Store-operated Ca2+ channels in human glomerular mesangial cells

Rong Ma, Sonja Smith, Angie Child, Pamela K. Carmines, and Steven C. Sansom

Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4575


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experiments were performed to identify the biophysical properties of store-operated Ca2+ channels (SOC) in cultured human glomerular mesangial cells (MC). A fluorometric technique (fura 2) was utilized to monitor the change in intracellular calcium concentration ([Ca2+]i) evoked by elevating external [Ca2+] from 10 nM to 1 mM (Delta [Ca2+]). Under control conditions, Delta [Ca2+] averaged 6 nM and was unaffected by elevating bath [K+]. After treatment with 1 µM thapsigargin to deplete the intracellular Ca2+ store, the change in [Ca2+]i (Delta [Ca2+]th) averaged 147 ± 16 nM. In thapsigargin-treated MC studied under depolarizing conditions (75 mM bath K+), Delta [Ca2+]th was 45 ± 7 nM. The Delta [Ca2+]th response of thapsigargin-treated cells was inhibited by La3+ (IC50 = 335 nM) but was unaffected by 5 µM Cd2+. In patch clamp studies, inward currents were observed in cell-attached patches with either 90 mM Ba2+ or Ca2+ in the pipette and 140 mM KCl in the bathing solution. The single-channel conductance was 2.1 pS with Ba2+ and 0.7 pS with Ca2+. The estimated selectivities were Ca2+ > Ba2+ >> K+. These channels were sensitive to 2 µM La3+, insensitive to 5 µM Cd2+, and voltage independent, with an average channel activity (NPo) of 1.02 at command potential (-Vp) ranging from 0 to -80 mV. In summary, MC exhibited an electrogenic Ca2+ influx pathway that is suggestive of Ca2+ entry through SOC, as well as a small-conductance divalent-selective channel displaying biophysical properties consistent with SOC. Based on estimates of whole cell Ca2+ influx derived from our data, we conclude that SOC with low single-channel conductance must be highly abundant in MC to allow significant capacitative Ca2+ entry in response to depletion of the intracellular store.

cadmium; lanthanum; fura 2 fluorescence; patch clamp; thapsigargin; angiotensin II


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

HUMAN GLOMERULAR MESANGIAL cells (MC) have contractile properties similar to smooth muscle cells of the vasculature and other organs. In MC, angiotensin II (ANG II) evokes an initial inositol trisphosphate (IP3)-mediated release of calcium from intracellular stores and a secondary sustained elevation in intracellular calcium concentration ([Ca2+]i) due to entry of calcium into the cell through ion-selective channels. These ion-selective channels are voltage-gated (VGCC) (9, 28, 38) and have properties consistent with the L-type, dihydropyridine-sensitive, Ca2+-selective channels described in cardiac cells (18) and vascular smooth muscle (37).

In addition to VGCC, recent studies have shown that some smooth muscle cells (7, 26, 27) and MC (23) also possess store-operated calcium channels (SOC). Initially characterized in a variety of nonexcitable cells (1, 15, 19), SOC are activated upon release of calcium from intracellular stores. Elevated [Ca2+]i is rapidly reduced back toward baseline by the Na+/Ca2+ exchanger (21) and Ca2+-ATPase (plasma membrane calcium ATPase, PMCA) in the plasmalemmal membrane and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) (29). Efflux through the plasma membrane transporters yields a net loss of calcium from the cell. SOC activation provides a Ca2+ influx mechanism (capacitative Ca2+ entry) that, in concert with the action of SERCA, replenishes the intracellular Ca2+ store.

Although the store-operated macroscopic current has been studied extensively, the single-channel properties have varied depending on the cells of study. These findings may be the result of differential expression of splice variants or related to the multiple technical difficulties that arise when studying the single-channel properties of SOC. The single-channel currents are very small, and rundown occurs rapidly in excised patches. Moreover, it may be difficult to activate SOC in cell-attached patches given that physical proximity to the endoplasmic reticulum appears critical. Although blockers of SOC have been reported previously (5), a detailed pharmacological profile has not yet been established.

One method to distinguish types of calcium channels in cell membranes is to determine the differential blocking effects of other multivalent cations. Different types of calcium-selective channels are blocked by multivalent cations with varying potencies. For example, VGCC are more sensitive than receptor-operated channels to blockade by Cd2+ (32), whereas receptor-operated Ca2+ channels are more sensitive than VGCC to blockade by La3+. Although high concentrations of these multivalent cations will equally inhibit all Ca2+-selective channels, low concentrations can distinguish the activities of VGCC and SOC in MC.

