Ca²⁺-activated K⁺ channels regulate cell volume in human glioblastoma cells

2.1 Cell culture

U87-MG (ICLC catalog code: HTL00013) GBM cell line was grown in high-glucose Dulbecco's minimum essential medium supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/mL penicillin G, 100 mg/mL streptomycin. The flasks were incubated at 37°C in a 5% CO₂-humidified atmosphere. The medium was changed twice a week, and the cells were sub-cultured when confluent. For electrophysiological experiments, cells were seeded in Petri dishes at 40,000/50,000 cells/mL and used at 2 or 3 days after seeding. For cell volume measurements, cells were trypsinized the day of the experiment and then plated on dishes and allowed to settle for 30 min before conducting cell volume recordings. This procedure allowed the cells to obtain a spherical shape, ideal for extrapolating the cell volume changes from the relative area measurements (see “Cell volume measurements” paragraph for more details).

2.2 Electrophysiology

The whole-cell perforated configuration was used for electrophysiological recordings. Currents were amplified with a HEKA EPC-10 amplifier (List Medical), digitized with a 12-bit A/D converter (TL-1, DMA interface; Axon Instruments), and analyzed with the software Patch-Master package (version 2_60, ELEKTRONIK) and Microcal Origin 8.0. For on-line data collection, macroscopic currents were filtered at 3 kHz and sampled at 200 µs/point. The external standard Ringer's solution contained: NaCl 140 mM, KCl 5 mM, CaCl₂ 2 mM, MgCl₂ 2 mM, MOPS 5 mM, glucose 10 mM (pH 7.4). The external Ca²⁺-free solution contained: NaCl 140 mM, KCl 5 mM, MgCl₂ 4 mM, MOPS 5 mM, glucose 10 mM (pH 7.4). The internal solution contained: K₂SO₄ 57.5 mM, KCl 55 mM, MgCl₂ 5 mM, MOPS 10 mM (pH 7.2). Electrical access to the cytoplasm was achieved by adding amphotericin B (200 μM) to the pipette solution. Access resistances ranging between 15 and 30 MΩ were achieved within 10–15 min from seal formation and were actively compensated to approximately 50%. Octanol (1 mM) was added to all external solutions to block the gap-junctions (Eskandari et al., 2002). All chemicals used were of analytical grade. Dimethyl sulfoxide (DMSO), amphotericin B, and gadolinium chloride (GdCl₃) were purchased from Sigma. 4-[(2-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy] butanoic acid (DCPIB), TRAM-34, paxilline, and Yoda1 were purchased from Tocris Biosciences. GdCl₃ was dissolved in water at the stock concentration of 100 mM and used at the final concentration of 10 μM. Amphotericin B, DCPIB, TRAM-34, paxilline, and Yoda1 were dissolved in DMSO at the stock concentration of 50, 10, 6, 10, and 10 mM, and used at the final concentration of 200, 10, 3, 1, and 10 μM, respectively. The maximal DMSO concentration in the recording solutions was 0.1%, which did not have effects on membrane currents (data not shown). The hypotonic solution was prepared by adding 30% distilled water to the extracellular Ringer's solution. All reagents were fresh daily solubilized at the concentrations stated, and bath applied with a gravity perfusion system. Experiments were carried out at room temperature (18–22°C).

2.3 Cell volume measurements

Cells were perfused with the identical extracellular Ringer's solution used for electrophysiological experiments. To study the hypotonic-induced cell swelling effect, a 30% hypotonic solution was applied. Relative projection area of cells was measured using a video-imaging technique, as previously described (Sforna et al., 2017, 2022). The cells were observed through a ×40 objective lens of a microscope connected to a video camera (Axiocam/Cm1, Zeiss). Images of the cells were saved as TIFF files and the area of each image was subsequently measured using ImageJ. Images were acquired every 2 min for the experiments shown in Figure 6a,b. Relative cell area (A_rel) was calculated by dividing the area of the cell at each time point (A_t) upon exposure to hypotonic solution by the initial area (A₀) taken under isotonic conditions (more precisely the average of the three time points recorded in the first 4 min). The extent of RVD was measured 62 min after the onset of the regulatory response, which started soon after the cell reached the peak of swelling under hypotonic solution, and was calculated as follows: %RVD = ((A_rel,peak − A_rel,62)/(A_rel,peak)) × 100, where A_rel,peak and A_rel,62 are the relative areas at the peak of cell swelling and at the end of the experiment, respectively (Sforna et al., 2022).

