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Am J Physiol Renal Physiol 294: F1279-F1286, 2008. First published April 16, 2008; doi:10.1152/ajprenal.00348.2007
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TRANSLATIONAL PHYSIOLOGY

Heterologous expression of polycystin-1 inhibits endoplasmic reticulum calcium leak in stably transfected MDCK cells

Kimberly H. Weber,1 Eun Kyung Lee,1 Uma Basavanna,1 Sabina Lindley,1 Roy C. Ziegelstein,2 Gregory G. Germino,1 and Michael Sutters1

Departments of 1Nephrology and 2Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland

Submitted 25 July 2007 ; accepted in final form 5 April 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We previously found that polycystin-1 accelerated the decay of ligand-activated cytoplasmic calcium transients through enhanced reuptake of calcium into the endoplasmic reticulum (ER; Hooper KM, Boletta A, Germino GG, Hu Q, Ziegelstein RC, Sutters M. Am J Physiol Renal Physiol 289: F521–F530, 2005). Calcium flux across the ER membrane is determined by the balance of active uptake and passive leak. In the present study, we show that polycystin-1 inhibited calcium leak across the ER membrane, an effect that would explain the capacity of this protein to accelerate clearance of calcium from the cytoplasm following a calcium release response. Calcium leak was detected by measurement of the accumulation of calcium in the cytoplasm following treatment with thapsigargin. Heterologous polycystin-1, stably expressed in Madin-Darby canine kidney cells, attenuated the thapsigargin-induced calcium peak with no effect on basal calcium stores, mitochondrial calcium uptake, or extrusion of calcium across the plasma membrane. The capacity of polycystin-1 to limit the rate of decay of ER luminal calcium following inhibition of the pump was shown indirectly using the calcium ionophore ionomycin, and directly by loading the ER with a low-affinity calcium indicator. We conclude that disruption of ER luminal calcium homeostasis may contribute to the cyst phenotype in autosomal dominant polycystic kidney disease.

cell calcium; thapsigargin; autosomal dominant polycystic kidney disease; Madin-Darby canine kidney cells

AUTOSOMAL DOMINANT POLYCYSTIC kidney disease (ADPKD) is caused by loss of functional polycystin-1 or polycystin-2 (pc1 or pc2) (8, 9, 27). In affected individuals, renal function is gradually lost as normal renal parenchyma is progressively replaced by numerous cysts. A great deal has been learned about the functions of the polycystins in the decade since the identification of the two genes, but the exact way that loss of functional protein leads to cyst formation remains uncertain. Much of the experimental data points to a role for the polycystins in regulation of cell calcium (33). Polycystin-2 is a nonselective cation channel (22) that conducts calcium across the plasma membrane in concert with pc1 (19) and serves as a calcium release mediator across the endoplasmic reticulum (ER) membrane (22). pc1 has been implicated in the regulation of the pc2 channel (19, 36), and, with pc2, in the cell calcium response to deflection of the apical cilium (29). The fact that the majority of genes implicated in human and animal cystic kidney diseases also encode proteins associated with cilia raises the possibility that cilial dysfunction plays a central role in disease pathogenesis (10, 18). On the other hand, cystic disease of the liver is triggered by mutations in two noncilial proteins important in the processing of nascent polypeptide chains in the ER (11, 12), and loss of the ER-associated aquaporin-11 causes polycystic kidney disease (28). These observations suggest that abnormal ER function might play a role in cystic transformation. From preceding studies, we found that pc1 accelerated the decay of the ligand-activated cell calcium response through enhanced ER calcium reuptake (20). This effect of pc1 might have been through activation of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) or inhibition of leak across the ER membrane. In the present series of studies, we show for the first time that pc1 inhibits calcium leak across the ER membrane of distal renal epithelial cells. Since maintenance of an optimal ER calcium level is essential for normal ER function (5), our findings suggest that ER calcium dysregulation may play a role in ADPKD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture conditions. Stably transfected Madin-Darby canine kidney cell lines expressing either full-length pc1 or vector alone (control) were created and cultured as previously described (4). For intracellular calcium measurements, cells were seeded onto Matrigel-coated glass coverslips as previously described (20).