Previous studies have described with fluorescence microscopy the presence of SOC in MC (23, 24); however, the biophysical properties have not been defined. The present study was performed to describe some of the properties of SOC in cultured MC and determine whether multivalent cations can be used to differentiate SOC from VGCC. Results of experiments using fura 2 to measure [Ca2+]i indicate that thapsigargin depletes intracellular stores of Ca2+ in MC and evokes a Ca2+ cell entry pathway that does not involve VGCC. Single-channel patch clamp experiments provide a description of a Ca2+-selective channel with biophysical properties consistent with SOC. Information regarding the blocking effects of these multivalent cations can be useful for distinguishing the functional roles of these channels during the response to a contractile agonist.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mesangial Cell Cultures

Human MC were subcultured to no more than 10 generations by standard methods previously described (13). Cells were cultured in DMEM supplemented with 10 mM HEPES (pH 7.4), 2.0 mM glutamine, 0.66 U/ml insulin, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, and 20% fetal bovine serum. Upon reaching confluence, cells were studied within 56 h of passage onto 22 × 22-mm cover glasses (Fisher, Pittsburgh, PA).

Fura 2 Measurements of [Ca2+]i

Measurements of [Ca2+]i in MC using fura 2 were performed using dual excitation wavelength fluorescence microscopy as previously described (3, 8). In brief, [Ca2+]i was monitored in individual MC placed in a perfusion chamber (Warner model RC-2OH) mounted on the stage of a Nikon Diaphot model 300 inverted microscope. The cells were illuminated alternately at 340- and 380-nm wavelengths (3-nm bandwidths) with light provided by a Deltascan dual monochromator system (Photon Technology International, Monmouth Junction, NJ). To monitor fluorescence emission (510 nm, 20-nm bandpass) from a single cell, an adjustable optical sampling window was positioned within the light path prior to detection by a photon-counting photomultiplier. Background-corrected data were collected (5 points/s), stored, and processed using the FeliX software package (Photon Technologies). Calibration of the fura 2 signal was performed according to established methods previously described (3, 8). Cells were loaded with fura 2 by incubation for 70-90 min (20°C) in PSS (135 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1 mM CaCl2 and 10 mM HEPES) containing 7 µM of the acetoxymethyl ester of fura 2 (fura 2-AM), 0.09 g/dl DMSO, and 0.018 g/dl Pluronic F-127 (Molecular Probes, Eugene, OR). In all experiments, the initial bathing solution contained (in mM) 135 NaCl, 5 KCl, 10 HEPES, and 1 CaCl2. In some experiments, the free Ca2+ concentration of the bath was adjusted to 1.0 µM by buffering with 1.08 mM EGTA, according to the calcium concentration program by MTK Software. Bath [Ca2+] was reduced to less than 10 nM by addition of EGTA.

Patch Clamp Procedure

Patch clamp experiments were performed with the pipette attached to the membrane (cell attached). The bath solution contained (in mM) 140 KCl, 10 HEPES, and 1 CaCl2. The pipette solution contained either 90 mM CaCl2 or 90 mM BaCl2 plus 10 mM HEPES, pH 7.4.

Single-channel analysis was made at 23°C using standard patch clamp techniques (10, 34). The patch pipette, partially filled with solution, was in contact with a Ag-AgCl wire on a polycarbonate holder connected to the head stage of a patch clamp (model 501A; Warner Instrument, Hamden, CT). The pipettes were lowered onto the cell membrane, and suction was applied to obtain a high-resistance (>5 GOmega ) seal. The unitary current (i), defined as zero for the closed state (C), was determined as the mean of the best-fit Gaussian distribution of the amplitude histograms. Channels were considered to be in an open state (S) when the total current (I) was >(n - 1/2)I and <(n + 1/2)I; n is the maximum number of current levels observed. The open probability (Po) was defined as the time spent in an open state divided by the total time of the analyzed record. When multiple channels occupied a patch, the channel activity was calculated as NPo = Sigma nPn, where Pn is the probability of finding n channels open. The Axoscope acquisition program and pClamp program set 6.02 (Axon Instruments, Foster City, CA) were used to record and analyze currents.

Thapsigargin was obtained from Calbiochem. Differences among groups of data were determined using the one-way ANOVA plus Student-Newman-Keuls test. P < 0.05 was considered statistically significant. Data are reported as means ± SE (n = number of cells).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Fura 2 Experiments