2.4 Statistical analysis

All statistical analyses were performed using the Origin v.8 software. To evaluate the significance of a difference, we used either the one-sample or two-sample t-test, depending on whether we compared one normally distributed data to unity (Figures 1e and 5c) or two normally distributed populations, respectively. For multiple comparisons referred to a single control group (Figure 6c), we used a one-way analysis of variance test with post hoc Tukey's correction. For all experiments, p < 0.05 was considered statistically significant and data are shown as mean ± SEM.

3.1 Hypotonic stimulus activates both IK_Ca and BK_Ca currents in U87-MG cells

As a first step, we assessed whether the osmotic stress could activate K⁺ selective currents in U87-MG cells by performing electrophysiologic experiments using the patch-clamp technique in the whole-cell perforated configuration. In these experiments the extracellular solution was constantly supplemented with 10 µM DCPIB to block the I_Cl,swell. Under these conditions, the application of an extracellular 30% hypotonic solution was able to activate an outward current (Figure 1a,b) whose reversal potential was close to the equilibrium potential of K⁺ ions as calculated by the Nernst equation (Figure 1b). The hypotonic-activated K⁺ current evoked by voltage ramps, appeared to be composed of a voltage-independent and a voltage-dependent component, with features reminiscent of the highly expressed IK_Ca and BK_Ca currents in GBM cells (Figure 1b; Fioretti et al., 2006; Rosa et al., 2017). In accordance, the hypotonic-activated current was partially blocked by the BK_Ca inhibitor paxilline (1 µM) that completely removed the voltage-dependent component of the current (Figure 1a,b). Subsequent addition of the IK_Ca channel inhibitor TRAM-34 (3 µM) eliminated also the voltage-independent component, dropping the current to isotonic condition level. These results strongly suggested that in U87-MG cells the only K⁺ currents activated by the hypotonic stimulus are the IK_Ca and the BK_Ca currents.

We next studied separately the two hypotonic-activated K_Ca currents, starting with the IK_Ca current. To isolate the IK_Ca current, the extracellular solution was supplemented with 10 µM DCPIB and 1 µM paxilline, inhibitors of VRAC and BK_Ca channels, respectively. Under these conditions, the hypotonic solution was able to activate an outward current at 0 mV, as shown by the representative time course in Figure 1c. The hypotonic-current showed the typical features of the IK_Ca current, such as voltage-independence and a reversal potential at −80 mV, (Figure 1d). In addition, the current was fully blocked by TRAM-34 (Figure 1c–e), as also confirmed by the quantitative analysis of the residual current in the presence of the inhibitor (Figure 1e). The TRAM-34-sensitive current elicited by the hypotonic stress, as measured at 0 mV of the applied potential, displayed an average density of 5.5 ± 0.5 pA/pF (Figure 1d, Inset).

To isolate the BK_Ca current, the extracellular solution was supplemented with 10 µM DCPIB and 3 µM TRAM-34. As shown by the time course of the current density in Figure 1f, constructed from the current ramps as shown in Figure 1g, taking the current at +50 mV, the application of a hypotonic solution elicited an outward current which exhibited the typical biophysical properties of the BK_Ca current (i.e., a strong voltage dependence and a relatively high current noise), including full block by 1 µM paxilline (Figure 1f,g). To quantify the hypotonic-induced activation of the BK_Ca current, we applied 200 ms voltage steps from −60 to +140 mV, from a holding potential of −40 mV, with increment steps of 20 mV (Figure 1h). Application of depolarizing steps under these conditions elicited an outward current associated with a high noise, typical of currents passing through the high-conductance BK_Ca. The quantitative analysis of the current-voltage relationship constructed from these voltage steps, showed that the BK_Ca current triggered by the osmotic stress was more than twice of that recorded under isotonic conditions (at +140 mV, the current density was 35.0 ± 4.6 vs. 85.7 ± 10.7 pA/pF under isotonic and hypotonic conditions, respectively).

