Ca2+ Dependence of Volume-Regulated VRAC/LRRC8 and TMEM16A Cl– Channels

All vertebrate cells activate Cl– currents (ICl,swell) when swollen by hypotonic bath solution. The volume-regulated anion channel VRAC has now been identified as LRRC8/SWELL1. However, apart from VRAC, the Ca2+-activated Cl– channel (CaCC) TMEM16A and the phospholipid scramblase and ion channel TMEM16F were suggested to contribute to cell swelling-activated whole-cell currents. Cell swelling was shown to induce Ca2+ release from the endoplasmic reticulum and to cause subsequent Ca2+ influx. It is suggested that TMEM16A/F support intracellular Ca2+ signaling and thus Ca2+-dependent activation of VRAC. In the present study, we tried to clarify the contribution of TMEM16A to ICl,swell. In HEK293 cells coexpressing LRRC8A and LRRC8C, we found that activation of ICl,swell by hypotonic bath solution (Hypo; 200 mosm/l) was Ca2+ dependent. TMEM16A augmented the activation of LRRC8A/C by enhancing swelling-induced local intracellular Ca2+ concentrations. In HT29 cells, knockdown of endogenous TMEM16A attenuated ICl,swell and changed time-independent swelling-activated currents to VRAC-typical time-dependent currents. Activation of ICl,swell by Hypo was attenuated by blocking receptors for inositol trisphosphate and ryanodine (IP3R; RyR), as well as by inhibiting Ca2+ influx. The data suggest that TMEM16A contributes directly to ICl,swell as it is activated through swelling-induced Ca2+ increase. As activation of VRAC is shown to be Ca2+-dependent, TMEM16A augments VRAC currents by facilitating Hypo-induced Ca2+ increase in submembraneous signaling compartments by means of ER tethering.

Previous studies did not answer the question whether LRRC8 and TMEM16A/F are activated in parallel to give rise to I Cl,swell , or whether TMEM16 proteins, particularly TMEM16A, support activation of LRRC8 by facilitating Ca 2+ signals near the plasma membrane (Benedetto et al., 2016;Cabrita et al., 2017). For comparison, such a Ca 2+ -modulating effect of TMEM16A is fundamental for activation of CFTR as demonstrated in vitro and in vivo in mouse and human (Von Kleist et al., 1975;Benedetto et al., 2017Benedetto et al., , 2019bPark et al., 2020), which express both proteins endogenously. The data provide evidence that TMEM16A supports Ca 2+ release from endoplasmic reticulum (ER) and cause Ca 2+ influx that activates TMEM16A and supports activation of LRRC8.
Coverslips were mounted in a perfused bath chamber on the stage of an inverted microscope (IM35, Zeiss) and kept at 37 • C. The bath was perfused continuously with Ringer solution at a rate of 8 ml/min. For activation of volume-dependent Cl − currents, isotonic Ringer bath solution (300 mosm/l; mM: 145 NaCl, 0.4 KH2PO4, 1.6 K2HPO, 4.6 D-glucose, 1 MgCl2, 1.3 Ca 2+ gluconate, pH 7.4) was changed to hypotonic bath solution (Hypo; 200 mosm/l) by removing 50 mM NaCl from Ringer solution. Patch-clamp experiments were performed in the fast whole-cell configuration. Patch pipettes had an input resistance of 4-6 M when filled with the cytosolic-like (physiological) solution. Currents were corrected for serial resistance. The access conductance was measured continuously and was 60-140 nS. Currents (voltage clamp) and voltages (current clamp) were recorded using a patch-clamp amplifier (EPC 7, List Medical Electronics, Darmstadt, Germany), the LIH1600 interface, and PULSE software (HEKA, Lambrecht, Germany) as well as Chart software (AD Instruments, Spechbach, Germany). Data were stored continuously on a computer hard disc and analyzed using PULSE software. In regular intervals, membrane voltage (Vc) was clamped in steps of 20 mV from −100 to +100 mV from a holding voltage of −100 mV. Current density was calculated by dividing whole-cell currents by cell capacitance.