Voltage dependence of thapsigargin-induced Ca2+ entry pathway. Thapsigargin, a potent and selective SERCA inhibitor (35), was utilized in experiments designed to unveil and characterize capacitative Ca2+ influx in MC. The effect of 1 µM thapsigargin on [Ca2+]i is illustrated by the tracing shown in Fig. 1A. Thapsigargin typically evoked a transient increase in [Ca2+]i to a peak value of 250 nM, which subsided to achieve a sustained plateau at 100 nM. Subsequent imposition of a decrease in bath [Ca2+] from 1 mM to <10 nM caused a decline in [Ca2+]i to 35 nM, a situation that promotes depletion of the intracellular store during SERCA inhibition. When bath [Ca2+] was then restored to 1 mM, [Ca2+]i rapidly increased to 110 nM. In contrast, MC not subjected to thapsigargin treatment displayed very small responses to raising bath [Ca2+]. In the typical tracing shown in Fig. 1B, [Ca2+]i only rose from 29 to 33 nM when bath [Ca2+] was increased from <10 nM to 1 mM in the absence of SERCA inhibition. Accordingly, the change in [Ca2+]i evoked by raising bath [Ca2+] from <10 nM to 1 mM in thapsigargin-treated cells was viewed as an indication of Ca2+ influx resulting from depletion of the intracellular store (capacitative Ca2+ entry). In high-K+ (75 mM) solution, thapsigargin evoked an elevation in [Ca2+]i from 50 to 250 nM, a value similar to the response observed in PSS containing 5 mM K+. However, the change in [Ca2+]i caused by thapsigargin (Delta [Ca2+]th) increased by only 40 nM under depolarizing conditions (Fig. 1C). As shown in the summary bar graph of Fig. 1D, Delta [Ca2+]th was significantly greater in both PSS (5 mM K+) and high KCl after intracellular stores were depleted by the application of thapsigargin. Furthermore, after thapsigargin-evoked store depletion, Delta [Ca2+]th was greater in the presence of 5 mM K+ than that observed under depolarizing conditions (75 mM K+). In the absence of thapsigargin, Delta [Ca2+]i was very small and was unaffected by bath [K+]. These data indicate that the thapsigargin-induced Ca2+ entry pathway is affected by cell potential. This pathway is marked by an inward flux of (positive) calcium ions evoked by the thapsigargin depletion of intracellular stores. The Ca2+ entry pathway is not likely VGCC, because the inward flux of Ca2+ was decreased rather than exaggerated by membrane depolarization. Moreover, the major component of this pathway is not Na+/Ca2+ exchange, because in this study Ca2+ entry is elevated with high external Na+.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1.   Fura 2 fluorescence measurements of [Ca2+]i demonstrating the voltage-dependence of thapsigargin-induced capacitative Ca2+ entry pathway. A: Typical MC [Ca2+]i response to thapsigargin and subsequent manipulation of bath [Ca2+] in the presence of 5 mM K+. B: Tracing showing the effect of manipulating bath [Ca2+] on MC studied in the absence of thapsigargin. C: Effect of bath [Ca2+] on thapsigargin-treated MC studied under depolarizing conditions (75 mM bath K+). D: Summary bar graph showing the effects of membrane depolarization on changes in [Ca2+]i evoked by increasing bath [Ca2+] from <10 nM to 1 mM in absence (Delta [Ca2+]) and presence of thapsigargin (Delta [Ca2+])th. * P < 0.05 vs control; ¶ P < 0.05 vs 5 mM K+.

Use of multivalent cations to distinguish Ca2+ influx through VGCC and SOC. Additional experiments probed the ability of multivalent cations to block capacitative Ca2+ entry through SOC. Typical tracings of [Ca2+]i and a dose-dependent curve for the blocking effects of La3+ on Delta [Ca2+]th in thapsigargin-treated MC are presented in Fig. 2. As shown in the [Ca2+]i tracings of Fig. 2A, La3+ dose-dependently reduced Delta [Ca2+]th in thapsigargin-treated MC. The dose-response curve (Fig. 2B) reveals a maximal blocking concentration of La3+ between 2 and 20 µM and an IC50 of 335 nM. Figure 3 shows the effects of 5 µM Cd2+ on capacitative Ca2+ entry. As shown in the tracing of [Ca2+]i (Fig. 3A) and the summary bar graph (Fig. 3B), compared with La3+, Cd2+ was a relatively ineffective blocker of Delta [Ca2+]th in thapsigargin-treated MC.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2.   A: Fura 2 fluorescence measurements of [Ca2+] showing the concentration-dependent effects of 20 nM (upper), 200 nM (middle), and 20 µM (lower) La3+ on the response to raising external [Ca2+] from <10 nM to 1 mM in thapsigargin-treated cells. B: Concentration-response relationship for log [La3+] vs. % inhibition of Delta [Ca2+]th.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 3.   Fura 2 fluorescence measurements of [Ca2+] showing the effects of Cd2+ on capacitative Ca2+ entry into MC. A: [Ca2+]i tracing in a typical experiment. B: Summary plot depicting Delta [Ca2+]th in the absence (control) and presence of 5 µM Cd2+.