3.2 Activation of IK_Ca and BK_Ca currents by hypotonicity requires the influx of extracellular Ca²⁺

Both IK_Ca and BK_Ca channels require an increase of intracellular Ca²⁺ to open. Many studies have shown that the hypotonic-induced cell swelling promotes cytosolic Ca²⁺ raises by triggering extracellular Ca²⁺ influx (Liu & Zhang, Men, et al., 2019; Park et al., 2002; Sheader et al., 2001a). Therefore, we hypothesized that the activation of both K_Ca channels upon exposure to osmotic stress might derive from cell swelling-induced Ca²⁺ entry. To verify this notion, we performed electrophysiological experiments as those shown in Figure 1 but initially using extracellular solutions devoid of Ca²⁺ ions. First, we probed the IK_Ca current, using the same pharmacological approach illustrated before (i.e., using extracellular solution constantly supplemented with 10 µM DCPIB and 1 µM paxilline). We began with a stabilization period of 5 min in 0-Ca²⁺ isotonic solution, after which cells were exposed to hypotonic solution, still in the absence of external Ca²⁺, to assess the activation of an outward current. After a stable period in 0-Ca²⁺ hypotonic solution, cells were probed with a hypotonic solution containing 2 mM Ca²⁺, which evoked a major increase of outward current, arguably due to the entry of external Ca²⁺, that was fully blocked by the IK_Ca current antagonist TRAM-34 (Figure 2a,b). Results from these experiments revealed that the hypotonic stimulus was able to activate a TRAM-34-sensitive current only upon the addition of external Ca²⁺ (Figure 2a,b). The quantitative analysis of the TRAM-sensitive current revealed that the hypotonic-activated current density was more than seven times greater when Ca²⁺ was added to the extracellular solution: 0.6 ± 0.2 versus 4.6 ± 0.3 pA/pF at 0 mV in the absence and presence of external Ca²⁺, respectively (Figure 2c).

We then performed electrophysiological experiments aimed at measuring the BK_Ca current (with 10 µM DCPIB and 3 µM TRAM-34 constantly present in the extracellular solution). As observed for the IK_Ca current, removal of external Ca²⁺ prevented the activation of BK_Ca current by the hypotonic stimulus, as shown by both the representative current traces obtained in response to voltage steps and the average current-voltage relationship (Figure 2d,e), showing no difference in current density at any given voltage. Taken together, these results indicate that an influx of external Ca²⁺ is required for the hypotonic-induced activation of both IK_Ca and BK_Ca currents.

3.3 Ca²⁺ influx required for the activation of IK_Ca and BK_Ca channels goes through mechanosensitive Ca²⁺ channels

The influx of external Ca²⁺ during hypotonic stress may occur through mechanosensitive Ca²⁺ permeable channels activated by cell swelling. To test this notion, we applied hypotonic solutions containing gadolinium (Gd³⁺, 10 µM), a potent inhibitor of mechanosensitive channels (Hamill & McBride, 1996; Yang & Sachs, 1989), on cells already equilibrated for 5 min with Gd³⁺ (in isotonic solution) (Figure 3a). Also in this case, we recorded IK_Ca and BK_Ca currents separately. First, we probed the IK_Ca current using extracellular solution constantly supplemented with 10 µM DCPIB and 1 µM paxilline. As found with zero external Ca²⁺ (Figure 2a–c), in the presence of Gd³⁺ only a very small outward current induced by hypotonicity was observed, while a significant TRAM-sensitive current was elicited upon removal of the mechanosensitive channel blocker Gd³⁺ (Figure 3a,b). Specifically, the average current density recorded at 0 mV, taken from the representative current ramps (Figure 1b), was 0.6 ± 0.3 vs. 4.5 ± 0.7 pA/pF in the presence and absence of Gd³⁺, respectively (Figure 3c).

We then recorded the BK_Ca current by using an extracellular solution supplemented with both DCPIB and TRAM-34. Results from these experiments revealed that also the activation of the BK_Ca current was fully prevented or blocked when the entry of Ca²⁺ through mechanosensitive channels was not allowed (Figure 3d,e).

3.4 Piezo1 selective agonist Yoda1 activates a current virtually identical to that elicited by hypotonic-induced cell swelling

