Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322-3030
Submitted 23 January 2003 ; accepted in final form 24 April 2003
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ABSTRACT |
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patch clamp; calcium; chloride channel; biophysics; pharmacology
The kidney plays a critical role in salt secretion and absorption, and Cl channels are an important component of this process. Determining the roles of different types of Cl channels is an area of active investigation. The inner medullary collecting duct (IMCD) is the final segment of the kidney involved in determining acid-base balance and urinary salt composition. In the IMCD, both electrogenic Na+ absorbtion and electrogenic Cl secretion participate in the regulation of overall NaCl balance (41, 42). Na+ readsorption in the IMCD occurs predominantly via apical amiloride-sensitive Na+ channels and a basolateral Na+-K+-ATPase. The activities of other transporters such as Na+-K+-2Cl and Na+-Cl cotransporters and the Na+/H+ exchanger may also be involved in Na+ homeostasis (50). The IMCD also has the capacity to secrete anions. The CFTR (21, 48), ClCa channels, swelling-activated Cl currents (3, 4, 45), and ClC channels (24) have been implicated in the process. Na+ absorption and Cl secretion in the IMCD are controlled by a wide variety of hormones and renal autacoids (20, 22, 41, 42, 50). For instance, mineralocorticoids augment the activities of both apical Na+ channels and basolateral Na-K+-ATPase, whereas atrial natriuretic peptides bind to guanylate cyclase-linked receptors, leading to inhibition of apical Na+ channel function (50). Anion secretion can be affected by changes in intracellular cAMP or Ca2+ (4, 21).
The purpose of this study was to characterize in detail the ClCa currents in IMCD-K2 cells. IMCD-K2 cells were established from the initial portion of the IMCD of a mouse transgenic for SV40 (27). The cell line retains features typical of IMCD: cyclic nucleotide-gated cation channels in the apical membrane mediate mineralocorticoid-sensitive Na+ absorption that is inhibited by amiloride (27). Moreover, this cell line exhibits electrogenic Cl secretion (27). A recent study presented evidence that mouse IMCD-K2 cells possess two separate apical Cl conductances that are activated by intracellular cAMP or Ca2+, respectively (3). ClCa currents in IMCD-K2 cells showed voltage-dependent kinetic changes in whole cell recording. After the induction of slow ramps in [Ca2+]i produced by exposing BAPTA-loaded IMCD-K2 cells to ionomycin, whole cell currents exhibited pronounced outward rectification with time dependence. ClCa currents in another IMCD cell line, IMCD-K3, exhibited somewhat different properties (40) (see DISCUSSION).
Our laboratory has studied ClCa channels in Xenopus laevis oocytes extensively (18, 28, 29, 31, 32, 38, 39). ClCa channels in oocytes resemble Ca2+-activated Cl channels expressed in some mammalian cells such as parotid acinar cells, lacrimal gland cells, and pancreatic acinar cells (33). They are activated directly by intracellular Ca2+, with a Kd in the 1 µM range, exhibit different voltage-dependent kinetics at low and high Ca2+ concentrations ([Ca2+]) and possess an anion selectivity sequence of I > Br > Cl. However, ClCa channels in other cells are different (33). Ca2+ activation of ClCa currents in colonic T84 and Jurkat T cells is mediated by Ca2+/calmodulin-regulated protein kinase II, whereas ClCa currents in olfactory receptors have a low Ca2+ affinity, with a Kd of 26 µM and an anion selectivity sequence of Cl > F > I > Br. Therefore, it seems that different types of ClCa channels exist in different cell types. We have described the "signature properties" of the X. laevis oocyte-type of ClCa channels (see Table 1 in Ref. 33). Stated simply, Ca2+ directly activates the channel without phosphorylation. The channel has a voltage-dependent Ca2+ affinity (in the 1 µM range) such that, at a [Ca2+]i of <1 µM, the currents strongly outwardly rectify and are time dependent. However, at high [Ca2+], the currents show a linear current-voltage (I-V) relationship and are time independent. The channel is more permeant to larger halide anions than to smaller ones (I > Br > Cl). The Cl channel blockers anthracene-9-carboxylic acid (A9C), diphenylamine-2-carboxylic acid (DPC), and DIDS block the channel from the outside in a voltage-dependent manner.