It was previously shown that the sustained (plateau) response to ANG II was the result of Ca2+ entering the cell through L-type VGCC (9). Figure 4 shows the ANG II-evoked changes in MC [Ca2+]i observed in the absence and presence of 5 µM Cd2+. In the absence of Cd2+ (Fig. 4A), the sustained elevation of [Ca2+]i evoked by 1 µM ANG II was 50% higher than baseline. In the presence of Cd2+, no ANG II-evoked sustained response was evident (Fig. 4B). Upon flushing of Cd2+ from the ANG II-containing bath, [Ca2+]i increased toward an elevated sustained level. As shown in the summary bar graph (Fig. 4C), 5 µM Cd2+ completely eliminated the sustained [Ca2+]i response evoked by ANG II. Thus 5 µM Cd2+ (which did not affect the thapsigargin-evoked capacitative Ca2+ entry) inhibited the VGCC of MC. It is concluded that the capacitative Ca2+ entry evident in thapsigargin-treated MC reflects activation of only SOC and not VGCC. Moreover, La3+ and Cd2+ at concentrations of 2 µM and 5 µM, respectively, can be used to distinguish these Ca2+ influx pathways.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4.   Measurements of [Ca2+] using fura 2 fluorescence showing the effects of 5 µM Cd2+ on the ANGII-induced [Ca2+]i responses in MC. A: Typical [Ca2+]i response to 1 µM ANGII, characterized by a prominent transient peak which then subsided to a sustained but elevated level. B: In the presence of 5 µM Cd2+, the sustained response to ANG II was eliminated. However, [Ca2+]i increased upon removal of Cd2+ from the ANG II-containing bathing solution. C: Bar graph summary of the effects of 5 µM Cd2+ on the sustained ANGII-induced % Delta [Ca2+]i relative to baseline. * P < 0.05 vs Control (no Cd2+).

Patch Clamp Experiments

Patch clamp experiments were performed to determine whether MC contained Ca2+ channels with properties consistent with the capacitative Ca2+ entry pathway evident in the fura 2 experiments. Figure 5A shows single-channel (inward) current tracings of a cell-attached patch at various holding potentials. These channels were in groups of at least three per patch and were spontaneously active in cell-attached patches with either Ca2+ or Ba2+ as the charge carrier.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5.   A: Single channel (inward) tracings of Ba2+ currents at various holding potentials and Ca2+ current at -Vp = -60 mV in cell-attached patches. (Outward currents were too small to resolve.) The pipette contained either 90 mM BaCl2 or 90 mM CaCl2. The bath contained 140 mM KCl. B: Current-voltage relationship for Ca2+ and Ba2+ currents. A total of 7 cells are represented by the calcium I-V plot (open circles) and 9 cells are represented by the barium I-V plot (closed circles). Each point represents current measurements from 3 to 8 patches at the given potential. The slope conductance was 0.7 pS for Ca2+ and 2.1 pS for Ba2+, and the extrapolated reversal potentials of 123 mV and 63 mV were consistent with Ca2+/K+ and Ba2+/K+ selectivities of 87 and 8.2, respectively. C: Effects of command potential (-Vp) on NPo. There were no significant differences in NPo between potentials ranging from 0 to -80 mV when using either Ca2+ or Ba2+ as the charge carrier.

Only inward currents were observed at command potentials (-Vp) ranging from 0 to -80 mV. The current-voltage relations (Fig. 5B) revealed a 0.7-pS channel with calcium as the charge carrier and a 2.1-pS calcium channel with barium as the charge carrier. The selectivity values of 87 and 8.2 for Ca2+/K+ and Ba2+/K+ were estimated by the Goldman-Hodgkin-Katz equation, using reversal potentials of 123 mV (Ca2+) and 63 mV (Ba2+), and assuming [K+]i = 120 mM (14). The membrane potential was near zero with 140 mM KCl in the bathing solution. As shown in Fig. 5C, the NPo of these channels did not change significantly when varying the holding potential.

The differential blocking effects of La3+ and Cd2+ on the calcium channel are presented in Fig. 6. As shown in the tracings of Fig. 6A, addition of 5 µM Cd2+ to the pipette solution containing 90 mM BaCl2 (-Vp = -60 mV) did not affect NPo of the calcium channels. However, when La3+ at concentrations of 200 nM and 2 µM was added to the pipette solution, channel activity diminished to 0.40 and 0.16, respectively. As shown in the summary bar graph (Fig. 6B), there was no significant effect of Cd2+ on NPo [5 µM Cd2+, 0.94 ± 0.16 (n = 7) vs. control, 1.01 ± 0.06 (n = 13)]. However, 200 nM La3+ decreased NPo to a value (0.4 ± 0.06, n = 5) significantly lower than control, and 2 µM La3+ decreased NPo to a value (0.16 ± 0.06, n = 5) significantly lower than control and 200 nM La3+. The degree of inhibition of the putative SOC in the patch clamp experiments was 42% with 200 nM La3+ and 82% with 2 µM La3+. These values were close to the percentage inhibition of SOC by these concentrations of La3+ as determined by the fura 2 experiments (38 ± 14% with 200 nM La3+ and 67 ± 10% with 2 µM La3+, see Fig. 2B).


