typical current development at -100 mV (b) in whole-cell mode. Voltage ramps ranging from +100 to -140 mV lasting 250 ms are applied every 2 s. First, a DVF application (before AA addition) is performed to gauge the leak (more...)

11.3.2. Calcium Imaging

ARC channels are highly Ca²⁺-selective channels, and fluorescence imaging microscopy is also a useful tool for studying Ca²⁺ influx through these channels in living cells. Typical imaging workstations, solutions, dyes, and protocols are described elsewhere [45–48] (see also Chapters 1 and 16).

11.3.2.1. Equipment Setup for Fluorescence Calcium Measurement

Figure 11.3 shows a schematic of the typical fluorescence calcium imaging setup used for Ca²⁺ measurements in cells.

Figure 11.3

Fluorescence calcium imaging setup typically used to measure intracellular Ca²⁺. Typically, a light source providing light with 340 and 380 nm Fura-2 excitation filters. A light guide connects the light source to cells viewed under a microscope equipped (more...)

11.3.2.1.1. Light Source

A xenon lamp as a light source is a cost-effective choice for a calcium imaging system. The Lambda LS (Sutter) xenon lamp has a built-in motor-driven six-positioned filter wheel; switching between neighboring filters (e.g., between 340 and 380 nm, as required for Fura-2) is relatively rapid, completed within 55 ms. A shutter control facilitates graded power output; an external controller enables manual switching. Filtered light from Lambda is transmitted to the fluorescence microscope via a 2 m long liquid light guide to avoid transmission of heat and vibration to the microscope. The Lambda LS xenon lamp produces light between 330 and 650 nm wavelength, which is suitable for a wide variety of dyes, including most fluorescent proteins (e.g., GFP, YFP, and RFP). It is necessary to allow the xenon lamp to warm up for at least 30 min before taking measurements. The light bulb of the Lambda LS lamp can last between 400 and 2000 h, depending on maintenance and the number of on/off switches.

11.3.2.1.2. Fluorescence Microscope

For basic Fura-2-based calcium imaging experiments, it is not necessary to purchase a high-end fluorescence microscope. For example, the Nikon ECLIPSE TS-100 (Nikon) fluorescence microscope is a good choice. The Nikon ECLIPSE TS-100 should be equipped with a 20x fluorescence objective and a Fura-2 filter set (Chroma 74500—with a dichroic mirror and an emission filter, the 340 and 380 nm excitation filters placed in the Lambda filter wheel).

11.3.2.1.3. The Detector

Detection is achieved by a CCD camera (BASLER scA640, Basler AG). The BASLER scA640 camera resolution is 658 x 492 pixels, and it is equipped with an ICX414 sensor, which has a frame rate of 79 fps. The free pylon software can be downloaded at http://www.baslerweb.com/de/produkte/software. Between the CCD camera and the inverted microscope, there is a HR055-CMT 0.55x High Resolution C-Mount Adapter (Diagnostic Instruments).

11.3.2.1.4. Computer-Controlled Filter Changer

The Lambda 10-B Optical Filter Changer (Sutter) is ideal for imaging applications requiring a single filter wheel. Lambda 10-B uses advanced motor technology to achieve 40 ms switching times between adjacent filters. It features USB and serial port interfaces, as well as keypad control.

11.3.2.1.5. Computer and Software

The computer system requirements of fluorescence calcium image system: Intel Pentium-4 processor or later, Microsoft Windows XP or later, CD-ROM drive, 1 GB or more system memory (RAM), 128 GB or more free disk space, and 24-bit graphics display. Data acquisition and analysis is achieved by specialized commercially available software.

11.3.2.2. Ca²⁺ Indicators

Fura-2 and Indo-1 are widely used UV-excitable fluorescence Ca²⁺ indicators (see Table 11.2). The synthesis and properties of Indo-1 and Fura-2 were presented by Tsien and colleagues in 1985 [49,50]. Fura-2 is a ratiometric Ca²⁺ indicator, which is one of the most popular Ca²⁺ indicators and is widely used for quantitative intracellular Ca²⁺ measurements. Its peak absorbance shifts from 335 to 363 nm in the Ca²⁺-bound and Ca²⁺-free state, respectively. Fluorescence emission occurs at a peak wavelength of 512 nm for excitation at either UV wavelength. The use of the ratio automatically cancels out confounding variables, such as variable dye concentration and cell thickness, making Fura-2 one of the most appreciated tools to quantify Ca²⁺levels. Fura-2 has a Ca²⁺ affinity (Kd ∼145 nM) that is comparable to endogenous resting Ca²⁺ levels [51,52].

