The Number and Position of Orai3 Units within Heteromeric Store-Operated Ca2+ Channels Alter the Pharmacology of ICRAC.

Store-operated heteromeric Orai1/Orai3 channels have been discussed in the context of aging, cancer, and immune cell differentiation. In contrast to homomeric Orai1 channels, they exhibit a different pharmacology upon application of reactive oxygen species (ROS) or 2-aminoethoxydiphenyl borate (2-APB) in various cell types. In endogenous cells, subunit composition and arrangement may vary and cannot be defined precisely. In this study, we used patch-clamp electrophysiology to investigate the 2-APB profile of store-operated and store-independent homomeric Orai1 and heteromeric Orai1/Orai3 concatenated channels with defined subunit compositions. As has been shown previous, one or more Orai3 subunit(s) within the channel result(s) in decreased Ca2+ release activated Ca2+ current (ICRAC). Upon application of 50 µM 2-APB, channels with two or more Orai3 subunits exhibit large outward currents and can be activated by 2-APB independent from storedepletion and/or the presence of STIM1. The number and position of Orai3 subunits within the heteromeric store-operated channel change ion conductivity of 2-APB-activated outward current. Compared to homomeric Orai1 channels, one Orai3 subunit within the channel does not alter 2-APB pharmacology. None of the concatenated channel constructs were able to exactly simulate the complex 2-APB pharmacology observed in prostate cancer cells. However, 2-APB profiles of prostate cancer cells are similar to those of concatenated channels with Orai3 subunit(s). Considering the presented and previous results, this indicates that distinct subtypes of heteromeric SOCE channels may be selectively activated or blocked. In the future, targeting distinct heteromeric SOCE channel subtypes may be the key to tailored SOCE-based therapies.


Introduction
In the last 14 years, many different groups advanced the understanding of structure-function relationships of store-operated Ca 2+ entry (SOCE) channels. Numerous extracellular stimuli produce intracellular second messenger inositol 1,4,5-trisphosphate (IP 3 ) that subsequently releases Ca 2+ via IP 3 receptors from intracellular Ca 2+ stores, i.e., the endoplasmic reticulum (ER). Upon ER depletion, Ca 2+ dissociates from stromal interaction molecule 1 s (STIM1) EF hand motif in the ER lumen. Consequently, STIM1 cluster in the ER membrane recruit and activate Orai1 channels in the plasma membrane. Studies in the past proposed a tetramer of Orai1 proteins to form a functional SOCE channel [1][2][3][4]. Later on, functional and structural studies suggested hexameric Orai1 channels [5][6][7]. In endogenous cells, large Orai1 channel complexes have been observed in liquid-phase scanning transmission electron microscopy and in native gels that may have been of tetrameric or hexameric

Store-Operated Orai1, Orai3 and Concatenated Orai1/Orai3 Channels
We transfected HEK293 cells stably expressing STIM1 (HEK STIM1) with Orai1 ( Figure 1A) or Orai3 ( Figure 1B) and evoked I CRAC with 50 µM IP 3 and 20 mM BAPTA in the patch pipette. Whole cell currents were extracted at -130 mV and +130 mV and plotted versus time. Upon I CRAC development, we applied 50 µM 2-APB from 120-180 s. Under these conditions STIM1/Orai1-mediated I CRAC is first amplified and then blocked, while STIM1/Orai3-mediated I CRAC (CD at t = 116 s~11 pA/pF) was amplified and a large outward current developed. Current-voltage relationships (IV) at the indicated time points are shown as insets ( Figure 1A,B). These findings are in line with previously published results [50,60].
In order to investigate the 2-APB-induced pharmacological profile of heteromeric Orai1/Orai3 CRAC channels, we transfected HEK STIM1 with Orai1 and Orai3 with a ratio of 4:1, assuming that in most cells Orai1 is the predominant endogenously expressed family member. We evoked I CRAC and applied 50 µM 2-APB as described above and CD was plotted versus time ( Figure 1C). IVs extracted at the indicated time points are shown as inset ( Figure 1C). Under these conditions, Orai1/Orai3-mediated I CRAC is reduced compared to Orai1-mediated currents. Upon application of 2-APB, the average CD via Orai1/Orai3 ( Figure 1C) is reduced to a lesser extent than Orai1 ( Figure 1A) and an outward current developed that was lower when compared to Orai3-mediated currents ( Figure 1B). Using our experimental conditions, cellular responses varied as cells expressed different ratios of Orai1/Orai3 subunits and within one cell, channels may have been assembled in varying ratios (light grey traces in Figure 1C). We next transfected a construct expressing a concatenated Orai1/Orai3 channel composed of one Orai3 and three Orai1 subunits (3-1-1-1) to investigate channels with a defined subunit composition and arrangement ( Figure 1D, single cell responses light grey traces). Under these conditions, a reduced I CRAC and no outward current developed. The IVs are shown as inset in Figure 1D. Analysis of IP 3 -induced inward currents and inward currents when 2-APB was applied extracted from single cells from Figure 1C,D is shown in Figure 1E. Both parameters display a higher variance when channels were transfected with Orai1 and Orai3 using a ratio of 4:1, compared to responses given by cells transfected with the concatenated channel (3-1-1-1). Figure 1F compares outward CD from both systems. Whilst variation of outward CD before 2-APB application is similar, outward currents of Orai1/Orai3 transfected cells show a larger scattering (s = 44.2) compared to 3-1-1-1 (s = 1.3) transfected cells.
Using this experimental set up, 50 µM 2-APB induced a small outward current in cells expressing Orai1 ( Figure 3A), while cells expressing Orai3 developed a large outward current ( Figure 3B) in line with previously published results [60]. IVs at the indicated time points are displayed as insets ( Figure 3A,B). We next assessed 2-APB-induced currents from cells expressing concatenated channels, when store depletion was omitted ( Figure 3C). All IVs are displayed in Figure 3D and quantification of CDs is given in Figure 3E. 2-ABP induced no currents in cells expressing concatenated channels containing no or one Orai3 subunit. 2-APB-induced outward currents were observed when two or more Orai3 subunits were present in the channel and an increasing number of Orai3 channels led to increased outward currents. The order of Orai3 subunits within the concatenated channel apparently determines the size of the outward current as the current size is larger in 3-1-3-1 compared to 3-1-1-3.
Similar to the experiments with HEK293 STIM1 cells ( Figure 3C-E) the data in Figure 4 demonstrate that two or more Orai3 subunits in the concatenated channel are sufficient to mediate a 2-APB-induced outward current, when endogenous STIM1 is not recruited to the plasma membrane ( Figure 4A-C) or may assemble with the channel (Figure 4D-F).