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 6.   A: Tracings demonstrating effects of 5 µM Cd2+, 200 nM La3+ and 2 µM La3+ on Ba2+ currents at -Vp = -60 mV. B: Summary plots demonstrating the effects of 5 µM Cd2+, 200 nM La3+ and 2 µM La3+ on the NPo of barium currents.

Thus the voltage independence and the sensitivity to block by La3+ > Cd2+ was distinct from VGCC and was consistent with the properties described for capacitative Ca2+ entry through SOC in the fura 2 experiments. Moreover, the similarities in the sensitivities to block by La3+ strongly suggest that the calcium channel in the patch clamp experiments is the same SOC described for capacitative Ca2+ entry in the fura 2 experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results of this study revealed that MC in culture possess a Ca2+ cell entry pathway activated upon release of intracellular stores and reduced by depolarizing potentials. The properties of this pathway are consistent with the capacitative Ca2+ entry pathway through SOC previously described in several types of nonexcitable cells (30). The relative La3+ > Cd2+ sensitivity, low conductance, absence of voltage gating, and relative selectivity of Ca2+ > Ba2+ observed in MC using patch clamp methods is consistent with previously described properties of SOC in a variety of nonexcitable cells (2, 11) and distinct from the VGCC recently described in these cells (9).

Differentiating Ca2+ Influx Pathways with La3+ and Cd2+

The differential sensitivities of Ca2+ channels to La3+ and Cd2+ can be used to determine the roles of Ca2+ channels in cells. For example, although 5 µM Cd2+ did not inhibit SOC in the present study, it was previously shown that <10 µM Cd2+ is an effective inhibitor of L-type VGCC (25). We therefore determined whether 5 µM Cd2+ could inhibit VGCC of MC. Electrophysiological and fura 2 evidence has shown that MC contain L-type (dihydropyridine-sensitive) VGCC that are activated by depolarizing potentials and ANG II (9, 28, 38). As described by results from patch clamp studies, these channels are 11 pS with barium as the charge carrier and are steeply voltage dependent between potentials of 0 and -30 mV (9). When [Ca]i is monitored with fura 2 fluorescence, ANG II causes an initial peak response of Ca2+ and a secondary sustained elevation in [Ca2+]i. Because the sustained response was sensitive to diltiazem, it was concluded that this pathway was the result of activating VGCC (9). Our observation that 5 µM Cd2+ inhibits the sustained response to ANG II in MC is consistent with previous findings that <10 µM Cd2+ inhibits L-type VGCC (25). However, since ANG II depletes internal stores of Ca2+, it is likely that SOC is also activated and is partially responsible for the sustained response.

The failure of 5 µM Cd2+ to block the thapsigargin-evoked Ca2+ influx pathway in MC is consistent with the notion that VGCC does not have a role in replenishing calcium stores. Moreover, the lack of effect of 5 µM Cd2+ is consistent with previous findings by Hoth and Penner (12) that the IC50 for block of SOC by Cd2+ is 240 µM. Lanthanum, on the other hand, is a more powerful blocker of SOC than VGCC (12). The differential sensitivities of VGCC and SOC to these multivalent cations allows pharmacological assessment of capacitative Ca2+ entry through SOC.

Single-Channel Properties of SOC

Although several studies using fura 2 fluorescence have indicated the presence of SOC in a variety of cells (2), patch clamp studies have yielded disparate descriptions of the single-channel properties. There are several reasons for the difficulties in determining the properties of SOC. First, the single-channel conductance of SOC is relatively small and difficult to resolve with patch clamp analysis. Second, in excised patches, channel activity runs down within ~5 s (20). Third, the coupling mechanism for sensing store depletion by SOC is likely spatially restricted (2, 30) and disrupted upon obtaining a cell-attached patch in which the cell membrane is pulled into the patch pipette (33). Therefore, regulatory properties of SOC determined with whole-cell methods of analysis may not coincide with results from single-channel analysis.

Earlier experiments utilized fluctuation analysis to estimate that the single-channel conductance of SOC is only 24 fS in 100 mM extracellular Ca2+ (39). However, the unitary conductance of SOC in A431 cells was measured at 2 pS with 200 mM CaCl2 and 10 pS with 80 mM BaCl2 (20). A more recent study determined that the conductance of SOC in Jurkat T lymphocytes was 36-40 pS when all Ca2+ was removed and Na+ was used as a charge carrier (15). On the basis of macroscopic Ca2+ current and the ratio of Na+/Ca2+ current, the single-channel current has been estimated to be as high as 1.6 pS with 20 mM Ca2+ as the charge carrier. Therefore, the single-channel conductances evident in MC (0.7 pS with Ca2+ as the charge carrier and 2.1 pS with Ba2+ as the charge carrier) are within the range of measurements obtained for SOC in A431 cells and Jurkat T lymphocytes.