Table 11.2

Properties of Ca²⁺ Indicators for Fluorescence Calcium Measurement.

Indo-1 is also widely used ratiometric Ca²⁺ indicator. It differs from Fura-2 in that it is single excitation and dual emission. When Ca²⁺ binding occurs, its emission exhibits a large change, which shifts from 485 nm without Ca²⁺ to 405 nm with Ca²⁺ when excited at about 338 nm. The use of the 405/485 nm emission ratio for indo-1 allows accurate measurements of the intracellular Ca²⁺ concentration. Both Fura-2 and Indo-1 have been used to measure calcium entry induced by arachidonic acid [19,53].

11.3.2.3. Solutions

For measuring calcium entry through ARC channels, the following bathing solutions are used:

Solution 1.	Ca2+ free Hanks’ balanced salt solution (HBSS) solution (in mM): 140 NaCl, 1.13 MgCl2, 4.7 KCl, 10 d-glucose, and 10 HEPES, with pH adjusted to 7.4 with NaOH (20 mL)
Solution 2.	2 mM Ca2+ HBSS solution (in mM): 140 NaCl, 1.13 MgCl2, 2 mM CaCl2, 4.7 KCl, 10 d-glucose, and 10 HEPES, with pH adjusted to 7.4 with NaOH (50 mL)
Solution 3.	Ca2+-free HBSS solution + 8 μM AA (20 mL)
Solution 4.	2 mM Ca2+ HBSS solution + 8 μM AA (20 mL)
Solution 5.	2 mM Ca2+ HBSS solution + 50 μM 2-APB (20 mL)
Solution 6.	2 mM Ca2+ HBSS solution + 10 μM ionomycin (20 mL)

11.3.2.4. Experimental Procedures

11.3.2.4.1. Seeding Cells

Twelve to twenty-four hours before performing calcium imaging experiments, cells are seeded onto 35 mm glass bottom dishes (MatTek Corporation) or 30 mm round glass coverslips in 6-well tissue culture plates (VWR).

11.3.2.4.2. Loading Cells

• One milliliter of media from the culture dish is transferred to a 15 mL centrifuge tube (Corning), then 1 μL, 1000x, 2 mM Fura-2AM (dissolved in DMSO, Life Technologies) is added to a centrifuge tube and mixed to achieve a 2 μM final concentration.
• A coverslip with attached cells is transferred to an imaging chamber and incubated at 37°C for 40 min to 1 h (incubation times depend on cell types) in culture media containing 2 μM Fura-2AM.
• The coverslip is washed with solution 2 for 3–4 times, then 1 mL of solution 2 is added, and the cells are left in the dark at room temperature for 10 min to allow cellular esterases to cleave the acetoxymethyl ester groups in Fura-2AM. Fura-2 acid capable of binding calcium is produced and trapped in the cytosol.

11.3.2.4.3. Performing Calcium Imaging Experiments

Before starting the recording, cells of interest are chosen. Excitation filter is switched between F340 and F380, and fluorescence intensities are measured. The measurement of Ca²⁺ influx through ARC channels is performed as described as follows:

• At 1 min, gently remove Solution 2 by suction, and add 1 mL Solution 4.
• At 4 min, switch to Solution 5.
• At 7 min, switch to Solution 6, and wait another 1 min, then end the experiment.