In this study, we applied whole cell recording to IMCD-K2 cells and observed Ca2+ activation by directly changing [Ca2+]i. Lower Ca2+ (<800 nM) activated outwardly rectifying Cl currents, whereas high [Ca2+]i elicited nearly linear Cl currents. The currents activated at low [Ca2+]i were time dependent for activation and deactivation. The Ca2+ activation and the Ca2+ affinity of the channel were voltage dependent. The Cl channel blocker niflumic acid (NFA) in the bath equally inhibited both inward and outward currents reversibly, with a Ki = 7.6 µM. DIDS, DPC, and A9C reversibly inhibited outward currents in a voltage-dependent manner. These biophysical and pharmacological characteristics are so similar to those in X. laevis oocytes that we conclude that the ClCa channels expressed in IMCD-K2 cells and in X. laevis oocytes are very similar.
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METHODS |
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Mouse kidney IMCD-K2 cells were kindly provided by Dr. Bruce A. Stanton (Dartmouth Medical School, Hanover, NH), routinely cultured (passages 1436) in PC-1 medium (Bio-Whittaker, Walkersville, MD) supplemented with PC-1 supplement (Bio-Whittaker), 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 2 mM L-glutamine (Life Technologies, Grand Island, NY), 50 U/ml penicillin, and 50 µg/ml streptomycin (Life Technologies) in tissue culture flasks (Costar, Cambridge, MA) coated with Vitrogen plating media containing DMEM (Life Technologies), human fibronectin (10 µg/ml, Collaborative Biomedical, Bedford, MA), 1% Vitrogen 100 (purified collagen; Cohesion, Palo Alto, CA), and BSA (fraction V, 10 µg/ml; Sigma, St. Louis, MO), and maintained in a 37°C and 5% CO2 incubator. For experiments, cells were seeded onto glass coverslips (Fisher Scientific, Pittsburgh, PA) and used 13 days later.
Whole Cell Patch-Clamp Recordings
Current recordings from mammalian cells were made using a whole cell
configuration. Whole cell currents were recorded with borosilicate glass
electrodes (Sutter Instrument), pulled by a Sutter P-2000 puller, and fire
polished. Pipette resistances were 25 M. Whole cell patch-clamp
data were acquired with an Axopatch 200A amplifier controlled by a Clampex 8.1
via a Digidata 1322A analog-to-digital and digital-to-analog converter (Axon
Instruments), sampled at 2 kHz, and filtered at 1 kHz with a four-pole
low-pass Bessel filter. The bath was grounded via a 3 M KCl-agarose bridge
connected to an Ag-AgCl reference electrode. Bath solution changes were
performed with a group of sewer pipes, having an 100-µm internal diameter,
connected to the gravity-fed solution containers so that the solution bathing
the cells could be changed in
2 ml/min.
Solutions
Symmetrical Cl solutions containing 150 mM Cl were
usually used for the whole cell recordings. Pipette solutions contained (in
mM) 150 N-methyl-D-glucamine chloride (NMDG)-Cl, 10
HEPES-NMDG (pH 7.3), and 10 EGTA-NMDG or 10
Ca2+-EGTA-NMDG. The bath solution contained (in mM) 140
NMDG-Cl, 4.5 KCl, 1 MgCl2, 2 CaCl2, and 10 HEPES-NMDG
(pH 7.4). Sucrose was used to adjust osmolarity. In some experiments,
NMDG+ was replaced with K+ in the pipette solution and
with Na+ in the bath solution. Mg-ATP (2 mM; Sigma) was also added
to the pipette solution.
To obtain submicromolar concentrations of free Ca2+, we buffered solutions with EGTA using the method of Tsien and Pozzan (47). The stock solution of Ca2+-EGTA was made by the pH-metric method (47). Working solutions having different free Ca2+ were prepared by mixing the 0Ca2+-EGTA solution with the Ca2+-EGTA solution (Molecular Probes) in various ratios. The free [Ca2+] was calculated from the equation [Ca2+] = Kd x [Ca2+-EGTA]/[EGTA], where Kd is the Kd of EGTA (Kd = 1.0 x 107 M at 24°C, pH 7.3, ionic strength 0.16 M). The calculated Ca2+ concentrations were confirmed in each solution by fura 2 (Molecular Probes) measurements using an LS-50B luminescence spectrophotometer (PerkinElmer, Norwalk CT).