Materials and Statistical Analysis
Suramin, U73122, 2-APB, dantrolene, and NS3728 were from Sigma-Aldrich, St. Louis, Missouri, United States. Niclosamide, Ani9, and DCPIB were from Tocris, Bristol, United Kingdom. Xestospongin C and niclosamide ethanolamine were from Cayman Chemical, Ann Arbor, Michigan, United States. Probenecid was from MP Biomedicals, Irvine, California, United States. Data are shown as individual traces or as summaries with mean values ± SEM and number of experiments given in each figure's legend. For statistical analysis, paired or unpaired Student's t-test was used as appropriate. A p-value of < 0.05 was accepted as a statistically significant difference (indicated by # for unpaired data and by * for paired data).

Activation of Endogenous and Overexpressed VRAC Is Ca 2+ Dependent
Earlier studies suggested a requirement of Ca 2+ for activation of I Cl,swell and stimulation of endogenous LRRC8/Swell1 Benedetto et al., 2016). Here we directly validated the Ca 2+ dependence of LRRC8 currents by coexpressing both LRRC8A and LRRC8C in HEK293 cells, using a bicistronic expression plasmid. Figure 1A shows strong increase of LRRC8A expression when compared with endogenous LRRC8A expression (overexpression of LRRC8C not shown). Overexpression of LRRC8A/C largely augmented the whole-cell currents activated by hypotonic (200 mosm/l) bath solution (Hypo) in the presence of an intracellular (pipette, 290 mosm/l) Ca 2+ concentration of 100 nM. Overexpressed LRRC8A/C was very rapidly activated by Hypo-induced cell swelling ( Figure 1B). Gradual decrease of intracellular (i.e., patch pipette) Ca 2+ concentrations to 10 and to 0 nM gradually inhibited hypo-activation of the endogenous I Cl,swell , as well as the overexpressed LRRC8A/C currents (Figures 1C,D). The experiments indicate a requirement of Ca 2+ for activation of VRAC/LRRC8. TMEM16A Supports Activation of I Cl,swell in HEK293 Cells Overexpressing LRRC8A/C It has been reported that TMEM16A (and TMEM16F) take part in whole-cell current activated through hypotonic cell swelling (I Cl,swell ), although these proteins are not known to be directly activated by cell swelling (Almaca et al., 2009;Benedetto et al., 2016;Sirianant et al., 2016a;. Here we examined the impact of TMEM16A on activation of VRAC at different intracellular Ca 2+ concentrations. At an intracellular resting Ca 2+ concentration of 100 nM, endogenous VRAC (mock) and overexpressed LRRC8A/C were readily activated by osmotic cell swelling (200 mosm/l; Hypo). However, activation was compromised at 10 nM intracellular Ca 2+ ([Ca 2+ ] i ) (Figure 2A). Even at low (10 nM) [Ca 2+ ] i , overexpression of TMEM16A increased hypotonic activation of endogenous VRAC and overexpressed LRRC8A/C (Figures 2A,B). This could suggest that Ca 2+ influx is also relevant for activation of VRAC, as suggested earlier (Sirianant et al., 2016a).
We intended to perform similar experiments with overexpressed TMEM16A at 100 nM pipette Ca 2+ , to examine how the presence of TMEM16A would affect volume activation of VRAC. However, we found overexpressed TMEM16A to be active even at this basal intracellular Ca 2+ concentration and without any additional Ca 2+ increase by hormonal stimulation or by Ca 2+ ionophores (Figure 3). In these experiments, we used CsCl buffer as patch pipette filling solution (100 nM Ca 2+ ; 290 mosm/l) and an extracellular CsCl buffer (1.3 mM Ca 2+ ; 300 mosm/l), to exclude any possible contribution of K + currents. In contrast to mock-transfected cells, TMEM16A-overexpressing cells demonstrated a large basal Cl − inward current that was inhibited by removal of extracellular Cl − , causing a right shift of the reversal potential (Figures 3A,B). Only overexpressed, but not endogenous TMEM16A, was found to be active at basal intracellular [Ca 2+ ] levels, which has been examined in detail earlier (Sirianant et al., 2016a;Schreiber et al., 2018). Thus, it was difficult to assess the contribution of overexpressed TMEM16A to swelling activation of VRAC in HEK293 cells at [Ca 2+ ] i of 100 nM.
augmented I Cl,swell , but it remained unclear whether TMEM16A itself is activated during cell swelling (by Ca 2+ store release or Ca 2+ influx) or whether TMEM16A supports activation of LRRC8A/C. We therefore performed additional experiments in HT 29 cells, which express both ion channels endogenously.