Western blotting and immunofluorescence imaging. Cells were harvested and lysed in a HEPES-buffered solution containing protease inhibitors and 1% Triton X-100, for SDS-PAGE analysis. In the immunofluorescence experiments, cells were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100. Antibodies against protein disulfide isomerase (PDI), actin, and SERCA were obtained from Sigma, Santa Cruz, and Biomol, respectively. Anti-PDI antibody was used at 1:4,000 for probing Western blots and at 1:200 for immunofluorescence. The actin antibody was used at 1:2,000 and anti-SERCA at 1:2,500 for Western blotting.

Fluorescent calcium imaging. Cells on coverslips were incubated with 5 µM fura 2-AM or 10 µM mag-fura 2-AM at room temperature for 45 or 75 min, respectively. Cells were loaded with fura 2-AM in the presence of 5 mM probenecid to prevent dye export. Calcium measurements were performed as described previously in a chamber perfused at 2 ml/min at room temperature, mounted on a Nikon Diaphot 300 microscope (20). The cells were excited at 340 and 380 nm (±10), and emission was measured at 510 nm. Thapsigargin and ionomycin studies were performed in a zero-calcium buffer. Increases in cytoplasmic calcium following thapsigargin arise as a consequence of calcium leak from the ER lumen, revealed after inhibition of the calcium pump. As discussed below, the magnitude of the thapsigargin-induced calcium peak will be influenced not only by the activity of leak pathways but also by multiple other potential calcium fluxes. Increases in cytoplasmic calcium following treatment with ionomycin, a calcium ionophore, result from the immediate release of calcium from all membrane-sequestered intracellular calcium stores. Since the ER is the predominant intracellular calcium store, the ionomycin peak response is an indicator of the magnitude of ER calcium stores. Where required to exclude plasma membrane calcium transport, bath Na+ was replaced with choline to inhibit Na+/Ca2+ exchange, and 5 mM lanthanum was added to inhibit the plasma membrane calcium pump. Ionomycin peaks were used to indicate residual cell calcium stores 2 min after treatment with thapsigargin. To abolish mitochondrial calcium uptake, cells were preincubated in 10 µM Ru360 for 45 min (21). Cytoplasmic calcium data in the fura 2 experiments are presented as calcium concentration. Fura 2 fluorescent ratios were converted to calcium concentration using the formula of Grynkiewicz et al. (17), [Ca2+] = Kd [(R – Rmin)/(Rmax – R)] (Sf/Sb), where the Kd for fura 2 is 220 nM. Rmin and Rmax (the minimum and maximum 340/380 ratios) and Sf and Sb (the 380 nM values for calcium-free and calcium-saturated dye) were determined by perfusing fura 2-loaded cells with 0 and 10 mM calcium buffer in the presence of 10 µM ionomycin.

Evaluation of ER calcium. Cells were loaded with Mag-fura 2-AM and perfused with zero-calcium buffer. To release the dye from the cytoplasm, the cells were switched into the plasma membrane permeabilization buffer so that the only dye remaining was sequestered within membrane-bound organelles, predominantly in the ER. Once the fluorescence signal dropped, signifying the release of cytoplasmic dye, the cells were switched into the intracellular buffer. Refill was initiated by the addition of the intracellular buffer supplemented with magnesium and ATP. Calibration of mag-fura 2 in the ER lumen was not possible because ER luminal calcium concentration could not be elevated to levels sufficient to saturate the dye; a difficulty that has been reported by others (37). Therefore, the data for ER calcium are presented as 340/380 ratios.

Buffer composition. The buffers used in these experiments were made up as follows (in mM): zero-calcium EGTA buffer (140 NaCl, 5 KCl, 1.8 MgCl2, 10 HEPES, 0.1 EGTA, 5 probenecid, pH 7.4 with NaOH); 2 mM calcium buffer (140 NaCl, 5 KCl, 2 CaCl2, 1.8 MgCl2, 10 HEPES, 5 probenecid, pH 7.4 with NaOH); choline/lanthanum buffer (140 C5H14NOCl, 5 LaCl3, 5 KCl, 1.8 MgCl2, 10 HEPES, pH 7.4 with KOH); permeabilization buffer (19 NaCl, 125 KCl, 10 HEPES, 1 EGTA, 0.33 CaCl2, 8.14 digitonin, pH 7.2 with KOH); intracellular buffer (19 NaCl, 125 KCl, 10 HEPES, 1 EGTA, 0.33 CaCl2, pH 7.2 with KOH); intracellular buffer with MgCl2 and ATP (95 nM free calcium concentration; 19 NaCl, 125 KCl, 10 HEPES, 1 EGTA, 0.33 CaCl2, 1.4 MgCl2, 3 ATP, pH 7.2 with KOH); and intracellular buffer with MgCl2 and ATP (372 nM free calcium concentration; 19 NaCl, 125 KCl, 10 HEPES, 1 EGTA, 0.66 CaCl2, 1.4 MgCl2, 3 ATP, pH 7.2 with KOH).

