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1 Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK2 Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK3 School of Biomedical Sciences, University of Leeds, Leeds, LS2 9JT, UK4 Department of Physiology and Neuroscience, St George's University, True Blue Campus, P. O. Box 7, St George's, Grenada
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(Received 8 October 2003;
accepted after revision 4 December 2003)
Corresponding author L. Robson: Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK. Email: l.robson{at}sheffield.ac.uk
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Introduction |
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KCNE1 is a K+ channel ß subunit that was initially cloned from rat kidney (Takumi et al. 1988) and is expressed in the heart and a number of epithelial cells (Demolombe et al. 2001; Warth & Barhanin, 2002). KCNE1 regulates the voltage-sensitivity of the K+ channel KCNQ1, and the complex formed by these two proteins has been shown to play an important role in cardiac repolarization (Schulze-Bahr et al. 1997; Shalaby et al. 1997). KCNQ1 has also been localized to a number of epithelial tissues, suggesting that these two proteins may be important in epithelial cell function (Demolombe et al. 2001). A role for KCNE1 could be the regulation of cell volume, as KCNE1 mediated currents in both vestibular dark cells of the inner ear (Wangemann et al. 1995) and in Xenopus oocytes (Busch et al. 1992) are activated by cell swelling. In addition, the ability of tracheal epithelial cells to recover their normal size following exposure to hypotonic solutions is lost in KCNE1 knockout mice (Lock & Valverde, 2000).
In the kidney KCNE1 is found in the cortex (Demolombe et al. 2001), specifically in the proximal tubule where it is localized to the apical membranes of the proximal tubule cells (Sugimoto et al. 1990; Vallon et al. 2001). An in vivo study using KCNE1 knockout mice has demonstrated a role for KCNE1 in K+ efflux across the apical cell membrane and therefore a role in maintaining resting membrane potential and electrogenic Na+ coupled transport (Vallon et al. 2001). The functional role of KCNQ1 is less clear, with evidence for and against colocalization with KCNE1 in the proximal tubule (Demolombe et al. 2001; Vallon et al. 2001). A recent study has suggested that KCNE1 is important in cell volume regulation in cultured renal proximal tubule cells (Barriere et al. 2003a). The aim of the following study was to determine the role of KCNE1 in volume regulation in freshly isolated renal proximal tubule cells.
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Methods |
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RVD in single cells was compared between wild-type (129/SV and C57/B6 background) and KCNE1 knockout mice of both sexes and over six weeks of age. KCNE1 knockout mice were a kind gift from Dr Douglas Vetter (Tufts University School of Medicine, Boston, USA). Homozygous KCNE1 knockout mice were confirmed by Southern analysis (Vetter et al. 1996).
Single Cell Isolation
Mice were killed humanely by cervical dislocation, according to UK legislation. Kidneys were removed into an ice-cold solution containing (in mM): 140 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES titrated to pH 7.4 with KOH and gassed with 100% O2. The kidneys were decapsulated and thin tangential cortical slices (< 0.5 mm) were taken. The cortical slices were finely minced with a scissoring action of two razor blades. The tissue fragments were further homogenized with two strokes of a glass/Teflon Dounce homogeniser, filtered through a fine plastic mesh (PP80, Millipore) and stored on ice until required. Single proximal tubule cells were identified as spherical cells of diameter 8.5–10.5 µm, given that such cells were previously found to exhibit swelling upon addition of 10 mML-alanine in the presence of sodium and in over 150 patch clamp recordings show no electrophysiological differences (Balloch et al. 2003).
Cell Diameter Measurement
Cell diameter was measured using two optical techniques. One technique was based on a photodiode array system that has been previously described (Mounfield & Robson, 1998). The second method utilized a digital camera based system using the software Soft Cell (Cairn Instruments, UK). Both these systems utilized the changes in light intensity that occur at the cell membrane–bath interface to provide markers for cell diameter. There was no significant difference between data obtained with the two methods and therefore these data have been pooled.
Peak cell diameter was defined as the maximum diameter recorded upon exposure to a swelling stimulus. Steady state diameter was the diameter attained by a cell for at least one minute in the case of a regulating cell and two minutes after reaching peak in the case of a non-regulating cell. Regulating cells were defined as those cells that reached a peak and showed a subsequent decrease in diameter, irrespective of the size of the decrease. Non-regulating cells were defined as those cells that reached a peak and showed no subsequent decrease in diameter.
Experimental protocol
Experimental solutions were either buffered with HEPES or HCO–3. The HEPES solution contained (in mM): 112 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 60 mannitol and 10 HEPES titrated to pH 7.4 with NaOH. The HCO–3 solution contained (in mM): 100 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 50 mannitol, 23.8 NaHCO3 and was continuously gassed with 95% O2/5% CO2 (pH 7.4). Cells were superfused with one of these solutions and then exposed to either a hypotonic shock (removal of 40 mM mannitol) or 10 mML-alanine (equimolar substitution of mannitol). In some experiments the effect of 1 µM valinomycin was assessed during a hyposmotic challenge in HCO–3 buffered solutions. Valinomycin was added to both control and hyposmotic solutions throughout. To investigate the role of K+ channels in RVD, cells were exposed to a hypotonic shock in the presence of a number of K+ channel blockers: (a) 5 mM barium (Ba2+) a generic K+ channel inhibitor (b) 100 µM clofilium, which inhibits the K+ channels KCNQ1 (Yang et al. 1997); hERG (Gessner & Heinemann, 2003) and TASK-2 (Niemeyer et al. 2001) or (c) 100 nM chromanol 293B, which inhibits KCNQ1 (Busch et al. 1997). Blockers were added to both the control and hypotonic solutions. All experiments were carried out at room temperature.