The relative selectivity of Ca2+/K+ for the putative SOC in MC was estimated at 87. This value is based on the assumption that the membrane potential was close to zero in cell-attached patches and that [K+]i was 120 mM, as previously measured in rat MC (14). A high selectivity for Ca2+/K+ is consistent with the view that the SOC is highly selective for divalent over monovalent cations (12). Although the relative Ca2+/Ba2+ selectivity was not determined directly, the relative Ca2+/K+ selectivity of 87 was much larger than Ba2+/K+ (estimated at 8.2). The higher selectivity for Ca2+ relative to Ba2+ is unique to SOC and was described earlier with whole cell current measurements for SOC of mast cells (11) and lymphocytes (39). Despite the finding that the single-channel conductance is larger when Ba2+ is the charge carrier, the putative SOC in MC is more selective for Ca2+. Similarly, SOC of lymphocytes are very conductive for Na+ ions but only when divalent cations are removed from the external solution (15). Thus divalent cations have more affinity for the SOC pore but permeate more slowly.

While several studies have provided estimates for the single-channel conductance and selectivity of SOC, there is less information regarding the multivalent blocking properties of SOC. In the present study, 2 µM La3+ blocked completely the activity of the barium-selective current. However, 5 µM Cd2+ was an ineffective blocker of the putative SOC. The relative blocking order of La3+ > Cd2+ in the patch and fura 2 experiments is consistent with other studies that have shown a greater La3+ compared with Cd2+ micromolar block of SOC (12, 32). Although a complete dose response for La3+ block of the putative SOC in the patch clamp experiments was not determined, the partial block by 200 nM and the complete block by 2 µM coincides well with the fura 2 experiments, which indicated an IC50 of ~335 nM for the block of SOC by La3+. This is strong evidence that the putative SOC is the same pathway for the capacitative entry of Ca2+ detected in the fura 2 experiments.

Previous observations have demonstrated that the SOC is not voltage gated (11). However, when membrane potential was depolarized with high K+ in the present study, thapsigargin-evoked Ca2+ entry was significantly decreased. Considering that membrane voltage (Vm) could be depolarized by as much as 70 mV when changing from 5 mM to 75 mM K+, the decreased capacitative Ca2+ entry could be the result of decreasing the driving force for Ca2+ entry through SOC. Interestingly, a previous study found that membrane depolarization by high K+ or blockers of K+ channels inhibit the rise in [Ca2+]i through SOC evoked by T cell mitogens (17). Moreover, membrane hyperpolarization by the potassium ionophore valinomycin reverses the effects of K+ channel blockers (31). These results show that membrane potential plays a role in maintaining the driving force for sustained Ca2+ entry during store depletion. Maintenance of a hyperpolarizing membrane potential would not be critical for Ca2+ entry into the cell during store depletion, because the chemical gradient would still be very large.

The genetic equivalent of SOC, like the channels in native cells, has been difficult to assess in expression systems. Several studies have suggested that transient receptor protein (trp) of Drosophila photoreceptors is the SOC channel (2, 36). Expressed in Sf9 cells, trp was activated by thapsigargin (36). However, in HEK-293 cells, Htrp3 was expressed as 17-pS and 66-pS channels that were activated by depolarizing potentials and were nonselective to monovalent and divalent cations (16). Therefore, because many of the single-channel properties of expressed trp are different from those of SOC in native cells, the genetic equivalent of SOC remains uncertain(6).

Ca2+ Channels in MC

At least three other Ca2+ channels have been described in either rat or human MC in culture. The L-type VGCC (11 pS) was recently described in MC (9). Chen et al. (4) described a 21-pS Ca2+-permeable and mechanosensitive channel in rat MC, and Matsunaga et al. (22) found a 1-pS receptor-operated Ca2+ channel in rat MC. Although the single-channel conductance of 1 pS was close to that of the putative SOC, the divalent/monovalent selectivity for the receptor-operated channels was close to unity and not likely responsible for the highly Ca2+-selective currents evident in the present study. The putative SOC of the present study is therefore distinct from these previously described channels.

On the basis of estimated surface area and an average of three channels per patch, the total number of channels present in a single MC is ~86,000. This value can be compared with an estimate based on the average rate of increase in [Ca2+]i (2.4 nM/s) upon elevating [Ca2+]i from <10 nM to 1 mM in the thapsigargin-treated cells. Using an estimated cell volume, the equivalent Ca2+ current that would account for such a rise in [Ca2+]i would be ~205 pA. If the channels were maximally activated to an average Po of 0.15 and the channel amplitude were 0.3 pA (estimated for 1 mM Ca2+ at a membrane potential of -40 mV), then the number of channels per cell would be approximated at 47,000. This is a minimum approximation because Ca2+ that enters the cell is immediately buffered, thereby blunting the measured rise in [Ca2+]i. These considerations suggest that the putative low-conductance SOC must be highly abundant in MC to provide the capacitative Ca2+ entry evident upon depletion.