It has been recently reported that Piezo1, a Ca²⁺-permeable channel identified as the essential component of the mechanically activated current that mediates mechanotransduction in vertebrates (Coste et al., 2010), is heavily overexpressed in high grade GBM (Chen et al., 2018; Qu et al., 2020). We verified its involvement in the activation of both IK_Ca and BK_Ca currents by assessing whether the hypotonic stimulus was able to activate a Piezo1 current. We first characterize the biophysical properties of Piezo1 in our U87-MG cells, by applying the highly selective Piezo1 synthetic agonist Yoda1 (Syeda et al., 2015). Experiments were carried out by either applying 1 s voltage ramps from −100 to +100 mV or 200 ms voltage steps from −100 to +100 mV protocols. As Piezo1 is a nonselective cation channel which mediates the flux of Ca²⁺, Na⁺, and K⁺ ions, currents were measured at both +80 mV (to appreciate K⁺ flux) and −80 mV (to appreciate both Ca²⁺ and Na⁺ fluxes). Activation of K_Ca and VRAC currents were prevented by supplementing the extracellular solution with TRAM-34, paxilline, and DCPIB. As shown in the representative time course in Figure 4a, acute application of 10 μM Yoda1 under isotonic conditions elicited an outward and an inward current (as measured at +80 and −80 mV, respectively, from the current ramps shown in (a, inset)) that were both fully blocked by the acute application of 10 μM Gd³⁺. The representative current ramps in Figure 4a (inset) showed that the Yoda1-activated current displayed a strong outward rectification, somehow expected because of its significant voltage dependence (in addition to stretch activation) (Moroni et al., 2018). Rectification and voltage modulation of Yoda1-mediated Piezo1 current were also clearly shown during the application of a voltage-step protocol ranging from −100 to +100 mV (20 mV increment steps) and by the corresponding I–V relationship (Figure 4b,c).

We then performed identical electrophysiological experiments, where cells were subjected to a 30% hypotonic solution instead of applying Yoda1 (Figure 4d–f). The extracellular solution was, as before, supplemented with TRAM-34, paxilline, and DCPIB. As can be seen from the current ramps, the step current traces and the related analysis, the biophysical and kinetic properties of the current elicited by the hypotonic solution were virtually identical to those of Yoda1-activated current, with a strong voltage-dependent outward rectification (Figure 4e,f). The hypotonic-induced currents were however significantly smaller than those elicited by Yoda1 (at 100 mV the hypotonic current and the Yoda1 current were 7.8 ± 0.6 pA/pF and 20.9 ± 2.1 pA/pF, respectively). Taken together, these results suggest that Piezo1 might be responsible for a large part of hypotonic induced Ca²⁺ influx in U87-MG cells.

3.5 Piezo1 channel agonist Yoda1 activates both IK_Ca and BK_Ca currents

To verify whether IK_Ca and BK_Ca channels may be activated as the result of Piezo1 opening, we performed experiments in which the specific Piezo1 activator Yoda1 was acutely applied under isotonic conditions. As clearly shown in Figure 5, acute application of Yoda1 under isotonic conditions was able to elicit currents exhibiting the typical biophysical and pharmacological features of both IK_Ca and BK_Ca currents (Figure 5a–g), indicating that both K_Ca channels can be activated by Ca²⁺ influx through Piezo1.

3.6 Both IK_Ca and BK_Ca channels are involved in RVD

Finally, we assessed the contribution of both IK_Ca and BK_Ca currents in the RVD process, by measuring changes in cell area in control conditions, and after either channel was pharmacologically blocked. We monitored cell volume changes with a phase-contrast microscope, and images were video recorded at 2 min intervals, over a long-time period (62 min). During the first 4 min of control recording, cells were exposed to isotonic solution, and then to 30% hypotonic solution for the remaining 58 min of the experiment. The average time course of the relative cell area showed that cell volume underwent a rapid and marked increase upon the application of the hypotonic solution. However, while in control conditions cells exhibited a marked ability to completely recover their original cell volume, the selective inhibition of IK_Ca or BK_Ca channels significantly impaired the re-establishment of the original cell volume (Figure 6a). The level of cell volume recovery was calculated as %RVD = ((A_rel,peak − A_rel,62)/(A_rel,peak)) × 100, where A_rel,peak and A_rel,62 are the relative areas at the peak of cell swelling and at the end of the experiment, respectively (see Section 2). %RVD was 97.6%, 24.1%, and 34.4% for cells in control conditions, in the presence of 3 µM TRAM-34, and in the presence of 1 µM paxilline, respectively (Figure 6c). Importantly, the RVD was also markedly affected by the absence of external Ca²⁺ (37.3%) or the presence of the mechanosensitive blocker 10 µM Gd³⁺ (40.3%), strongly suggesting the involvement of mechanosensitive-mediated Ca²⁺ influx in the RVD process. Collectively, these data indicated that K_Ca channels play a key role in the regulation of cell volume, as their selective inhibition strongly hampers the RVD process.