Reagents. Fluorescent dyes were from Molecular Probes, Ru360 was from Calbiochem, cell culture reagents were from Invitrogen, and all other reagents were from Sigma.

Calculations and analysis. Calculations for free calcium concentrations were made using WEBMAXCLITE. The significance of differences in responses was determined (using Graph Pad software) by t-test. Data are presented as means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Thapsigargin responses. The cytoplasmic calcium response to exposure to thapsigargin, due to leak of calcium from ER stores, was significantly attenuated in cells expressing pc1 (Fig. 1). Heterologous pc1 was found to have no effect on basal calcium stores (measured as the cytoplasmic calcium response to ionomycin in a zero-calcium bath) and could not therefore have reduced the thapsigargin responses by lowering ER calcium content. Concentration/normalized-response curves were established for both groups of cell lines and showed that the EC50 values for thapsigargin were similar in control and pc1 cells (control 77 nM, pc1 63 nM). Furthermore, the effect of pc1 to reduce the thapsigargin peak was even more evident at supramaximal concentrations of thapsigargin, where SERCA inhibition was likely to have been complete in all cell lines. The possibility that pc1 could have decreased the amplitude of the thapsigargin-induced cytoplasmic calcium response through a reduction in the ER to cytoplasmic volume ratio was addressed by immunostaining of the ER and quantitation of two ER-resident proteins with respect to total cell protein content. As can be seen in Fig. 2, heterologous expression of pc1 resulted in no obvious changes in either ER morphology or the relative abundance of SERCA or PDI.


Figure 1
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  		Fig. 1. Cytoplasmic calcium peak in response to thapsigargin (TG) is attenuated by heterologous expression of polycystin-1 (pc1). F6 and F8 are control cell lines for C8 and G7, which express heterologous intact pc1. A: traces show the cytoplasmic calcium responses to 1 µM TG measured with the ratiometric calcium indicator fura 2. Each trace is a composite of 5–6 experiments with each of the 4 cell lines. The thickness of the line indicates the SE. As shown in both traces and the bar charts to the right, the peak amplitude of the TG response was reduced by heterologous pc1 (*P = 0.0008). Basal endoplasmic reticulum (ER) calcium stores, as estimated by the peak amplitude of ionomycin responses, were no different between cell lines (n = 6 each cell line). B: from the TG concentration response study, pc1 did not influence the sensitivity of the TG-induced calcium release response, and the effect of pc1 to minimize the TG peak remained evident at supramaximal doses of TG. At each concentration, 5–6 experiments were performed for each of the control and pc1 cell line groups with equal representation of each individual cell line. *P < 0.05.

 

Figure 2
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  		Fig. 2. Heterologous expression of pc1 had no effect on ER morphology but resulted in downregulation of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) expression. A: anatomic distribution of the ER, identified by immunofluorescent staining for protein disulfide isomerase (PDI), was not obviously altered by heterologous pc1. B: abundance of the ER calcium pump, SERCA, was determined by Western blotting normalized for both actin and the ER-resident protein PDI. The abundance of SERCA, actin, and PDI was determined by successive probes of the same membrane. Data are presented for 6 independent experiments in each cell line with depiction of representative immunoblots.

