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Ca²⁺ modulation of volume-regulated anion channels: evidence for colocalization with store-operated channels ¹

Laboratoire de Physiologie Cellulaire, INSERM EPI 9938, Bâtiment SN3, USTL, 59655 Villeneuve d’Ascq, France

⁴Correspondence: Laboratoire de Physiologie Cellulaire, INSERM EPI 9938, Bâtiment SN3, Université des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq, France. E-mail: phycel{at}univ-lille1.fr

SPECIFIC AIM

The aim of this study was to explore the regulation of the Cl^- current activated by hypotonic cell swelling (I_Cl,swell) in the prostate cancer epithelial cells (LNCaP) by the most universal second messenger, Ca²⁺. We addressed the hypothesis about a possible functional link between plasma membrane volume-regulated anion channels (VRACs) that carry I_Cl,swell and the store-operated Ca²⁺ channels (SOCs).

PRINCIPAL FINDINGS

1. I_Cl,swell in human prostate cancer epithelial cells is not sensitive to the changes in global intra- or extracellular Ca²⁺
Under whole-cell patch clamp recording the control I_Cl,swell was evoked by cell exposure to the hypotonic solution containing standard extracellular Ca²⁺ concentration ([Ca²⁺]_out) of 2 mM and TEA as a major cation to block K⁺ currents (the so called 2/Ca Hypo-TEA solution) with the free Ca²⁺ concentration in the intracellular pipette solution ([Ca²⁺]_in) adjusted to 10^-8 M. Variations of [Ca²⁺]_out from 0 mM to 10 mM or increase of [Ca²⁺]_in < 1 µM did not noticeably affect either the temporary parameters of I_Cl,swell activation in response to hypotonicity or the size and rectification properties of I_Cl,swell compared with the control, allowing one to conclude that changes in global intra- or extracellular Ca²⁺ are not able to modulate I_Cl,swell in LNCaP cells.

Ca²⁺ measurements of Fura-2AM-loaded cells have shown that within the time span sufficient for I_Cl,swell activation, [Ca²⁺]_in stayed basically constant, suggesting that LNCaP cell swelling and concomitant I_Cl,swell activation do not interfere with intracellular Ca²⁺ homeostasis.

2. Thapsigargin confers I_Cl,swell sensitivity to regulation by extracellular Ca²⁺
We have recently shown that by using thapsigargin (TG), an inhibitor of the SERCA Ca²⁺ pump of endoplasmic reticulum (ER), we can raise intracellular Ca²⁺ in LNCaP cells by two physiologically relevant means: release from intracellular stores and entry via plasma membrane store-operated channels (SOCs). Therefore, our next series of experiments examined whether influencing intracellular Ca²⁺ homeostasis with TG could alter Ca²⁺ sensitivity of I_Cl,swell.

In the presence of TG, 10/Ca Hypo-TEA failed to evoke statistically significant elevation I_Cl,swell within < 8 min of exposure. However, complete removal of external Ca²⁺ in the continuing presence of TG (i.e., switching to TG-supplemented 0/Ca Hypo-TEA) elicited the development of typical I_Cl,swell. Depending on the extracellular Ca²⁺ content, TG inclusion also differentially affected the fully developed I_Cl,swell: if in the absence of Ca²⁺ inclusion of TG produced almost no change of I_Cl,swell, then in the presence of 10 mM Ca²⁺ it resulted in a dramatic I_Cl,swell inhibition. Lower concentrations of extracellular Ca²⁺ in the presence of TG produced less I_Cl,swell inhibition. In the presence of TG, the degree of inhibition at V_m = +50 mV increased from 37 ± 6% (n=4) to 50.5 ± 6.5% (n=7) in response to an elevation in [Ca²⁺]_out from 2 to 10 mM.

Our results unequivocally demonstrate that TG confers VRAC sensitivity to inhibition by extracellular Ca²⁺. The ineffectiveness of TG itself under 0 [Ca²⁺]_out argues against its possible direct action on VRAC; rather, it suggests the involvement of TG-induced changes in intracellular Ca²⁺ homeostasis.