4.1 Main findings

The present study examined the involvement of IK_Ca and BK_Ca channels in RVD, the cell response to hypotonic-induced cell swelling that ultimately results in the re-establishment of the original cell volume. We show that cell exposure to hypotonic solution in the constant presence of DCPIB activates a macroscopic current essentially sustained by IK_Ca and BK_Ca channels. The hypotonic-induced activation of IK_Ca and BK_Ca channels requires the influx of Ca²⁺ from the extracellular environment, as demonstrated by the result that removal of external Ca²⁺ prevents the activation of both channels. Moreover, the observation that the nonselective mechanosensitive channel blocker Gd³⁺ prevents the hypotonic-induced activation of both IK_Ca and BK_Ca channels indicates that the Ca²⁺ entry passes through mechanosensitive channels. Among the mechanosensitive channels that are activated by cell swelling, Piezo1 may represent an important component since: i) it is highly expressed in high grade gliomas; ii) Yoda1 can activate both IK_Ca and BK_Ca channels under isotonic conditions; iii) the Yoda1-activated current exhibits the biophysical and pharmacological properties of nonspecific mechanosensitive cation currents, such as a reversal potential close to 0 mV, strong outward rectification, and block by Gd³⁺. Worth of notice, these properties are fully shared by the current activated by the hypotonic stimulus in the presence of DCPIB, TRAM-34, and paxilline, congruent with Piezo1 as the underlying channel. On the other end, the observation that Yoda1, under isotonic conditions, can activate both IK_Ca and BK_Ca channels, shows that these channels are under the control of Piezo1 (although not necessarily exclusive).

We finally show that the recovery of the original cell volume (RVD) following exposure to hypotonic solution is significantly hampered by the selective inhibition of either IK_Ca or BK_Ca channel. As expected, a marked inhibition of RVD is also observed when the influx of Ca²⁺ from the extracellular space is prevented, either by removing the external Ca²⁺ or blocking its influx through mechanosensitive channels.

4.2 How the influx of Ca²⁺ determines the IK_Ca and BK_Ca channels activation

Although IK_Ca and BK_Ca channels are both activated by internal Ca²⁺, the impact that given cytoplasmic Ca²⁺ concentrations will have on their activation is strongly affected by two distinctive features of these channels: Ca²⁺ sensitivity and voltage dependence. IK_Ca channels are fully voltage independent but are activated by low concentrations of cytoplasmic Ca²⁺ (EC₅₀ 100–200 nM). By contrast, BK_Ca channels have a much lower Ca²⁺ sensitivity (EC₅₀ 1–5 µM, at 0 mV) but are sensitive to voltage. Their opening thus requires cytosolic Ca²⁺ increases in the micromolar range, even at moderate depolarizations. In the virtual absence of cytosolic Ca²⁺, BK_Ca channels can be activated only with extreme depolarizations, that can never be experienced by non-excitable cells such as GBM.

Several studies using fluorescent dyes have shown that hypotonic-induced cell swelling can raise the global cytoplasmic Ca²⁺ concentration in the range of 200-400 nM (Nitschke et al., 1993; Wong et al., 1990; Yellowley et al., 2002). While these Ca²⁺ concentrations would be sufficient to activate a large fraction of IK_Ca channels, they are way lower than those required for the activation of BK_Ca channels (in the range of physiological membrane potentials). This makes it difficult to explain how the hypotonic shock can activate BK_Ca channels, especially in intact cells, as in the RVD experiments, with negative (resting) voltage. Yet, our data clearly demonstrate that it does, as BK_Ca channels have been shown essential for the re-establishment of the original cell volume in the RVD process, and current mearurements indicate that they open at potentials close to the resting membrane potential (i.e., −30/−40 mV in our cell model) during the hypotonic shock.

These results compel us to consider the hypothesis that BK_Ca channels are confined in specialized compartments where they can sense a Ca²⁺ concentration, following a hypotonic shock, much higher than the Ca²⁺ bulk assessed with fluorescent probes. These compartments can be found near Ca²⁺ sources, such as mechanosensitive Ca²⁺-permeable channels in the plasma membrane where Ca²⁺ concentrations can increase up to several hundred micromolar. Several instances have been reported in the literature where BK_Ca channels associate, via a direct protein-protein interaction, with Ca²⁺ entry channels, as in rat brain where BK_Ca channels are linked to L-type voltage-gated Ca²⁺ channels (Berkefeld et al., 2006). In our specific case, we should consider also that BK_Ca channels could be located in specialized membrane patches in the plasma membrane, called lipid rafts, in close proximity with Ca²⁺-release channels of intracellular stores, as reported in glioma cells (Weaver et al., 2007). We did not investigate these aspects of channel coupling and Ca²⁺ sensing in the present study, which we count to address them in a future study, however.