 
Contribution of plasma membrane calcium transport to the thapsigargin response. The next series of experiments were designed to examine the possibility that the attenuated thapsigargin peak seen in pc1 cells was due to enhanced extrusion of calcium across the plasma membrane. Thapsigargin responses were examined following substitution of choline for extracellular sodium (to inactivate Na+/Ca2+ exchange), and the addition of lanthanum to the bath (to broadly inhibit calcium channels and the plasma membrane calcium ATPase PMCA) (Fig. 3). On the switch to the lanthanum choline buffer, fura 2 signal increased. Since there was no calcium in the bath, this was probably due to an increase in cytoplasmic calcium resulting from inhibition of basal calcium extrusion, as has been previously described (16). Thapsigargin was applied to the cells once a new steady-state cytoplasmic calcium concentration was attained. Under these conditions, the amplitude of the thapsigargin responses became comparable in the two groups of cell lines (0.42 vs. 0.46, P = 0.29), but the rate of increase of cytoplasmic calcium was significantly slowed in cells expressing heterologous pc1 (see the t20–80% data in Fig. 3). The thapsigargin-induced increases in cytoplasmic calcium seen in these experiments exhibited a stable plateau. This was best explained by efficient inhibition of SERCA by thapsigargin and of plasma membrane calcium transport by the manipulations of the bath components described above; calcium released from the ER into the cytoplasm stayed there simply because it had nowhere else to go. The similarity of the plateau level between the two groups of cell lines indicated that pc1 did not influence the total amount of calcium released from the thapsigargin-sensitive store. However, the different rates of increase in cytoplasmic calcium leading up to the plateau provided further evidence that pc1 was indeed slowing down the rate of calcium release via inhibition of leak pathways in the ER membrane.


Figure 3
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  		Fig. 3. Effect of heterologous pc1 on the TG response was not due to changes in total calcium released from the ER, nor to differences in the rate of calcium export from the cell. In these experiments, plasma membrane calcium transport was blocked by substitution of bath Na+ with choline and the addition of extracellular lanthanum. A: as shown in the representative traces, following TG-induced ER release, cytoplasmic calcium rose to a plateau, indicating abolition of calcium transport across the plasma and ER membranes. In the first bar chart in the summary data shown in B, cells expressing heterologous pc1 exhibited the usual attenuated TG peaks in the nonmodified zero-calcium bath (*P = 0.0001). By contrast, the maximal amplitude of the cytoplasmic calcium response to TG was similar in all cell lines in the lanthanum-choline bath. However, the rate of increase of cytoplasmic calcium under these conditions (expressed in C as the time taken to ascend from 20 to 80% of the maximal response) was significantly slowed by heterologous pc1. D: both basal cytoplasmic calcium and the calcium response to lanthanum-choline were reduced in pc1 cells. *P = 0.00003; n = 16 for control and 11 for pc1 cells.

 
Rate of decay of ER calcium stores as determined by ionomycin peaks. As a means of determining the leak-associated rate of decline of ER calcium content in intact cells, ionomycin peaks were examined at baseline and 2 min following exposure to thapsigargin. A thapsigargin concentration of 100 nm was selected for these experiments because calcium release from the ER was very rapid at higher concentrations of thapsigargin (1 µM), which resulted in ionomycin peaks that were too small to reliably measure amidst background noise. The results of this study (shown in Fig. 4) once again showed that basal calcium stores were the same in the two groups of cell lines. As expected, ionomycin peaks after thapsigargin were smaller than the basal peaks in all cell lines, because of calcium leak from the ER. However, the post thapsigargin ionomycin peaks were better sustained in cells expressing heterologous pc1, consistent with the idea that pc1 was inhibiting ER calcium leak. Some component of the ionomycin peak might have arisen from non-ER sources such as mitochondria (2). If so, then both the attenuation of the thapsigargin-induced cytoplasmic calcium peak by pc1 and the relative preservation of ionomycin peak amplitude following inhibition of SERCA might have been the result of translocation of calcium released from the ER lumen into some other compartment, rather than from inhibition of leak. To address this possibility, the thapsigargin-ionomycin experiment was repeated after inhibition of mitochondrial calcium uptake. As shown in Fig. 4, exposure to Ru360 resulted in a modest decline in the magnitude of ionomycin-induced calcium release under basal conditions, probably because of depletion of the mitochondrial calcium pool. Heterologous expression of pc1 had no effect on the magnitude of this Ru360-sensitive component under basal conditions. The degree of reduction in the ionomycin peak 2 min after exposure to thapsigargin was not affected by prior treatment with Ru360 in any of the cell lines. This indicated that, at least under these conditions, there was no appreciable transfer of calcium from ER to mitochondria in either of the cell groups. The capacity of polycystin-1 to attenuate the thapsigargin response was similarly unaffected by inhibition of mitochondrial calcium uptake.