Anion Channel Blockers
NFA, DPC, and tamoxifen were purchased from Sigma, A9C was from Aldrich Chemical, and DIDS was from Molecular Probes. DIDS was suspended in water at 0.3 M as a stock before working solutions were made. Other compounds were dissolved in DMSO at 0.3 M as stocks to keep the DMSO concentration in working solutions <0.1%. DL-DTT (Sigma) stock solution was made in H2O.
Display and Analysis of Data
For the calculations and graphical presentation, we used Origin 6.0 software (Microcal). Curve fitting was performed using the iterative algorithms in Origin. Results are presented as means ± SE, and n refers to the number of patches in each experiments. The significance of the difference between values was determined using Student's t-test (paired data) for tests between individual data pairs.
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RESULTS |
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Figure 1 shows the
biophysical characteristics of ClCa currents in an IMCD-K2 whole cell
recordings. Both extracellular and cytosolic solutions contained NMDG-Cl, so
that cation currents were minimized. The patches were held at 40 mV and
stepped to a membrane potential (Vm) between 100
and +100 mV for 0.75 s and then stepped to 40 mV for 0.3 s. At <20
nM Ca2+, only very small currents were recorded
(Fig. 1A). However,
when [Ca2+] was increased to 100 nM, a slowly developing
but sustained outward current was observed in response to depolarizing steps
(Fig. 1B).
Deactivating inward tail currents were observed on the return to 40 mV
from depolarized potentials (Fig.
1B). Outward currents observed with 100500 nM
Ca2+ were composed of a small instantaneous
time-independent component and a large slowly activating time-dependent
component (Fig. 1,
BD). Very little steady-state inward
current was observed at negative potentials at [Ca2+]
<500 nM (Fig. 1, B and
C). With [Ca2+] 500 nM, outward
currents were increasingly dominated by the time-independent component and
inward currents developed (Fig. 1,
D and E). At 21 µM
Ca2+, inward and outward currents were nearly equal in
amplitude, and the currents became essentially instantaneous
(Fig. 1F).
Figure 1G shows that
the steady-state I-V relationship changed from outwardly
rectifying at <800 nM Ca2+ to almost linear at 21
µM [Ca2+]. The currents reversed at
Cl equilibrium potential. Note that the outward currents at
800 nM and 21 µM were smaller than that at 500 nM. We do not know whether
this was caused by current rundown or whether the channel responded
biphasically to Ca2+. We prefer the explanation that
Ca2+ has a biphasic effect. A similar phenomenon was
observed in excised patches from X. laevis oocytes
(28).
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Similar experiments were done with 150 mM KCl on the intracellular side and
150 mM NaCl on the extracellular side. Similar results were obtained except
that reversal potentials were shifted from 0 with symmetrical NMDG
(Fig. 1G) to about
10 mV with K+/Na+ (data not shown), indicating
that nonselective cation conductance may have contaminated the records under
these conditions or that the channel is also slightly permeable for small
cations.
Voltage-Dependent Ca2+ Affinity of ClCa Currents
The data in Fig. 1G indicate that the activation of ClCa currents is voltage dependent at a [Ca2+] between 100 and 800 nM. For further analysis, the voltage dependence of the ClCa currents at different [Ca2+] was determined by plotting conductance vs. Vm. Conductance was determined by measuring the instantaneous tail currents (see Fig. 1) at the beginning of the 40-mV step after various voltage steps between +100 and 100 mV as shown in Fig. 1A and dividing by the driving force (40 mV). Figure 2A shows the voltage dependence of the ClCa conductance at different [Ca2+]i. The average conductance-voltage curves (n = 512 different whole cell patches for each [Ca2+]) show that increasing [Ca2+] from 100 to 500 nM shifted the conductance-voltage relationship strongly in the leftward direction and significantly increased the voltage dependence (increased the slope), indicating that the activation of the channel by Ca2+ is affected by Vm. We have previously shown that the effect of Vm on the Ca2+ activation of X. laevis oocyte ClCa channels is due to the voltage dependence of the channel for Ca2+ affinity.