4.3 Does the influx of Ca²⁺ needed for the activation of K_Ca channels goes through Piezo1 channels?

The mechanism of K_Ca channels activation upon cell swelling is poorly understood. A few reports claim that BK_Ca channels can be directly activated by membrane stretch during hypotonic-induced cell swelling (Allard et al., 2000; Christensen, 1987; Mallouk & Allard, 2000; Taniguchi & Guggino, 1989). Conversely, others reported that the BK_Ca channel activation was due to stretch-induced Ca²⁺ influx (Taniguchi & Imai, 1998). This study shows that the opening of BK_Ca channels upon osmotic shock requires cytosolic Ca²⁺ elevation and excludes their activation by increased membrane stretch per se. Rather, the hypotonic-induced activation of BK_Ca channels needs the opening of mechanosensitive channels, which they do respond to the increased membrane tension during cell swelling. Similarly, the activation of IK_Ca channels by the hypotonic stimulus, occurs only in the presence of extracellular Ca²⁺ influx and the resulting cytosolic Ca²⁺ elevation (Vázquez et al., 2001; Wang et al., 2003).

Most mechanosensitive channels are nonselective cation channels with high Ca²⁺ permeability, that convert mechanical force into Ca²⁺ influx. The results presented in Figures 2 and 3 show that the hypotonic-induced cell swelling activates IK_Ca and BK_Ca channels by inducing entry of Ca²⁺ through mechanosensitive channels. The involvement of mechanosensitive channels is suggested by the lack of hypotonic-induced K_Ca activation in presence of their nonselective blocker Gd³⁺. Clearly this pharmacological approach (i.e., the use of nonspecific blocker Gd³⁺) does not allow to identify the specific mechanosensitive channel(s) involved. Nor do their biophysical properties, as were inferred from the hypotonic-induced current recorded in presence of DCPIB, TRAM-34 and paxilline, as they are shared by many types of mechanosensitive channels (i.e., reversal potential at 0 mV, strong outward rectification).

Over the past years, several mechanosensitive channels have been considered as possible sensors of the stretching forces on plasma membrane during cell volume regulation. Members of the transient receptor potential (TRP) subfamilies, especially TRPV4 and TRPV7, have been shown to play important roles either in sensing or transducing mechanical stimuli acting on plasma membrane, but also in regulating cell volume (Arniges et al., 2004; Benfenati et al., 2011; Kim et al., 2017; Lanciotti et al., 2012; Liedtke, 2005; Numata et al., 2021; Toft-Bertelsen et al., 2017). The several discrepancies present in the conclusions reached by the different studies make the identification of the swelling-activated Ca²⁺ permeable channel, which serves as a route for Ca²⁺ influx, still elusive.

In 2010, a small family of evolutionarily conserved channels, encompassing Piezo1 and Piezo2, was identified as the essential component of the mechanically activated Ca²⁺-permeant channels that mediate mechanotransduction in vertebrates (Coste et al., 2010). Both channels are controlled by membrane stretch that results from hypotonic-induced cell volume increase, allowing the permeation of cations including Ca²⁺. Interestingly, it has been recently reported that Piezo1 overexpression correlates with the grade of GBM malignancy (Chen et al., 2018; Qu et al., 2020).

We recently reported that Piezo1 is activated by hypotonic stimuli and controls both cell volume and migration in HEK293 cells (Sforna et al., 2022), suggesting that Piezo1 works as trandrucer of mechanical forces into intracellular signals for volume regulation. These findings and the recognized properties of Piezo1 prompted us to verify if it represents the main mechanosensitive channel, responsible for the activation of IK_Ca and BK_Ca channels upon hypotonic-induced cell swelling in U87-MG cells. To this end we compared the hypotonic-activated current in presence of a cocktail of blockers for all the major known currents (DCPIB, TRAM-34, and paxilline), except for the mechanosensitive current and the current activated by the selective Piezo1 agonist Yoda1. Most interesting, the two currents shared important biophysical properties as voltage rectification and reversal potential, suggesting that they were sustained by the same channels. Unfortunately, other mechanosensitive channels share these properties, so we are not able to clarify the matter conclusively. However, combining the above observations with the result that activation of both IK_Ca and BK_Ca channels by the highly selective Piezo1 activator Yoda1 and the notion that Piezo1 expression is highly correlated with the grade of glioma malignancy (Chen et al., 2018; Qu et al., 2020), it is conceivable to infer that this channel may play a key role in mechanotransduction related to volume regulation in these cells. Dedicated studies using specific genetic approaches, lingering the absence of selective Piezo1 blockers, are needed to elucidate this unsolved issue.