Figure 4
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  		Fig. 4. pc1 slows down the rate of ER calcium depletion following exposure to TG. The same series of experiments was performed in the presence and absence of the mitochondrial calcium uptake inhibitor RU360. In the representative traces shown in panel A, cells were studied in the absence of RU360. The initial peak was the response to TG, followed 2 min later by the response to ionomycin. Control cells showed consistently bigger TG peaks and smaller ionomycin peaks compared with pc1 cells. Summary data, shown in B–E, demonstrated that 1) basal ionomycin peaks were not affected by pc1; 2) TG peaks were reduced by pc1 (*P = 0.0001 and 0.00005 in the presence and absence of RU360); and 3) the decline in amplitude of post-TG ionomycin peaks (relative to basal ionomycin responses) was significantly attenuated by pc1. *P = 0.01 and 0.04 in the presence and absence of RU360. The fact that none of these relationships were affected by treatment with RU360 indicates that pc1 was not acting through mitochondrial calcium buffering; n = 22 for control and 22 for pc1 groups in the presence of RU360, and 20 and 24, respectively, in the absence of RU360. Equal proportions of each individual cell line were included in each group.

 
Direct measurements of ER luminal calcium. To more directly evaluate the effect of pc1 on ER calcium leak, we used the low-affinity calcium indicator mag-fura 2 to report changes in calcium concentration within the ER lumen. We found that ER signal could be resolved once the emission at 340 nm fell below 50% of the starting value. As the interior of the cells equilibrated with the permeabilization buffer, ER calcium stores were run down because of inactivation of SERCA in the absence of ATP. Upon switching the perfusion solution to an intracellular buffer containing ATP and magnesium, there was a clear increase in the fluorescent ratio due to activation of SERCA-driven calcium uptake into the ER lumen. We refer to this increase in signal as "ER refill." Under the initial set of experimental conditions described above (with 95 nM calcium in the intracellular buffer), ER refill was always greater in the pc1-expressing cells than in controls (Fig. 5). Hence, subsequent thapsigargin responses were difficult to interpret when starting from such different baselines. However, ER calcium loading became comparable in the pc1 and control cells after bath calcium was ramped up to 372 nM, a level within the physiological range of the cytoplasmic calcium transients driven by ligand-activated calcium release in intact cells. The responses of the two groups of cells to thapsigargin were consequently examined in the permeabilized preparation with the higher calcium (372 nM) intracellular buffer. Under these conditions, the rate of decline of ER calcium signal was steeper in control cells compared with pc1-expressing cells (Fig. 6). Although the mean ER-refill amplitude appeared slightly greater in control cells, this was due to the influence of two outlying data points (shown in the scatter plot in Fig. 6). There was no statistical difference in the refill amplitudes between cell lines. Indeed, as shown in the scatter plot in Fig. 6, the refill amplitude was very similar in the majority of these experiments, and refill amplitude could not have been a confounder since, under these conditions, there was no correlation between refill amplitude and peak-20% times after exposure to thapsigargin.


Figure 5
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  		Fig. 5. pc1 increases the capacity of the ER membrane to sustain a large calcium gradient. In these experiments, cells were loaded with the low-affinity calcium-sensitive dye mag-fura 2-AM. As described in MATERIALS AND METHODS, cytoplasmic dye was removed by permeabilization of the plasma membrane, leaving residual dye within the ER. In the representative traces, ER refill with calcium was triggered by addition of Mg2+ and ATP to the intracellular (IC) buffer. Two IC buffer calcium concentrations were used, 95 and 372 nM. At the former, ER refill was significantly greater in pc1 cells (*P = 0.005). Upon ramping of the IC calcium to 372 nM, refill increased in control cells (**P = 0.006) but was not augmented in pc1 cells. As a consequence, refill became equivalent between cell lines at the elevated ambient calcium concentration; n = 5 for control and n = 6 for clones. All 4 cell lines are represented.