The contribution of TMEM16A to swelling activation of endogenous VRAC was examined in mock-transfected HEK293 cells (-T16A) or HEK293 cells overexpressing TMEM16A (+T16A) at different extracellular hypotonicities. VRAC was activated by extracellular bath solution of different hypotonicities Mean ± SEM; n = 9-14 for each series). # Significant decrease when compared to mock (p < 0.01 for all; unpaired t-tests).
(275, 240, 200, and 150 mosm/l). The data demonstrate an increase in hypotonic activation of VRAC by coexpression of TMEM16A, which was more significant at less severe hypotonicity (Supplementary Figure S1). These experiments were performed at 10 nM intracellular Ca 2+ concentration.

Contribution of TMEM16A to I Cl,swell Under Non-voltage Clamp Conditions
As cell swelling and volume regulation take place under non-voltage clamp conditions, we examined the role of TMEM16A for activation of I Cl,swell in iodide quenching experiments using halide-sensitive yellow fluorescent protein (YFP). When applying hypotonic bath solution (200 mosm/l), immediate increase in YFP-fluorescence is expected due to rapid uptake of water through aquaporins, dilutions of anions, and de-quenching of YFP fluorescence, which occurs within seconds (Benedetto et al., 2016). This is followed by iodide-induced quenching, as VRAC channels are swelling activated, thus allowing entry of iodide ( Figure 6B). Analysis of the maximal rate of quenching showed a clear activation of halide conductance by Hypo (Figure 6C). siRNA knockdown of TMEM16A or LRRC8A (Figures 6A,D) showed similar results as in patch-clamp experiments: Knockdown of TMEM16A attenuated the Hypo-induced quenching, while it did not further reduce quenching inhibited by siLRRC8A (Figures 6E,F). It should be noted that in previous studies we observed an inverse correlation between expression of TMEM16A and LRRC8A. For example, TMEM16A was found to be strongly upregulated in cells that lacked expression of LRRC8A (Benedetto et al., 2016). Thus, it may not be surprising to find that LRRC8A was less efficient downregulated with parallel knockdown of TMEM16A (Western blot in Figure 6A).

Hypotonicity Activates TMEM16A-Dependent Ca 2+ Increase
When analyzing instantaneous peak current densities, the VRAC blocker DCPIB inhibited I Cl,swell independent of siRNA-TMEM16A, while siRNA-LRRC8A strongly inhibited instantaneous peak currents ( Figure 5A). However, knockdown of TMEM16A induced pronounced time-dependent inactivation of I Cl,swell , and the same was observed for knockdown of TMEM16F (Sirianant et al., 2016a; Figure 5B). Notably, TMEM16F has also been reported to conduct Ca 2+ ions, apart from its ability to scramble phospholipids (Yang et al., 2012). Thus, TMEM16 proteins maintain I Cl,swell activity, possibly by supporting ER Ca 2+ release and through activation of Ca 2+ influx (Benedetto et al., 2016;Cabrita et al., 2017). To further elucidate the contribution of Ca 2+ store release to activation of I Cl,swell , cells were swollen in the presence of IP3R-inhibitor 2-ABP or the RyR inhibitor dantrolene, which both inhibited I Cl,swell ( Figure 5C). Activation of CaCC in HT 29 cells by the purinergic ligand ATP was entirely dependent on TMEM16A, as shown by siRNA-TMEM16A, and knockdown of LRRC8A did not compromise activation of CaCC (Figures 5D,E). This indicates that Ca 2+ increase alone is not sufficient to activate VRAC/LRRC8A. Unlike stimulation of I Cl,swell , activation of CaCC was only inhibited by 2-ABP, but not by dantrolene ( Figure 5F). Using the Ca 2+ sensor Fura-2, we examined how TMEM16A affects Ca 2+ increase induced by ATP and Hypo (200 mosm/l). ATP induced a typical peak (ER-Ca 2+ store release) and plateau (Ca 2+ influx; SOCE) response, which were both inhibited in the absence of TMEM16A ; Figure 5G). The Hypoinduced Ca 2+ increase was much smaller but was also inhibited by siRNA-TMEM16A ( Figure 5H). Taken together, the data demonstrate the role of TMEM16A for swelling-induced Ca 2+ increase, which is important for full activation of I Cl,swell .