3. Ca²⁺ entering via SOCs is primarily responsible for the inhibition of VRAC
Since TG confers sensitivity of VRAC to the extracellular Ca²⁺ and this sensitivity becomes [Ca²⁺]_out dependent, but at 0 mM [Ca²⁺]_out TG produces no effects, we hypothesized that Ca²⁺ entering from the extracellular space via activated SOCs is a primary player in VRAC regulation.

To test this hypothesis, we used Ni²⁺, a blocker of store depletion-activated Ca²⁺ entry in LNCaP cells. In contrast to what has been observed in 10/Ca Hypo-TEA with TG alone, inclusion of 2 mM Ni²⁺ completely prevented the downward trend of I_Cl,swell (data not shown), consistent with the idea that Ca²⁺ entering via SOCs is primarily responsible for the inhibition of VRAC. Moreover, in agreement with inward rectification of Ca²⁺ current carried through the SOCs, TG-conferred inhibition of I_Cl,swell was stronger at more negative holding potentials at which Ca²⁺ entry is enhanced (data not shown).

We also performed experiments using the Ca²⁺ ionophore ionomycin (IM), which affects Ca²⁺ homeostasis similar to TG. Again, inclusion of 1 µM IM in the 10/Ca Hypo-TEA solution caused I_Cl,swell inhibition but its addition to the 0/Ca Hypo-TEA was without effect (data not shown).

The bar graph of Fig. 1 A summarizes the effects of major interventions used to manipulate Ca²⁺ homeostasis on the density of I_Cl,swell in LNCaP cells. These results strongly suggest that Ca²⁺ entering the cell via plasma membrane SOCs is the most crucial for VRAC modulation.

View larger version (41K): [in this window] [in a new window]   Figure 1. Ca2+-dependent regulation of ICl,swell and RVD process in LNCaP cells. A) Summary of the effects (mean±SE, n=4–16) of major interventions used to manipulate Ca2+ homeostasis (indicated under each bar) on ICl,swell density at + 50 mV (upward bars) and -50 mV (downward bars). B) RVD process in LNCaP cells exposed to TG-supplemented (0.1 µM) 0/Ca (gray bars) and 2/Ca (light gray bars) Hypo-TEA solutions; inset graph shows RVD under control conditions (exposure to 2/Ca Hypo-TEA, gray bars) and in the presence of Cl- channel blocker NPPB (100 µM) (light gray bars).

4. Inhibition of I_Cl,swell by Ca²⁺ entry via SOCs significantly alters the RVD process
Having demonstrated that interventions that lead to the activation of SOCs confer Ca²⁺ sensitivity on VRAC, we wondered whether these interventions were also able to alter the RVD process, which is determined largely by activation of VRAC-mediated I_Cl,swell. To test this, we measured relative changes in cell volume different times after exposure to 0.1 µM TG-supplemented 0/Ca or 2/Ca Hypo-TEA solutions. Measurements used a flow cytometer, which allows a volume estimate of at least 5000 cells at a time. Exposure of the cells to 0/Ca Hypo-TEA + TG evoked a rapid increase in relative cell volume < 30%, followed by a slow decline due to development of I_Cl,swell-mediated RVD, which resulted in complete cell volume relaxation in 15 min (Fig. 1B , gray bars). The overall behavior of cell volume in this group of cells was not much different from the control group, which was treated with regular 2/Ca Hypo-TEA in the absence of any TG (Fig. 1B , inset, gray bars), suggesting that TG per se does not interfere with the normal course of RVD. In sharp contrast, inclusion of TG in 2/Ca Hypo-TEA resulted in a somewhat higher increase of maximal cell volume and, most important, in a significant slowdown of the volume relaxation phase, such that after 15 min it decreased from a peak value of 37% to only 26% (Fig. 1B , light gray bars). Such a dramatic difference with 0/Ca conditions indicates that only when extracellular Ca²⁺ is present is TG able to impair the RVD process, consistent with TG-conferred down-regulation of I_Cl,swell by extracellular Ca²⁺. Inhibition of RVD by TG was similar to that observed in the presence of the Cl^- channel blocker NPPB (100 µM) (Fig. 1B , inset, light gray bars), suggesting that both interventions target Cl^- efflux via VRACs.