11.4. SICE Channel Function in Health and Disease

Compared to SOCE channels, little is known about the role of STIM/Orai-mediated SICE pathways in cell functions and their contribution to disease. Pla and colleagues first reported that low concentrations of arachidonic acid are able to evoke a store-independent Ca²⁺ influx, exerting a mitogenic role in bovine aortic endothelial cells [54]. Endogenous ARC currents in primary murine parotid and pancreatic acinar cells were reported, and it was shown that they play a critical role in modulating calcium entry responses to physiological agonists [25]. Another study has proposed that ARC channels may play a role in the regulation of insulin secretion in rat pancreatic β cells [55]. A recent study reported that AA-activated ARC currents from airway smooth muscle (ASM) cells isolated from asthmatic individuals are significantly greater than in ASM cells of normal controls, suggesting that ARC channels could potentially contribute to dysregulated calcium signaling in diseases such as asthma [56]. Using immunofluorescence and biotinylation, it was demonstrated that Orai3 expression in the plasma membrane is triggered by vascular endothelial growth factor (VEGF) stimulation of endothelial cells. VEGF-mediated Orai3 membrane accumulation involves activation of phospholipase Cγ1, cytosolic group IV phospholipase A2α leading to AA and AA metabolism into LTC₄ [57]. Saliba and colleagues showed that adult cardiomyocytes express a calcium-permeable conductance activated by AA, mediated by Orai3, and regulated by STIM1. This LRC/ARC-like conductance is increased during cardiac hypertrophy and was proposed to mediate the effects of STIM1 in driving pathological remodeling in heart during cardiac hypertrophy [58]. Studies from our group showed upregulation of Orai3 and LRC/ARC currents during vascular smooth muscle remodeling in vivo, namely, in vessels of rats subjected to balloon angioplasty [29]. The knockdown of Orai3 in balloon-injured carotid arteries using lentivirus-encoding shRNA prevented Orai3 upregulation, inhibited LRC/ARC currents, and decreased neointima formation, supporting the idea that remodeling of Orai1/Orai3 LRC/ARC channels contributes to neointima formation after vascular injury [29]. In a subsequent study, we showed that the knockdown of either LTC₄S or Orai3 inhibits VSMC migration with no effect on proliferation and that in vivo knockdown of LTC₄S inhibits neointima formation [30]. A similar remodeling of Orai1/Orai3 expression was reported in prostate cancer [59]. This remodeling was proposed to involve increased expression of Orai3, favoring the formation of heteromultimers of Orai1/Orai3 to increase an ARC-like conductance and promote decrease in apoptosis and increase in proliferation of prostate cancer. Another study in prostate cancer proposed that Orai1/Orai3 heteromultimers are store-operated. They proposed that SOCE in human prostate epithelial cells and prostate cancer cells is mediated by Orai1/Orai3 heteromers and that there is a correlation between the Orai1/Orai3 ratio and the redox sensitivity of SOCE and therefore, cell viability. An increased Orai1/Orai3 ratio in cells derived from prostate cancer tumors may contribute to the higher sensitivity of these cells to reactive oxygen species (ROS) [60]. Indeed, earlier work by Bogeski and colleagues showed that Orai1 is more resistant to ROS-mediated inhibition of channel function than Orai3 [61].

In summary, much work is needed to fully understand the physiological functions of different Orai channel isoforms, their differential regulation and multimerization patterns, and their contribution to pathological conditions. Particularly, the regulation, exact subunit composition of Orai1/Orai3 SICE channels, and their dysregulation during disease are far from being completely understood. The existence of three Orai isoforms encoded by three independent genes and of translational variants (such as Orai1α and β) and likely yet to be identified splice variants suggest that various associations between these different isoforms likely contribute to enhancing the diversity as well as the subcellular localization of Orai channels for the purpose of selective calcium signaling. While Orai1 has been clearly implicated in the SOCE pathway in virtually all cell types, the exact functions of Orai2 channels remain mostly obscure, and Orai3 has been uniquely implicated in store-independent calcium entry, along with Orai1. The association of two exclusively mammalian proteins, Orai1α and Orai3, to form SICE channels likely constitutes a highly specialized conductance that mediates cellular responses to subtle environmental or humoral cues. The upregulation of Orai1 and Orai3 observed in various disease states suggests the potential use of channels formed by these two proteins as targets for therapy for those diseases. Future studies into the exact oligomeric state of native Orai1α/Orai3 channels, their cellular distribution, and mechanisms of regulation are likely to bring us closer to using these channels as targets in human ARC/LRC-related disease therapy.

References

1.

Putney, J.W., Jr. 1986. A model for receptor-regulated calcium entry. Cell Calcium 7:1-12. [PubMed: 2420465]

2.

Feske, S., Gwack, Y., Prakriya, M., Srikanth, S., Puppel, S.H., Tanasa, B., Hogan, P.G., Lewis, R.S., Daly, M., and Rao, A. 2006. A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature 441:179-185. [PubMed: 16582901]

3.

Vig, M., Peinelt, C., Beck, A., Koomoa, D.L., Rabah, D., Koblan-Huberson, M., Kraft, S., et al. 2006. CRACM1 is a plasma membrane protein essential for store-operated Ca²⁺ entry. Science 312:1220-1223. [PMC free article: PMC5685805] [PubMed: 16645049]

4.