In additional experiments, we varied the iodide concentration in the hypotonic bath solution between 0 and 50 mM (Figures 6G,H). It is shown that at 0 mM iodide, no quenching takes place but only cell swelling, as indicated by an increase in fluorescence. With increasing iodide concentrations in the extracellular buffer, the maximum of YFP dequenching is no longer reached and the rate of YFP quenching increases. The data suggest a very fast activation of VRAC, which probably parallels the decrease in intracellular ionic strength (Syeda et al., 2016). In contrast, the regulatory volume decrease (RVD) takes considerably longer as indicated by the delayed YFP-re-quenching in the presence of 0 mM iodide.
We examined concentration-dependent quenching by ATP and found pronounced inhibition of quenching by siRNA-TMEM16A (Figures 7A,B). Notably, at pronounced stimulation with saturating concentrations of ATP (50, 100 µM), ATPinduced quenching was slightly but significantly inhibited by siLRRC8A. This suggests a contribution of VRAC to ATPinduced halide permeability. We examined the effects of a number of different inhibitors on activation of I Cl,swell by testing them individually in HT 29 cells. We decided to analyze the effects of the various inhibitors in YFP-quenching experiments with non-dialyzed cells and under non-voltage clamp conditions (instead of whole-cell patch clamping), to leave intracellular Ca 2+ signaling untouched and to avoid artifacts due to voltage clamping.
Three different inhibitors of IP3R-mediated ER Ca 2+ -store release, xestospongin C, suramin, and probenecid, blocked ATPinduced activation of halide permeability ( Figure 7C). Hypo (200 mosm/l)-induced quenching was blocked by the VRAC blocker DCPIB, but not by the TMEM16A blocker Ani9 (Seo et al., 2016), suggesting that the presence of TMEM16A, but not its Cl − conductance, supports activation of I Cl,swell ( Figure 7D). Surprisingly, the inhibitory effect of DCPIB on Hypo-induced quenching, i.e., activation of VRAC, was rather weak. However, this is probably explained by the strong voltage dependence of VRAC inhibition by DCPIB. During YFP measurements, DCPIB was applied under non-voltage clamp conditions, i.e., at the intrinsic negative membrane voltage of the HT 29 cells, which is around −40 mV. In additional patch-clamp experiments, we found indeed weak inhibition of VRAC by DCPIB at negative clamp voltages but a much more pronounced inhibition at depolarized clamp voltages (Supplementary Figure S2). The cystic fibrosis transmembrane conductance regulator (CFTR), another Cl − channel expressed in HT 29 cells, is unlikely to contribute to I Cl,swell , as the CFTR inhibitor CFTRinh172 (30 µM) did not inhibit activation of VRAC (data not shown).
A number of compounds inhibiting IP3R-and RyR-mediated ER Ca 2+ -store release, such as suramin, U73122, probenecid, and dantrolene, as well as NS3728 (Helix et al., 2003), inhibited activation of I Cl,swell . HT 29 cells were found to express all three paralogs of the IP3 receptor as well as the ryanodine receptor RyR2, along with the TRPV4 channel, which is the Ca 2+ influx channel most frequently found to have a role in volume regulation (Pasantes-Morales, 2016; Supplementary Figure S3). The contribution of Ca 2+ influx for activation of VRAC was demonstrated by exposing the cells to hypotonic solution in the presence of low (1 µM) extracellular Ca 2+ , which attenuated the activation of VRAC (Figures 7E,F). Taken together, Ca 2+ may not be a prerequisite for activation of VRAC. However, Ca 2+ store release and Ca 2+ influx facilitate its activation, which is in line with earlier observations Sirianant et al., 2016a;. TMEM16A facilitates intracellular compartmentalized Ca 2+ increase and thus supports activation of VRAC. Along with swelling activation of VRAC, TMEM16A is activated through swelling-induced rise in intracellular Ca 2+ . The extent of this coregulation of I Cl,swell is cell dependent and depends on expression of TMEM16 proteins.