Comparison to Prostate Cancer Cells
In many cell types, e.g., T cells, B cells and mast cells, I CRAC is first amplified and then completely blocked when 50 µM 2-APB is applied [41][42][43].
We have previously shown that in the prostate cancer cell line LNCaP a low number of Orai3 subunits within store-operated CRAC channels alters the 2-APB response compared to Orai1-mediated currents when 50 µM was applied [18].
To compare data from LNCaP prostate cancer cells with concatenated channels, we normalized data from Figure 2 and data from LNCaP when 50 µM 2-APB were applied. Data were normalized to CD at 116 s when I CRAC had fully developed ( Figure 5A). A bar diagram of the 2-APB-induced block is shown in Figure 5B. We here omit all outward currents as outward current development was not observed in endogenous cells.
to CD at 116 s when ICRAC had fully developed ( Figure 5A). A bar diagram of the 2-APB-induced block is shown in Figure 5B. We here omit all outward currents as outward current development was not observed in endogenous cells.
None of the concatenated channels displayed the exact pharmacological profile of LNCaP cells. However, ICRAC from cells expressing concatenated channels exhibit a similar pharmacological profile as the prostate cancer cells. It is possible that the concatenation of subunits changes the complex pharmacological profile. Moreover, endogenous CRAC channels might exist as a mixed population of channels composed of varying numbers of Orai1/Orai3 subunits that cannot be simulated with one distinct channel composition.
Taken together, none of the concatenated channels displayed the exact pharmacological profile of LNCaP cells; however, here, we speculate that endogenous store-operated Orai1/Orai3 channels are formed with one or more Orai3 subunit(s).  None of the concatenated channels displayed the exact pharmacological profile of LNCaP cells. However, I CRAC from cells expressing concatenated channels exhibit a similar pharmacological profile as the prostate cancer cells. It is possible that the concatenation of subunits changes the complex pharmacological profile. Moreover, endogenous CRAC channels might exist as a mixed population of channels composed of varying numbers of Orai1/Orai3 subunits that cannot be simulated with one distinct channel composition.
Taken together, none of the concatenated channels displayed the exact pharmacological profile of LNCaP cells; however, here, we speculate that endogenous store-operated Orai1/Orai3 channels are formed with one or more Orai3 subunit(s).