Zhang, S.L., Yeromin, A.V., Zhang, X.H., Yu, Y., Safrina, O., Penna, A., Roos, J., Stauderman, K.A., and Cahalan, M.D. 2006. Genome-wide RNAi screen of Ca²⁺ influx identifies genes that regulate Ca²⁺ release-activated Ca²⁺ channel activity. Proc Natl Acad Sci USA 103:9357-9362. [PMC free article: PMC1482614] [PubMed: 16751269]

5.

Prakriya, M. and Lewis, R.S. 2015. Store-operated calcium channels. Physiol Rev 95:1383-1436. [PMC free article: PMC4600950] [PubMed: 26400989]

6.

Berridge, M.J. 1993. Inositol trisphosphate and calcium signalling. Nature 361:315-325. [PubMed: 8381210]

7.

Roos, J., DiGregorio, P.J., Yeromin, A.V., Ohlsen, K., Lioudyno, M., Zhang, S., Safrina, O., et al. 2005. STIM1, an essential and conserved component of store-operated Ca²⁺ channel function. J Cell Biol 169:435-445. [PMC free article: PMC2171946] [PubMed: 15866891]

8.

Liou, J., Kim, M.L., Heo, W.D., Jones, J.T., Myers, J.W., Ferrell, J.E., Jr., and Meyer, T. 2005. STIM is a Ca²⁺ sensor essential for Ca²⁺-store-depletion-triggered Ca²⁺ influx. Curr Biol 15:1235-1241. [PMC free article: PMC3186072] [PubMed: 16005298]

9.

Brandman, O., Liou, J., Park, W.S., and Meyer, T. 2007. STIM2 is a feedback regulator that stabilizes basal cytosolic and endoplasmic reticulum Ca²⁺ levels. Cell 131:1327-1339. [PMC free article: PMC2680164] [PubMed: 18160041]

10.

Trebak, M. 2012. STIM/Orai signalling complexes in vascular smooth muscle. J Physiol 590:4201-4208. [PMC free article: PMC3473279] [PubMed: 22641780]

11.

Motiani, R.K., Abdullaev, I.F., and Trebak, M. 2010. A novel native store-operated calcium channel encoded by Orai3: Selective requirement of Orai3 versus Orai1 in estrogen receptor-positive versus estrogen receptor-negative breast cancer cells. J Biol Chem 285:19173-19183. [PMC free article: PMC2885196] [PubMed: 20395295]

12.

Motiani, R.K., Zhang, X., Harmon, K.E., Keller, R.S., Matrougui, K., Bennett, J.A., and Trebak, M. 2013. Orai3 is an estrogen receptor alpha-regulated Ca²⁺ channel that promotes tumorigenesis. FASEB J 27:63-75. [PMC free article: PMC3528310] [PubMed: 22993197]

13.

Motiani, R.K., Stolwijk, J.A., Newton, R.L., Zhang, X., and Trebak, M. 2013. Emerging roles of Orai3 in pathophysiology. Channels (Austin) 7:392-401. [PMC free article: PMC3913762] [PubMed: 23695829]

14.

Shuttleworth, T.J. 1996. Arachidonic acid activates the noncapacitative entry of Ca²⁺ during [Ca²⁺]_i oscillations. J Biol Chem 271:21720-21725. [PubMed: 8702966]

15.

Shuttleworth, T.J. 2012. Orai3—The ‘exceptional’ Orai?J Physiol 590:241-257. [PMC free article: PMC3285062] [PubMed: 22041188]

16.

Shuttleworth, T.J. 2012. STIM and Orai proteins and the non-capacitative ARC channels. Front Biosci 17:847-860. [PMC free article: PMC3295580] [PubMed: 22201777]

17.

Thompson, J.L., Mignen, O., and Shuttleworth, T.J. 2013. The ARC channel—An endogenous store-independent Orai channel. Curr Top Membr 71:125-148. [PubMed: 23890114]

18.

Mignen, O. and Shuttleworth, T.J. 2000. I(ARC), a novel arachidonate-regulated, noncapacitative Ca²⁺ entry channel. J Biol Chem 275:9114-9119. [PubMed: 10734044]

19.

Mignen, O., Thompson, J.L., and Shuttleworth, T.J. 2003. Ca²⁺ selectivity and fatty acid specificity of the noncapacitative, arachidonate-regulated Ca²⁺ (ARC) channels. J Biol Chem 278:10174-10181. [PubMed: 12522216]

20.