 

Figure 6
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  		Fig. 6. pc1 slows the rate of ER calcium depletion following treatment with TG. Cells were loaded with mag-fura 2-AM and subjected to the permeabilization protocol. In these experiments, ER refill was completed in a 372 nM calcium IC buffer to ensure that ER repletion was comparable before the introduction of TG (see Fig. 5). Traces representative of the response of the ER to 1 µM TG are shown in A. In B, summary data show that there was no significant difference between groups in the degree of ER refill and that the rate of decline in ER signal (expressed as the time taken to fall from peak to 20% of maximal refill response) was significantly slowed by heterologous pc1. The scatter plot in C illustrates that there was no relationship between refill and decay time in these experiments. *P = 0.008; n = 14 for control group and 10 for pc1 group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In previous studies, we showed that heterologous expression of intact pc1 caused acceleration of the decay of the ATP-activated cell calcium response in stably transfected Madin-Darby canine kidney cells (20). These experiments indicated that pc1 was promoting reuptake of calcium by the ER. Active reuptake of calcium into the ER lumen is driven by the ER calcium pump, which has the capacity to create a very large calcium concentration gradient across the ER membrane. However, a calcium leak pathway(s) exists in the ER membrane (7), so an increase in the rate of net calcium uptake by the ER could have resulted from activation of the pump, minimizing of a leak, or a combination of both. In the experiments described above, data derived from multiple different approaches were consistent with the idea that pc1 accelerated ER calcium uptake, at least in part, by inhibition of calcium leak across the ER membrane.

The majority of the experiments were based on the use of thapsigargin to reveal calcium leak from the ER. Thapsigargin is a potent and very specific inhibitor of SERCA, so pc1 was certainly influencing events arising from abolition of active calcium pumping into the ER lumen. Since pc1 had no effect on basal cell calcium stores, mitochondrial calcium buffering, or extrusion of calcium from the cell, the most obvious conclusion was that the decline in the appearance of calcium in the cytoplasm in response to thapsigargin arose from reduced leak. This was supported indirectly by the ionomycin and choline-lanthanum studies (Figs. 3 and 4) and directly from measurements of the thapsigargin-induced decline in ER luminal calcium concentration (shown in Fig. 6). Two other observations, both derived from the choline-lanthanum studies, lend further support to the view that pc1 inhibits ER leak (Fig. 3D). First, basal cytoplasmic calcium concentration was significantly lower in the pc1 cells in the zero-calcium buffer (but not in the physiological calcium buffer). Second, the increase in cytoplasmic calcium in response to bath substitution with choline and lanthanum was significantly attenuated by heterologous pc1 expression. Both observations could be explained by diminished ER calcium leak, which would tend to buffer the decline in cytoplasmic calcium in a zero-calcium bath and to result in an attenuated increase in cytoplasmic calcium when plasma membrane calcium extrusion is blocked (see discussion above and Ref. 16). The calcium-ramping experiments in the permeabilized preparation (Fig. 5) also provided direct evidence that pc1 was regulating ER membrane calcium conductance. It is possible that heterologous pc1 was influencing ER calcium handling in a nonspecific way, as a consequence of protein loading of the ER lumen. However, two observations make this possibility unlikely; first, heterologous pc1 was expressed at very low levels in these cells (undetectable unless immunoprecipitated from very large quantities of cellular lysate), and, second, the ER refill phenotype induced by heterologous pc1 was reversed by coexpression of the dominant-negative cytoplasmic COOH-terminal 193 amino acids of pc1 (3). Our findings might also be explained by reduced effectiveness of thapsigargin in cells expressing heterologous pc1. This could occur through an effect of pc1 to reduce accumulation of drug at the site of activity or through changes in the responsiveness of SERCA. We think that neither of these alternative explanations is likely to explain our observations. With respect to the former, differences in thapsigargin responses were maintained at supramaximal concentrations of the drug (10 µM, Fig. 1) and were also seen in the permeabilized preparation where plasma membrane drug transport could no longer influence exposure of the ER to the drug. With respect to the latter, modifications of SERCA that alter responsiveness to thapsigargin would also likely be overwhelmed at the 10 µM concentration, and we observed no differences in the pharmacodynamics of the thapsigargin response in the two groups of cell lines (Fig. 1).