Discussion
The versatile role of SOCE channels in physiology/pathophysiology make them an attractive therapeutic target. Endogenous heteromeric store-operated Orai/Orai3 channels exhibit a complex but promising pharmacology [16,18,25,27,28,40,59], however, their investigation is challenging. In this work we used concatenated channels with defined Orai1 and Orai3 subunit stoichiometry and arrangement to systematically investigate their complex pharmacology using a widely used tool compound (2-APB).
Upon activation by STIM1, all channels exhibited the typical inwardly rectified I CRAC . When store depletion was prevented or only endogenous STIM1 was present at the channel, no I CRAC was observed.
In line with previous studies [15,61], the expression of one or more Orai3 subunit(s) within these channels result in a significant reduction of I CRAC that was also observed in endogenous systems [15][16][17][18]23,24]. In concatenated channels, expression of two or more Orai3 subunits resulted in 2-APB-induced outward currents in STIM1-dependent and store-independent 2-APB-activated channels. The amount of this outward current gradually increased with the number of Orai3 subunits (3-1-3-1 < 3-1-1-3 < 3-1-3-3). From experiments with channel constructs containing two Orai3 subunits, we can conclude that the pore size depends on the position of Orai1 and Orai3 subunits within the multimeric channel arrangement. To our knowledge, such outward currents were never observed in endogenous cells. Strikingly, presence of STIM1 at the channels increased 2-APB-induced outward component by 2-fold. This has been observed earlier [50] and may indicate that STIM1 is present at the 2-APB-activated channel and adds to ion channel conductivity. In contrast, it has been published that STIM1 dissociates from the channel before 2-APB-induced conductivity is observed [57]. One explanation might be that STIM1 activation before 2-APB application results in 2-APB-activated channels that differ from 2-APB-activated channels that originate from a closed (STIM1 free) state. In addition to STIM1/Orai1 and STIM1/Orai3, STIM2/Orai1 and STIM2/Orai3 can form store-operated channels. Without store depletion, STIM2/Orai1 overexpressing HEK293 cells give a transient current response upon application of 50 µM 2-APB that is absent in STIM1/Orai1-expressing cells [62]. Future studies may compare the 2-APB response from STIM2 and Orai1, Orai3, or concatenated channel overexpression systems to functionally analyze the pharmacology of STIM2-expressing endogenous cells.
The response of prostate cancer cells LNCaP to the application of 50 µM 2-APB could not be exactly simulated with any concatenated channel. Concatenated channels may differ in their response to 2-APB as subunits are linked by an additional intramolecular connection. In addition, endogenous channels may form mixed heteromeric populations in contrast to concatenated channels that represent one defined subunit composition. Furthermore, we cannot exclude that an additional 2-APB-sensitive component adds to heteromeric SOCE channels altering the Orai1/Orai3-mediated response such as TRPC [63,64].
The ROS-dependent block is abolished when one Orai3 subunit is present within the channel [15]. In contrast, no difference in current is observed between 1-1-1-1 and 3-1-1-1 in response to 2-APB. In conclusion, endogenous heteromeric Orai1/Orai3 channels with only one Orai3 subunit may be undistinguishable from homomeric Orai1 channels in their 2-APB profile. Development of tool compounds based on the known 2-APB or other scaffolds that distinguish homomeric Orai1 channels and heteromeric channels with one Orai3 subunit may help to identify heteromeric Orai channels in various endogenous cell types in the future. Blocking or activating distinct heteromeric store-operated channels may be the key for specific modulations of SOCE in cancer and immune therapy.

Electrophysiology
All patch-clamp experiments were performed in a whole-cell setting at 20-24 • C. Currents were acquired and filtered at 2.9 kHz with a HEKA EPC-10 amplifier (HEKA Elektronik, Lambrecht (Pfalz), Germany) and recorded with the HEKA Patchmaster (v2×53) software. Every 2 s, 50 ms voltage ramps spanning −150 mV to 150 mV from a holding potential of 0 mV were applied. All currents were corrected for a liquid junction potential of 10 mV. Capacitive currents were corrected before every voltage ramp delivery. For analysis, currents at −130 mV and 130 mV were extracted, normalized to cell capacitance, and plotted versus time. The error bars represent the standard error of the mean (SEM) if not indicated otherwise. Any additional normalizations, if used, are mentioned in the figure captions. Data was analyzed with Igor Pro 6.37 (Wavemetrics, Lake Oswego, OR, USA). Bath solution contained: 120 mM NaCl, 20 mM CaCl 2 , 10 mM TEA-Cl, 10 mM HEPES, 2 mM MgCl 2 . Store-depleting pipette solution contained: 120 mM Cs-glutamate, 20 mM BAPTA (10 mM for LNCaP), 10 mM HEPES, 3 mM MgCl 2 , 50 µM IP 3. In pipette solution that prevented store depletion, IP 3 was omitted and calcium was clamped to 200 nM with BAPTA. Calcium concentration was calculated according to WEBMAXC STANDARD [66]. Osmolarity was adjusted with glucose to 310 mOsm. Under these conditions, pipettes had resistances of 2-3 MΩ.

Statistical Analysis
Statistical tests were performed in GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA) as detailed in the figure captions.

Conclusions
Targeting endogenous heteromeric ion channels in therapeutic interventions is generally very challenging. Often their pharmacological behavior varies with subunit composition and arrangement. Here, we used concatenated SOCE channels in order to rationalize the action of 2-APB on different Orai1/Orai3 channel architectures. Our findings show that as Orai3 content is increased, in particular, 2-APB pharmacology digresses significantly from mostly Orai1 containing channels. Ultimately, our long-term goal is to generate SOCE channel subtype-dependent pharmacology profiles that can be exploited therapeutically in diseased native cells.
Funding: This research received no external funding.