In summary, we have described Ca2+ entry pathways using fura 2 and patch clamp methods of analysis. The fura 2 experiments revealed a capacitative Ca2+ entry pathway activated by thapsigargin-evoked depletion of intracellular stores and sensitive to blockade by La3+ over Cd2+. The patch clamp experiments disclosed a La3+-sensitive, Ca2+-selective current with voltage characteristics consistent with the SOC. Both measurements suggest that a typical mesangial cell contains at least 50,000 low-conductance SOC. Therefore, human glomerular MC, contractile cells previously shown to contain VGCC, contain an abundance of SOC, which likely function to replenish intracellular calcium after IP3-mediated release from the endoplasmic reticulum.


    ACKNOWLEDGEMENTS

We are grateful to Hanna Abboud of the University of Texas Health Science Center at San Antonio for providing us with cultures of human mesangial cells.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-49561 (to S. C. Sansom). R. Ma was supported by a fellowship grant from the American Heart Association (Heartland Affiliate).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. C. Sansom, Dept. of Physiology and Biophysics, Univ. of Nebraska Medical Center, 984575 Nebraska Medical Center, Omaha, NE 68198-4575 (E-mail: ssansom{at}unmc.edu).

Received 23 September 1999; accepted in final form 17 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Barritt, GJ. Receptor-activated Ca2+ inflow in animal cells: a variety of pathways tailored to meet different intracellular Ca2+ signalling requirements. Biochem J 337: 153-169, 1999[ISI][Medline].

2.   Berridge, MJ. Capacitative calcium entry. Biochem J 312: 1-11, 1995[ISI][Medline].

3.   Carmines, PK, Fowler BC, and Bell PD. Segmentally distinct effects of depolarization on intracellular [Ca2+] in renal arterioles. Am J Physiol Renal Fluid Electrolyte Physiol 265: F677-F685, 1993[Abstract/Free Full Text].

4.   Chen, V, Guber HA, and Palant CE. Mechanosensitive single channel calcium currents in rat mesangial cells. Biochem Biophys Res Commun 203: 773-779, 1994[ISI][Medline].

5.   Cho, JH, Balasubramanyam N, Chernaya G, Gardner JP, Aviv A, Reeves JP, and Dargis PG. Oligomycin inhibits store-operated channels by a mechanism independent of its effects on mitochondrial ATP. Biochem J 324: 971-980, 1999.

6.   Clapham, DE. TRP is cracked but is CRAC TRP? Neuron 16: 1069-1072, 1996[ISI][Medline].

7.   Gibson, A, McFadzean I, Wallace P, and Wayman CP. Capacitative Ca2+ entry and the regulation of smooth muscle tone. Trends Pharmacol Sci 19: 266-269, 1998[ISI][Medline].

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

9.   Hall, D, Carmines PK, and Sansom SC. Dihydropyridine-sensitive Ca2+ channels in human glomerular mesangial cells. Am J Physiol Renal Physiol 278: F97-F103, 2000[Abstract/Free Full Text].

10.   Hamill, OP, Marty A, Neher E, Sackmann B, and Sigworth FJ. Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391: 85-100, 1981[ISI][Medline].

11.   Hoth, M, and Penner R. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature 355: 353-356, 1992[ISI][Medline].

12.   Hoth, M, and Penner R. Calcium release-activated calcium current in rat mast cells. J Physiol (Lond) 465: 359-386, 1993[Abstract].

13.   Jaffer, F, Saunders C, Shultz P, Throckmorton D, Weinshell E, and Abboud HE. Regulation of mesangial cell growth by polypeptide mitogens. Am J Pathol 135: 261-269, 1989[Abstract].

14.   Kasner, SE, and Ganz MB. Regulation of intracellular potassium in mesangial cells: a fluorescence analysis using the dye, PBFI. Am J Physiol Renal Fluid Electrolyte Physiol 262: F462-F467, 1992[Abstract/Free Full Text].

15.   Kerschbaum, HH, and Cahalan MD. Single-channel recording of a store-operated Ca2+ channel in Jurkat T lymphocytes. Science 283: 836-839, 1999[Abstract/Free Full Text].

16.   Kiselyov, K, Xu X, Mozhayeva G, Kuo T, Pessah I, Mignery G, Zhu X, Birnbaumer L, and Muallem S. Functional interaction between InsP3 receptors and store-operated Htrp channels. Nature 396: 478-482, 1998[ISI][Medline].

17.   Lewis, RS, and Cahalan MD. Potassium and calcium channels in lymphocytes. Annu Rev Immunol 13: 623-653, 1995[ISI][Medline].

18.   Ling, BN, Seal EE, and Eaton DC. Regulation of mesangial cell ion channels by insulin and angiotensin II. Possible role in diabetic glomerular hyperfiltration. J Clin Invest 92: 2141-2151, 1993[ISI][Medline].

19.   Liu, XB, Rojas E, and Ambudkar IS. Regulation of KCa current by store-operated Ca2+ influx depends on internal Ca2+ release in HSG cells. Am J Physiol Cell Physiol 275: C571-C580, 1998[Abstract/Free Full Text].