Gonzalez-Cobos, J.C. and Trebak, M. 2010. TRPC channels in smooth muscle cells. Front Biosci (Landmark Ed) 15:1023-1039. [PMC free article: PMC3164555] [PubMed: 20515740]

21.

Trebak, M., Vazquez, G., Bird, G.S., and Putney, J.W., Jr. 2003. The TRPC3/6/7 subfamily of cation channels. Cell Calcium 33:451-461. [PubMed: 12765690]

22.

Mignen, O. and Shuttleworth, T.J. 2001. Permeation of monovalent cations through the non-capacitative arachidonate-regulated Ca²⁺ channels in HEK293 cells. Comparison with endogenous store-operated channels. J Biol Chem 276:21365-21374. [PubMed: 11285268]

23.

Zhang, X., Zhang, W., Gonzalez-Cobos, J.C., Jardin, I., Romanin, C., Matrougui, K., and Trebak, M. 2014. Complex role of STIM1 in the activation of store-independent Orai1/3 channels. J Gen Physiol 143:345-359. [PMC free article: PMC3933941] [PubMed: 24567509]

24.

Shuttleworth, T.J. and Mignen, O. 2003. Calcium entry and the control of calcium oscillations. Biochem Soc Trans 31:916-919. [PubMed: 14505448]

25.

Mignen, O., Thompson, J.L., Yule, D.I., and Shuttleworth, T.J. 2005. Agonist activation of arachidonate-regulated Ca²⁺-selective (ARC) channels in murine parotid and pancreatic acinar cells. J Physiol 564:791-801. [PMC free article: PMC1464460] [PubMed: 15760932]

26.

Mignen, O., Thompson, J.L., and Shuttleworth, T.J. 2001. Reciprocal regulation of capacitative and arachidonate-regulated noncapacitative Ca²⁺ entry pathways. J Biol Chem 276:35676-35683. [PubMed: 11470795]

27.

Mignen, O., Thompson, J.L., and Shuttleworth, T.J. 2008. Both Orai1 and Orai3 are essential components of the arachidonate-regulated Ca²⁺-selective (ARC) channels. J Physiol 586:185-195. [PMC free article: PMC2375546] [PubMed: 17991693]

28.

Mignen, O., Thompson, J.L., and Shuttleworth, T.J. 2007. STIM1 regulates Ca²⁺ entry via arachidonate-regulated Ca²⁺-selective (ARC) channels without store depletion or translocation to the plasma membrane. J Physiol 579:703-715. [PMC free article: PMC2151373] [PubMed: 17158173]

29.

Gonzalez-Cobos, J.C., Zhang, X., Zhang, W., Ruhle, B., Motiani, R.K., Schindl, R., Muik, M., et al. 2013. Store-independent Orai1/3 channels activated by intracrine leukotriene C4: Role in neointimal hyperplasia. Circ Res 112:1013-1025. [PMC free article: PMC3658112] [PubMed: 23349245]

30.

Zhang, W., Zhang, X., Gonzalez-Cobos, J.C., Stolwijk, J.A., Matrougui, K., and Trebak, M. 2015. Leukotriene-C4 synthase, a critical enzyme in the activation of store-independent Orai1/Orai3 channels, is required for neointimal hyperplasia. J Biol Chem 290:5015-5027. [PMC free article: PMC4335238] [PubMed: 25540197]

31.

Zhang, X., Gonzalez-Cobos, J.C., Schindl, R., Muik, M., Ruhle, B., Motiani, R.K., Bisaillon, J.M., et al. 2013. Mechanisms of STIM1 activation of store-independent leukotriene C4-regulated Ca²⁺ channels. Mol Cell Biol 33:3715-3723. [PMC free article: PMC3753864] [PubMed: 23878392]

32.

Fukushima, M., Tomita, T., Janoshazi, A., and Putney, J.W. 2012. Alternative translation initiation gives rise to two isoforms of Orai1 with distinct plasma membrane mobilities. J Cell Sci 125:4354-4361. [PMC free article: PMC3516440] [PubMed: 22641696]

33.

Desai, P.N., Zhang, X., Wu, S., Janoshazi, A., Bolimuntha, S., Putney, J.W., and Trebak, M. 2015. Multiple types of calcium channels arising from alternative translation initiation of the Orai1 message. Sci Signal 8:ra74. [PMC free article: PMC4583604] [PubMed: 26221052]

34.