CONCLUSIONS AND SIGNIFICANCE

In many cell types, osmotic cell swelling has been shown to be associated with an increase in [Ca²⁺]_in with cell-specific contributions from a variety of sources. Therefore, if intracellular Ca²⁺ had an effect on VRACs activity, this would affect the RVD process. However, in contrast to the clearly established Ca²⁺ dependence of K⁺ channels involved in RVD, activation of VRACs in most cell types was independent of intracellular Ca²⁺.

In experiments with prostate cancer epithelial cells, we were unable to demonstrate any significant influence of global [Ca²⁺]_in and [Ca²⁺]_out on the characteristics of hypotonicity-evoked I_Cl,swell. However, under conditions that allow Ca²⁺ influx via plasma membrane SOCs, the sensitivity of VRAC to extracellular Ca²⁺ became obvious. Our results suggest that Ca²⁺ entering the cell via SOCs has preferred access to VRACs and imply close colocalization of these two channel types in the plasma membrane. Figure 2 demonstrates an OC-Ca²⁺–VRAC interaction hypothesis consistent with our data.

View larger version (40K): [in this window] [in a new window]   Figure 2. Simplified schematic diagram of the SOC-Ca2+–VRAC interaction hypothesis. Under normal conditions (left panel), the activity of the SERCA pump in counteracting Ca2+ leak maintains optimal filling of the ER Ca2+ store, keeping the plasma membrane SOCs closed; the cell swelling stimulus is able to trigger a baseline VRAC-carried ICl,swell. Emptying the ER Ca2+ store via the leak channels after blockade of the SERCA pump with TG (right panel) activates SOCs, and Ca2+ ions entering through them from the extracellular space interact with closely colocalized VRAC to inhibit its function. The SOC-Ca2+–VRAC interaction occurs in the microdomain from the inner surface of the membrane, which is not accessible to the changes in global [Ca2+]in. The bottom panel illustrates typical time course of TG-conferred ICl,swell inhibition by extracellular Ca2+.

Ca²⁺ is a universal intracellular messenger regulating many processes. Therefore, spatial compartmentalization of the structures subjected to Ca²⁺ regulation is a prerequisite for the specificity of its action. The physiological significance of an inverse correlation between the Ca²⁺ influx and I_Cl,swell as well as of underlying colocalization of SOCs and VRACs in prostate cancer epithelial cells is not yet clear. As shown here, depletion of intracellular Ca²⁺ stores and concomitant activation of SOCs by TG strongly impair the normal course of RVD process. One can assume that in the presence of any hormonal stimuli acting through cell surface receptors and resulting in liberation of Ca²⁺ and store depletion, the VRAC-mediated RVD process would be inhibited as well. We have demonstrated that emptying of intracellular stores and not the rise in [Ca²⁺]_in is the primary reason for apoptotic LNCaP cells death in a TG-induced apoptosis model. Emptying of stores during progression to apoptosis would activate Ca²⁺ entry via SOCs, increase cytosolic Ca²⁺, and down-regulate VRACs. Concomitant inhibition of a VRAC-mediated RVD process would lead to enhanced vesiculation and formation of apoptotic blebs in response to any osmotic perturbations and, together with increased cytosolic Ca², would result in accelerated apoptotic cell death.

It has recently been shown that expression of a highly Ca²⁺-permeable CaT1 channel in prostate carcinoma cells (including LNCaP cells) correlates with the tumor grade of prostate cancers; with functional properties similar to the native SOCs, this channel is a likely candidate for mediating store-operated Ca²⁺ influx. Thus, a functional interaction between SOC-mediated Ca²⁺ entry, tumor growth, and activity of VRACs may provide novel targets for therapy and diagnostic of prostate cancers.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0383fje; to cite this article, use FASEB J. (December 14, 2001) 10.1096/fj.01-0383fje

² Permanent address: Bogomoletz Institute of Physiology, Bogomoletz Str., 4, 01024 Kiev-24, Ukraine.

³ Permanent address: KU Leuven, Laboratorium voor Fysiologie, B-3000 Leuven, Belgium

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