4.4 The role of K_Ca channels in the hypotonic induced RVD

A key result of this study is that both IK_Ca and BK_Ca channels control RVD. Our interpretation is that these channels respond to both global (IK_Ca) and local (BK_Ca) intracellular Ca²⁺ elevation mediated by the opening of mechanosensitive channels (such as Piezo1) upon an increase of lateral membrane tension induced by cell swelling. The resulting K⁺ efflux through K_Ca channels, in conjunction with the Cl⁻ efflux through VRAC, is essential not only for the extrusion of KCl necessary for the obliged loss of water, but also for the maintenance of membrane potential at values between the equilibrium potentials of Cl⁻ (E_Cl = −20 mV) and K⁺ (E_k = −80 mV), which guarantees the driving force for the efflux of both ions throughout the RVD process. However, the reason why GBM cells use two Ca²⁺-activated K⁺ channels, remains unsolved. Although the involvement of K_Ca channels in RVD has been reported by many studies in different cell types, none of these works reported that K_Ca channels are concomitantly required for volume regulation. For instance, some authors showed that RVD is under the control of IK_Ca but not BK_Ca channels (Barfod et al., 2007; Vázquez et al., 2001; Wang et al., 2003). Others reported, instead, a predominant role of the BK_Ca channel (Fernández-Fernández et al., 2002; Lionetto et al., 2010a, 2010b; Sheader et al., 2001b). The discrepancies observed among these studies suggest that regulation of cell volume is a quite complex process and the type of channels involved strongly depend on the type of cell. We plan to investigate this issue in future works.

Another important aspect that emerges from our data and that deserves discussion is that the blockade of both K_Ca channels is not able to fully abolish the RVD process. The lack of a complete inhibition of RVD occurs also when VRAC channels are blocked with DCPIB (Serpe et al., 2022). Why blockade of both K_Ca (and VRAC) channels is not able to fully abolish the RVD process remains an unsolved question for which we can only try to give a plausible explanation. RVD is a quite complex process in which many membrane transporters (besides K_Ca and VRAC channels) participate. An example is the Cl/K cotransporter (KCC) which promotes a nonelectrogenic efflux of K⁺ and Cl⁻ out of the cell whose activity depends on the amount of cell swelling. This transporter has a very important role in the regulation of cell volume in erythrocytes and together with other transporters could explain the residual percentage of RVD when K_Ca and VRAC channels are blocked.

4.5 Conclusion

We have shown that the influx of Ca²⁺ through mechanosensitive channels upon hypotonic-induced cell swelling is an essential step for opening both IK_Ca and BK_Ca channels, whose activation is crucial for the efflux of K⁺ ions and recovery of the original cell volume (i.e., RVD). Although the critical contribution of K⁺ and Cl⁻ channels in cell volume regulation is well established, their role in GBM cell migration and invasion remains a more debated issue (Liu & Stauber, 2019; Rosa et al., 2017, 2018). However, our study performed on an established GBM cell model, could be of relevance for understanding the mechanisms underlying GBM tumor cell invasion through the narrow extracellular space of healthy brain parenchima, where cell volume regulation seems to be a key process.

To this regard we would like to speculate that the same mechanosensitive channel activation/Ca²⁺ influx/K_Ca activation pathway is triggered by more localized mechanical stimuli. This occurrence would greatly help explaining the GBM cells behavior and underlying mechanism they use to modify their shape and steer their movements to avoid obstacles that they can encounter in vivo, and pass through the narrow spaces of the brain parenchyma.

Several open questions remain unanswered, however. One regards the mechanosensitive channel(s) involved in the above pathway. Another is the mechanism through which the entry of Ca²⁺ induced by hypotonicity can activate two channels (IK_Ca and BK_Ca) that exhibit such a marked difference in Ca²⁺ sensitivity. Future studies will be designed to seek answers to these open questions.