Possible leak pathway-pc1 interaction. Polycystin-1 appears to be expressed within the ER (15) and thus could be influencing ER calcium dynamics directly. However, even if pc1 is restricted to the plasma membrane, interactions between ER luminal proteins and pc1 might still be possible through conformational coupling, a process whereby the ER and plasma membrane systems can be brought into close apposition (30). It is also possible that pc1 is influencing ER function indirectly through signal transduction, but little is known of the physiological regulation of ER leak. An increasing number of ER proteins are being identified that are potential leak pathways (7), but the physiological role and regulation of calcium leak from the endoplasmic reticulum remain poorly understood. pc2, an ER membrane cation channel with significant calcium conductance (13, 22, 26), would be an obvious candidate for the leak pathway because of its known functional interactions with pc1 (31, 35). However, it would be difficult to explain how an inhibitory interaction between pc1 and pc2 could be consistent with the fact that loss of either of these two proteins leads to the same clinical syndrome. An alternative model might invoke pc2 as a regulator of ER leak (rather than the channel mediating this phenomenon). This is not too far fetched, since there are precedents for channels acting as regulators of other channels. A regulatory function for pc2 is suggested by the observation that haploinsufficiency of pc2 caused a reduction in ER luminal calcium content (32), and that loss of pc2 in Caenorhabditis elegans (23) caused a prolongation of cell calcium responses (not due to elevation of ER calcium content), which suggested a delay in reuptake (as our data predict would be the case with loss of pc1). Also, pc2 has been described as a regulator of both inositol 1,4,5-trisphosphate and ryanodine receptors (1, 25), which might constitute leak pathways in addition to their primary roles as mediators of calcium release. Nonetheless, the possibility that pc2 is an ER leak channel (or regulator of such a channel) remains speculative, and there is direct experimental evidence that a number of other proteins mediate ER calcium leak and might therefore be potential mediators of the effect of pc1 (7).

Significance. ER luminal calcium is important in fundamental cellular processes such as posttranslational folding and modification of nascent polypeptide chains (5). ER calcium content is regulated and maintained within ranges that are probably cell type specific. The transient dramatic shifts in ER calcium content that occur in relation to calcium release triggered by signal transduction events would seem to threaten the stability of processes requiring a constant intraluminal calcium level, especially if recovery is delayed as our experiments suggest might be the case in ADPKD. The consequences of abnormal ER calcium homeostasis are becoming clear. Excessive elevation of basal calcium is associated with increased calcium release, which leads to mitochondrial calcium loading and apoptotic cell death (34). On the other hand, reductions in ER luminal calcium disturb posttranslational processing of newly synthesized proteins destined for insertion into cellular membranes, or for secretion (5). The cell is thus presented with an apparent dilemma in maintaining ER calcium homeostasis in the face of sporadic episodes of calcium release from this compartment. This suggests that processes regulating ER calcium content are probably rather complex and may operate with spatial and temporal heterogeneity. Perhaps because they are both complex and difficult to study, the mechanisms of ER calcium regulation remain poorly understood but probably involve concerted modulation of pump and leak pathways. Others have shown that SERCA expression is upregulated by ER calcium depletion (24), which might explain our own data indicating that the effect of pc1 in minimizing ER leak is linked with a significant decrease in SERCA abundance. Although it would therefore seem unlikely that pc1 is accelerating ER refill through increased SERCA activity, this possibility has not been addressed in the present studies.

In the light of the experimental data presented above, pc1 should probably be added to the list of proteins involved in the regulation of calcium leak across the ER membrane. Since pc1 appears to influence ER luminal calcium homeostasis, the obvious question is whether loss of this function could play a role in cystogenesis. As mentioned above, the fact that cystic diseases arise as a consequence of mutations in three proteins known to reside in the ER provides a plausible basis for the view that pc1 might similarly influence cyst formation through effects on ER function. Since cells in a physiological environment are constantly subjected to ligand activation dependent on the homeostatic demands placed on the kidney, loss of the capacity to swiftly replete ER calcium content may exert cumulative stress on ER luminal homeostasis. This might result in altered biosynthesis of membrane and secreted proteins, apoptosis, and dedifferentiation, and thereby ultimately engender a cellular phenotype that contributes to cyst formation (6, 14).


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant KO8-DK-066323-01 and grants from the PKD Foundation (105a2r) and the National Kidney Foundation, Maryland Chapter.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Sutters, Rm. B2N, Div. of Renal Medicine, Johns Hopkins Bayview Medical Center, 4940 Eastern Ave., Baltimore, MD 21224 (e-mail: msutters{at}jhmi.edu)

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. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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