per se, previous data suggest a role in I Cl,swell (Almaca et al., 2009;Juul et al., 2014;Benedetto et al., 2016;Sirianant et al., 2016a;Zhang et al., 2020). Analysis of I Cl,swell in tissues from mice lacking expression of TMEM16A (Almaca et al., 2009), TMEM16F (Ousingsawat et al., 2015;Sirianant et al., 2016a), and TMEM16K (Hammer et al., 2015;Wanitchakool et al., 2017) shows reduced I Cl,swell ) and regulatory volume decrease (RVD). The contribution of TMEM16A and other TMEM16proteins to I Cl,swell and volume regulation is cell dependent and may be particularly relevant in highly differentiated native (non-cultured) cells. Equally important appear the patch-clamp conditions under which I Cl,swell are measured, which may explain some of the earlier controversial findings regarding the role of TMEM16F (Almaca et al., 2009;Shimizu et al., 2013;Juul et al., 2014;Sirianant et al., 2016a).
non-voltage clamp conditions. Nevertheless, even at negative membrane voltages the impact of TMEM16A was found to be significant (Figure 6 and Supplementary Figure S4). While a number of transport properties were ascribed to LRRC8/VRAC (Planells-Cases et al., 2015;Lutter et al., 2017;Kang et al., 2018;Stuhlmann et al., 2018;Zhou et al., 2020), its physiological relevance in terms of volume regulation is still a matter of debate. We found that cells are able to perform RVD also in the absence of LRRC8A (Milenkovic et al., 2015;Benedetto et al., 2016;Sirianant et al., 2016b), while other different members of the TMEM16A family, CFTR, and the KCl cotransporter KCC clearly contribute to volume regulation (Sirianant et al., 2016b;Wanitchakool et al., 2016). We found that activation of I Cl,swell was fast (within 1 min), which, however, was still somewhat delayed when compared to immediate cell swelling (within seconds) (Benedetto et al., 2016). This suggested additional regulatory steps, apart from direct opening of VRAC by low ionic strength (Syeda et al., 2016). Others and we proposed Ca 2+ -dependent unfolding of caveolae-like membrane reserves upon cell swelling and activation of I Cl,swell (Groulx et al., 2006;Kozera et al., 2009;Benedetto et al., 2016).
This additional mechanism may require Ca 2+ increase in an intracellular compartment close to the plasma membrane Benedetto et al., 2016;. Liu et al. showed that intracellular Ca 2+ was necessary but not sufficient to activate LRRC8A-mediated currents . Lemonnier and coworkers provided evidence for a colocalization of VRAC with store-operated Ca 2+ channels and showed that activation of VRAC was strongly dependent on Ca 2+ release through IP3R (Lemonnier et al., 2002). They concluded that VRAC is regulated within Ca 2+ microdomains. Similarly, Akita and collaborators suggested that VRAC/VSOR channels can be activated by PLCcoupled GPCRs, which depends on Ca 2+ store release in close vicinity of the channel  and proposed a Ca 2+ nanodomain-mediated component of VRAC .
Our present data may help to clarify the role of TMEM16 proteins for Ca 2+ -dependent activation of VRAC. The role of TMEM16A and other members of the TMEM16-family for ER Ca 2+ release was found meanwhile in numerous tissues (Jin et al., 2016;Cabrita et al., 2017;Wanitchakool et al., 2017;Benedetto et al., 2019a;Centeio et al., 2020;Park et al., 2020). TMEM16A-controlled Ca 2+ release is also essential for activation of CFTR (Benedetto et al., , 2019bPark et al., 2020). Thus, cell swelling-induced Ca 2+ release from the ER and activation of VRAC is facilitated in the presence of TMEM16 proteins. Because knockdown of TMEM16A but not inhibition of TMEM16A by Ani9 attenuated activation of VRAC, it suggests that ER-tethering by TMEM16A rather than Cl − transport supports activation of VRAC (Jin et al., 2013;Cabrita et al., 2017).
Along this line, TMC8, a member of the closely related family of transmembrane channel-like TMC proteins, also controlled activation of VRAC and volume regulation (Sirianant et al., 2014). Depending on the cell type, swelling-induced Ca 2+ release will activate TMEM16 proteins and CFTR (Thiele et al., 1998;Wanitchakool et al., 2016), which parallels activation of VRAC. This circumstance may explain many earlier findings, such as the overlapping Cl − channel pharmacology (Koslowsky et al., 1994;Rottgen et al., 2018;Centeio et al., 2020).

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

AUTHOR CONTRIBUTIONS
RC, JO, and RS performed the experiments and analyzed the data. RC, RS, and KK wrote the manuscript. All authors contributed to the article and approved the submitted version.