Solutions
All chemicals were obtained from Sigma (Dorset, UK) except for chromanol 293B, which was obtained from Tocris (Bristol, UK). Osmolality of the experimental solutions was checked using a Roebling (Camlab, Cambridge, UK) osmometer and adjusted to 300 ± 1 mOsm.kg–1H2O using mannitol or water as appropriate.
Statistics
Results are presented as mean ±S.E.M.. Comparison of the proportion of cells exhibiting RVD between different genotypes or treatments was achieved using Fisher's exact probability test. Effects of experimental interventions on RVD responses were assessed by ANOVA and Student's t test. Significance was assumed at the 5% level.
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Results |
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In HEPES buffered solution two types of response to hypotonic shock were observed. In 64% of cells tested (18 from 28) cell diameter increased to a peak followed by RVD (Figs 1 and 2). In the remaining cells no RVD was observed and these cells were classified as non-regulators (Figs 1 and 2). There was no significant difference between either the initial or peak diameters of regulating and non-regulating cells. However, the steady state diameters of regulating cells, expressed relative to the initial diameter, were significantly smaller than the non-regulating cells. In marked contrast, in the presence of HCO–3 only one type of response to hypotonic shock was observed. All WT cells tested demonstrated RVD (Fig. 2).
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Volume regulation of proximal tubule cells isolated from knockout mice was initially investigated in HEPES buffered solutions. As described for the WT cells two types of response were observed in response to hypotonic shock. 56% of cells (10 from 18) demonstrated RVD (Fig. 2). This was not significantly different to the number of WT cells demonstrating RVD. The number of cells demonstrating RVD in response to L-alanine (Fig. 3) was also not significantly different to the WT cells (50%: 7 from 14), However, in marked contrast, when KO cells were exposed to either hypotonic shock or L-alanine in the presence of HCO–3 (Figs 2 and 3) a proportion of cells still failed to show RVD. In response to hypotonic shock 61% of cells (14 from 23) demonstrated RVD. In response to L-alanine 57% of cells (8 from 14) demonstrated RVD. These were significantly different to the number of WT cells demonstrating RVD under the same circumstances.
Role of K+ in hypotonic shock induced RVD
The participation of K+ movement in impaired volume regulation in KO proximal tubule cells was investigated by adding 1 µM valinomycin to HCO3– buffers. When challenged with hypotonic shock in the presence of valinomycin all KO cells demonstrated RVD (n= 9), Fig. 2. This was significantly greater than the fraction of regulating KO cells observed in the absence of valinomycin. Comparing cells displaying RVD, there was no significant difference in the magnitude of RVD in the absence and presence of valinomycin.
To investigate the role of K+ channels in the response of HCO–3 dependent cells, WT cells were exposed to different K+ channel inhibitors in both HEPES and HCO–3 buffered solutions. The rationale for these experiments was that examining RVD in HEPES buffered solution would show the effects of the inhibitors on the non-KCNE1 dependent cells, while examining RVD in the presence of HCO–3 would allow both cell populations to be examined. In this way the effects of the inhibitors on the KCNE1 dependent cells could be determined.
Figure 4 shows that in contrast to data obtained in the absence of K+ channel inhibitors, both Ba2+ and clofilium produced regulating and non-regulating WT cells in HCO–3 solution. However, chromanol had no effect on the proportion of cells demonstrating RVD. In cells displaying RVD, there was no significant difference between the initial diameters, increase in diameter and steady-state diameters comparing Ba2+, clofilium or chromanol treatment with control.
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Table 1 summarizes the proportion of regulators versus non-regulators for all the cell diameter experiments.
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Discussion |
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The presence of two populations of cells with respect to RVD complicates the examination of what effect loss of KCNE1 function has on RVD. Our conclusions are based largely on the observation of changing proportions of cells that did or did not undergo an RVD response. Whilst there is a degree of subjectivity in the definition of a regulator or non-regulator (see Fig. 1), analysis of the actual changes in cell volume within each category provide validation for the approach. For example taking cells categorized as volume regulators in Fig. 2 or 3 cell diameter at steady state was significantly less than the peak volume and the magnitude of RVD response was not significantly different irrespective of whether cells were WT or KO or were in HEPES or bicarbonate solutions. In the case of non-regulators there was no significant RVD response in any group and no significant difference between groups. In addition, the steady state diameter, expressed relative to the initial level, was significantly smaller in regulating cells compared to non-regulating cells. The same clear categorization of cells was apparent in experiments with K+ channel inhibitors (Fig. 4), if cells volume regulated, the response was of the same magnitude as control. If a drug had an effect it was to prevent RVD completely in a proportion of cells only.