Hoth, M. and Penner, R. 1993. Calcium release-activated calcium current in rat mast cells. J Physiol 465:359-386. [PMC free article: PMC1175434] [PubMed: 8229840]

35.

Kerschbaum, H.H. and Cahalan, M.D. 1998. Monovalent permeability, rectification, and ionic block of store-operated calcium channels in Jurkat T lymphocytes. J Gen Physiol 111:521-537. [PMC free article: PMC2217122] [PubMed: 9524136]

36.

Prakriya, M. and Lewis, R.S. 2003. CRAC channels: Activation, permeation, and the search for a molecular identity. Cell Calcium 33:311-321. [PubMed: 12765678]

37.

Zhang, S.L., Kozak, J.A., Jiang, W., Yeromin, A.V., Chen, J., Yu, Y., Penna, A., Shen, W., Chi, V., and Cahalan, M.D. 2008. Store-dependent and -independent modes regulating Ca²⁺ release-activated Ca²⁺ channel activity of human Orai1 and Orai3. J Biol Chem 283:17662-17671. [PMC free article: PMC2427323] [PubMed: 18420579]

38.

Thompson, J., Mignen, O., and Shuttleworth, T.J. 2010. The N-terminal domain of Orai3 determines selectivity for activation of the store-independent ARC channel by arachidonic acid. Channels (Austin) 4:398-410. [PMC free article: PMC3037817] [PubMed: 20818184]

39.

Hou, X., Pedi, L., Diver, M.M., and Long, S.B. 2012. Crystal structure of the calcium release-activated calcium channel Orai. Science 338:1308-1313. [PMC free article: PMC3695727] [PubMed: 23180775]

40.

Mignen, O., Thompson, J.L., and Shuttleworth, T.J. 2008. Orai1 subunit stoichiometry of the mammalian CRAC channel pore. J Physiol 586:419-425. [PMC free article: PMC2375595] [PubMed: 18006576]

41.

Mignen, O., Thompson, J.L., and Shuttleworth, T.J. 2009. The molecular architecture of the arachidonate-regulated Ca²⁺-selective ARC channel is a pentameric assembly of Orai1 and Orai3 subunits. J Physiol 587:4181-4197. [PMC free article: PMC2754359] [PubMed: 19622606]

42.

Thompson, J.L. and Shuttleworth, T.J. 2013. How many Orai’s does it take to make a CRAC channel?Sci Rep 3:1961. [PMC free article: PMC3675454] [PubMed: 23743658]

43.

Zweifach, A. and Lewis, R.S. 1993. Mitogen-regulated Ca²⁺ current of T lymphocytes is activated by depletion of intracellular Ca²⁺ stores. Proc Natl Acad Sci USA 90:6295-6299. [PMC free article: PMC46915] [PubMed: 8392195]

44.

Dehaven, W., Jones, B., Petranka, J., Smyth, J., Tomita, T., Bird, G., and Putney, J. 2009. TRPC channels function independently of STIM1 and Orai1. J Physiol 587:2275-2298. [PMC free article: PMC2697298] [PubMed: 19332491]

45.

Trebak, M., Bird, G.S., McKay, R.R., and Putney, J.W., Jr. 2002. Comparison of human TRPC3 channels in receptor-activated and store-operated modes. Differential sensitivity to channel blockers suggests fundamental differences in channel composition. J Biol Chem 277:21617-21623. [PubMed: 11943785]

46.

Trebak, M., St, J.B.G., McKay, R.R., Birnbaumer, L., and Putney, J.W., Jr. 2003. Signaling mechanism for receptor-activated canonical transient receptor potential 3 (TRPC3) channels. J Biol Chem 278:16244-16252. [PubMed: 12606542]

47.

Trebak, M., Lemonnier, L., DeHaven, W.I., Wedel, B.J., Bird, G.S., and Putney, J.W., Jr. 2009. Complex functions of phosphatidylinositol 4,5-bisphosphate in regulation of TRPC5 cation channels. Pflugers Arch 457:757-769. [PMC free article: PMC2802130] [PubMed: 18665391]

48.

Lemonnier, L., Trebak, M., Lievremont, J.P., Bird, G.S., and Putney, J.W., Jr. 2006. Protection of TRPC7 cation channels from calcium inhibition by closely associated SERCA pumps. FASEB J 20:503-505. [PubMed: 16401641]

49.