20.   Luckhoff, A, and Clapham DE. Calcium channels activated by depletion of internal calcium stores in A431 cells. Biophys J 67: 177-182, 1994[Abstract].

21.   Mashburn, NA, Unlap MT, Runquist J, Alderman A, Johnson GVW, and Bell PD. Altered protein kinase C activation of Na+/Ca2+ exchange in mesangial cells from salt-sensitive rats. Am J Physiol Renal Physiol 276: F574-F580, 1999[Abstract/Free Full Text].

22.   Matsunaga, H, Ling BN, and Eaton DC. Ca2+-permeable channel associated with platelet-derived growth factor receptor in mesangial cells. Am J Physiol Cell Physiol 267: C456-C465, 1994[Abstract/Free Full Text].

23.   Mené, P, Teti A, Pugliese F, and Cinotti GA. Calcium release-activated calcium influx in cultured human mesangial cells. Kidney Int 46: 122-128, 1994[ISI][Medline].

24.   Mene, P, Pugliese F, and Cinotti GA. Regulation of capacitative calcium influx in cultured human mesangial cells: roles of protein kinase C and calmodulin. J Am Soc Nephrol 7: 983-990, 1996[Abstract].

25.   Miller, RJ. Multiple calcium channels and neuronal function. Science 235: 46-52, 1987[ISI][Medline].

26.   Missiaen, L, De Smedt H, Droogmans G, Himpens B, and Casteels R. Calcium ion homeostasis in smooth muscle. Pharmacol Ther 56: 191-231, 1992[ISI][Medline].

27.   Morel, JL, Macrez-Leprêtre N, and Mironneau J. Angiotensin II-activated Ca2+ entry-induced release of Ca2+ from intracellular stores in rat portal vein myocytes. Br J Pharmacol 118: 73-78, 1996[Abstract].

28.   Orth, SR, Nobiling R, Bönisch S, and Ritz E. Inhibitory effect of calcium channel blockers on human mesangial cell growth: evidence for actions independent of L-type Ca2+ channels. Kidney Int 49: 868-879, 1996[ISI][Medline].

29.   Parekh, AB, and Penner R. Store depletion and calcium influx. Physiol Rev 77: 901-930, 1997[Abstract/Free Full Text].

30.   Putney, JW, Jr, and McKay RR. Capacitative calcium entry channels. Bioessays 21: 38-46, 1999[ISI][Medline].

31.   Randriamampita, C, Bismuth G, Debre P, and Trautmann A. Nitrendipine-induced inhibition of calcium influx in a human T-cell clone: role of cell depolarization. Cell Calcium 12: 313-323, 1991[ISI][Medline].

32.   Ruegg, UT, Wallnofer A, Weir S, and Cauvin C. Receptor-operated calcium-permeable channels in vascular smooth muscle. J Cardiovasc Pharmacol 14: S49-S58, 1989[ISI][Medline].

33.   Sokabe, M, Sachs F, and Jing Z. Quantitative video microscopy of patch clamped membranes stress, strain, capacitance, and stretch channel activation. Biophys J 59: 722-728, 1991[Abstract].

34.   Stockand, JD, and Sansom SC. Large Ca2+-activated K+ channels responsive to angiotensin II in cultured human mesangial cells. Am J Physiol Cell Physiol 267: C1080-C1086, 1994[Abstract/Free Full Text].

35.   Thastrup, O, Cullen PJ, Drobak BK, Hanley MR, and Dawson AP. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Natl Acad Sci USA 87: 2466-2470, 1990[Abstract].

36.   Vaca, L, Sinkins WG, Hu Y, Kunze DL, and Schilling WP. Activation of recombinant trp by thapsigargin in Sf9 cells. Am J Physiol Cell Physiol 267: C1501-C1505, 1994[Abstract/Free Full Text].

37.   Xiong, Z, Sperelakis N, and Fenoglio-Preiser C. Regulation of L-type calcium channels by cyclic nucleotides and phosphorylation in smooth muscle cells from rabbit portal vein. J Vasc Res 31: 271-279, 1994[ISI][Medline].

38.   Yu, YM, Lermioglu F, and Hassid A. Modulation of Ca by agents affecting voltage-sensitive Ca channels in mesangial cells. Am J Physiol Renal Fluid Electrolyte Physiol 257: F1094-F1099, 1989[Abstract/Free Full Text].

39.   Zweifach, A, and Lewis RS. Mitogen-regulated Ca2+ current of T lymphocytes is activated by depletion of intracellular Ca2+ stores. Proc Natl Acad Sci USA 90: 6295-6299, 1993[Abstract].


Am J Physiol Renal Fluid Electrolyte Physiol 278(6):F954-F961
0363-6127/00 $5.00 Copyright © 2000 the American Physiological Society