Null mutation of the KCNE1 gene had no effect on the pattern of volume regulatory responses observed in HEPES buffered solution. As in the WT cells approximately half of the knockout cells demonstrated RVD in response to cell swelling induced by either hypotonic shock or stimulation of Na+-alanine cotransport. This suggests that RVD in Cl– dependent cells was unaffected by loss of KCNE1. In contrast, a different pattern of regulators versus non-regulators was observed in the KO cells compared to the WT cells in the presence of HCO–3 buffered solution. While all of the WT cells demonstrated RVD with HCO–3 solution, only half of the KCNE1 null cells were capable of RVD. This suggests that the RVD in the HCO–3 dependent cells was inhibited by knockout of KCNE1. This is in contrast to a previous study on cultured proximal tubule cells, where only a single proximal tubule cell population was observed, all cells demonstrated RVD in the absence of HCO–3 and all cells required the expression of KCNE1 protein for RVD (Barriere et al. 2003a). The reason for the difference between this earlier study and the current study is unclear, but may reflect differences in the preparation (freshly isolated cells versus cultured cells) or may be due to a difference in the part of the kidney used for isolation of the cells.
Previous studies have demonstrated that KCNE1 regulates KCNQ1 (Busch et al. 1997). It has also been suggested that KCNE1 can regulate a Cl– channel found in Xenopus oocytes, although this regulation may be an artefact of over expression (Attali et al. 1993). A recent study in cultured proximal tubule cells also suggests that KCNE1 regulates volume-sensitive K+ and Cl– channels (Barriere et al. 2003a). The current study supports a role for KCNE1 in regulating K+ efflux during RVD in a population of renal proximal tubule cells. The evidence for this comes from rescue of RVD by the K+ ionophore valinomycin, which introduces an exogenous K+ efflux pathway into cells. If the lack of RVD in the knockout cells is a consequence of defective K+ efflux, then valinomycin should rescue RVD. However, if KCNE1 regulates another part of the RVD mechanism then valinomycin should be without effect. The data in the study shows that valinomycin rescued RVD in the knockout cells, indicating that KCNE1 is required for a K+ efflux pathway during RVD. The response of the HCO–3 dependent cells is similar to gerbil vestibular dark cells and Xenopus oocytes expressing rat KCNE1, where hyposmolality increased potassium currents, implicating KCNE1 in mediating RVD (Busch et al. 1992; Wangemann et al. 1995). However, the effect of KCNE1 knockout on RVD appears to be tissue specific, since RVD was compromised in tracheal cells (Lock & Valverde, 2000), but not vestibular dark cells of the inner ear (Vetter et al. 1996). Interestingly, in the study of Lock & Valverde (2000) KCNE1 knockout led only to a partial reduction in the hyposmotically induced RVD response, suggesting multiple RVD pathways within the same cell. However, the present study found that loss of KCNE1 led to a total absence of RVD in susceptible cells. This suggests that at least certain proximal tubule cells possess only one volume sensitive K+ efflux pathway, or multiple pathways that are all regulated by KCNE1.
What could be the identity of the KCNE1 regulated K+ channel? The most obvious candidate is KCNQ1, since this is regulated by KCNE1 and it is also expressed in the proximal tubule. However, chromanol 293B, an inhibitor of KCNQ1, was without effect on RVD in either the Cl– or HCO–3 dependent cell populations. This suggests that, although the mRNA for KCNQ1 is present in the proximal tubule (Haigh et al. 2003), the KCNQ1 channel protein does not play an important role in KCNE1-dependent RVD. In contrast, the non-selective K+ channel inhibitor Ba2+ prevented RVD in the HCO–3 dependent cells, providing further evidence for a role for conductive K+ efflux in RVD. Finally, clofilium, another K+ channel inhibitor, prevented RVD in the HCO–3 dependent cells, suggesting that a clofilium sensitive K+ channels may mediate RVD induced K+ efflux. One candidate is the clofilium-sensitive K+ channel TASK-2, as volume regulation is compromised in cultured proximal tubule cells derived from TASK-2 knockout mice (Barriere et al. 2003b).
In conclusion, the mouse proximal tubule is composed of two cell populations. One cell population has the ability to undergo RVD in Cl– containing HEPES buffered solution, suggesting that RVD may utilize Cl– efflux mechanisms. The other cell population only undergoes RVD in HCO–3 buffered solution, suggesting that HCO–3 plays an important role in RVD in these cells. In KCNE1 knockout mice only the HCO–3 dependent cells were affected, with loss of KCNE1 inhibiting RVD induced K+ efflux. The identity of the K+ channel mediating KCNE1 regulated K+ efflux is currently unknown, but the lack of effect of chromanol 293B would suggest that KCNQ1 is not important in proximal tubule RVD.
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References |
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represents regulating cell data, while • represents non-regulating cell data. The n numbers are given for each group. * indicates a significant difference to peak diameter.