Tsien, R.Y., Rink, T.J., and Poenie, M. 1985. Measurement of cytosolic free Ca²⁺ in individual small cells using fluorescence microscopy with dual excitation wavelengths. Cell Calcium 6:145-157. [PubMed: 3874696]

50.

Grynkiewicz, G., Poenie, M., and Tsien, R.Y. 1985. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440-3450. [PubMed: 3838314]

51.

Hurley, T.W., Ryan, M.P., and Brinck, R.W. 1992. Changes of cytosolic Ca²⁺ interfere with measurements of cytosolic Mg²⁺ using mag-fura-2. Am J Physiol 263:C300-C307. [PubMed: 1514577]

52.

Pesco, J., Salmon, J.M., Vigo, J., and Viallet, P. 2001. Mag-indo1 affinity for Ca²⁺, compartmentalization and binding to proteins: The challenge of measuring Mg²⁺ concentrations in living cells. Anal Biochem 290:221-231. [PubMed: 11237323]

53.

Mignen, O., Thompson, J.L., and Shuttleworth, T.J. 2003. Calcineurin directs the reciprocal regulation of calcium entry pathways in nonexcitable cells. J Biol Chem 278:40088-40096. [PubMed: 12874277]

54.

Fiorio Pla, A. and Munaron, L. 2001. Calcium influx, arachidonic acid, and control of endothelial cell proliferation. Cell Calcium 30:235-244. [PubMed: 11587547]

55.

Yeung-Yam-Wah, V., Lee, A.K., Tse, F.W., and Tse, A. 2010. Arachidonic acid stimulates extracellular Ca²⁺ entry in rat pancreatic beta cells via activation of the noncapacitative arachidonate-regulated Ca²⁺ (ARC) channels. Cell Calcium 47:77-83. [PubMed: 20018371]

56.

Thompson, M.A., Prakash, Y.S., and Pabelick, C.M. 2014. Arachidonate-regulated Ca influx in human airway smooth muscle. Am J Respir Cell Mol Biol 51:68-76. [PMC free article: PMC4091853] [PubMed: 24471656]

57.

Li, J., Bruns, A.F., Hou, B., Rode, B., Webster, P.J., Bailey, M.A., Appleby, H.L., et al. 2015. Orai3 surface accumulation and calcium entry evoked by vascular endothelial growth factor. Arterioscler Thromb Vasc Biol 35:1987-1994. [PMC free article: PMC4548547] [PubMed: 26160956]

58.

Saliba, Y., Keck, M., Marchand, A., Atassi, F., Ouille, A., Cazorla, O., Trebak, M., et al. 2015. Emergence of Orai3 activity during cardiac hypertrophy. Cardiovasc Res 105:248-259. [PMC free article: PMC4351368] [PubMed: 25213556]

59.

Dubois, C., Vanden Abeele, F., Lehen’kyi, V., Gkika, D., Guarmit, B., Lepage, G., Slomianny, C., et al. 2014. Remodeling of channel-forming ORAI proteins determines an oncogenic switch in prostate cancer. Cancer Cell 26:19-32. [PubMed: 24954132]

60.

Holzmann, C., Kilch, T., Kappel, S., Dorr, K., Jung, V., Stockle, M., Bogeski, I., and Peinelt, C. 2015. Differential redox regulation of Ca²⁺ signaling and viability in normal and malignant prostate cells. Biophys J 109:1410-1419. [PMC free article: PMC4601090] [PubMed: 26445441]

61.

Bogeski, I., Kummerow, C., Al-Ansary, D., Schwarz, E.C., Koehler, R., Kozai, D., Takahashi, N., et al. 2010. Differential redox regulation of ORAI ion channels: A mechanism to tune cellular calcium signaling. Sci Signal 3:ra24. [PubMed: 20354224]

62.

Paredes, R.M., Etzler, J.C., Watts, L.T., Zheng, W., and Lechleiter, J.D. 2008. Chemical calcium indicators. Methods 46:143-151. [PMC free article: PMC2666335] [PubMed: 18929663]

Xuexin Zhang

Department of Cellular and Molecular Physiology

Pennsylvania State University College of Medicine

Hershey, Pennsylvania

Maxime Gueguinou

Department of Cellular and Molecular Physiology

Pennsylvania State University College of Medicine

Hershey, Pennsylvania

Mohamed Trebak

Department of Cellular and Molecular Physiology

Pennsylvania State University College of Medicine

Hershey, Pennsylvania