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To quantify the voltage dependence of the Ca2+ affinity of the channel, we plotted the mean conductance vs. [Ca2+] for each voltage (Fig. 2B). Only the data between <20 and 500 nM Ca2+ were fitted to the Hill equation. Data for 800 nM and 21 µM Ca2+ were not included when the average data were fitted to the Hill equation, because of the rundown of the current at these Ca2+ concentrations. The analysis shows that the apparent affinity of the channel for Ca2+ (EC50) increased about threefold from 950 nM at 100 mV to 350 nM at +100 mV (Fig. 2C). The Hill coefficient ranged from 2 to 3 from 100 to +100 mV, indicating that more than one Ca2+ ion bound to the channel to activate it.
To fit the data to the Hill equation, we calculated a predicted maximum conductance (Gmax) by fitting the conductance data for +100 mV and allowing all the parameters to float. We then assumed that this predicted Gmax was the same for all voltages. The data for all voltages were then fit by fixing the Gmax to this predicted Gmax value. This approach assumes that the predicted Gmax at 21 µM Ca2+ is larger than what is actually recorded. The recorded currents are smaller because of rundown or some secondary process. Using this method, the EC50 is estimated to be 350 nM at 100 mV and 950 nM at 100 mV. As an alternative method for estimating EC50 values, we measured the [Ca2+] that produced 50% of the observed conductance at 21 µM Ca2+. This concentration was 169 nM at +100 mV and 550 nM at 100 mV. Therefore, although the EC50 values calculated by these two methods differ by about twofold, the conclusion is the same: the EC50 for Ca2+ is smaller at positive potentials than it is at negative potentials.
Pharmacological Properties of ClCa Currents in IMCD-K2 Cells
NFA. Figure 3 shows the block of ClCa currents by NFA applied to whole cell patched IMCD-K2 cells in the bath. NFA is one of the most commonly used Cl channel inhibitors. Cells were patched in the whole cell configuration with symmetrical NMDG-Cl solutions applied to intra- and extracellular sides and perfused with bath solutions containing different NFA concentrations. To observe the inhibition of inward and outward currents by blockers, a high-Ca2+ (21 µM Ca2+) pipette solution was used to fully activate inward and outward currents.
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Figure 3A shows
I-V relationships recorded in the absence and presence of
various concentrations of NFA in the bath. The whole cell patch was voltage
clamped from the holding potential of 40 mV with a 200-ms-duration
voltage ramp from 100 to +100 mV. Inward and outward currents were
almost equally blocked in a concentration-dependent manner. The current traces
did not show a significant voltage-dependent block. In
Fig. 3B, the averaged
currents inhibited by each NFA concentration at +100 mV were expressed as a
fraction of the current at +100 mV in the absence of NFA
(I/INFA = 0) and plotted as a function of NFA
concentration. The data were fitted to the equation of the form
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DIDS, DPC, and A9C. Three other Cl channel blockers, namely, DIDS, DPC, and A9C, exhibited a voltage-dependent block. Figure 4A shows the effect of DIDS on IMCD ClCa currents. DIDS, unlike NFA, blocks in a voltage-dependent manner. The outward current was blocked at positive voltages more than the inward currents at negative voltages. At 300 µM, DIDS blocked outward currents by 80.8% at +100 mV but inward currents by 54.9% at 100 mV (Fig. 4, D and E). The block was reversible. DPC behaved in a similar way. It also inhibited ClCa currents in a voltage-dependent manner (Fig. 4B). DPC (300 µM) blocked outward currents by 70.0% at +100 mV but inward currents by 44.4% at 100 mV (Fig. 4, D and E). The inhibition was also reversible. A9C reversibly blocked ClCa currents in a voltage-dependent manner (Fig. 4C). A9C (300 µM) blocked outward currents by 68.3% at +100 mV but inward currents only by 26.8% at 100 mV (Fig. 4, D and E).
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DTT and tamoxifen. DTT, a reducing agent, has been reported to strongly block heterologously expressed CLCA currents (10, 14, 16). Figure 5A shows that 2 mM DTT has only a slightly inhibitory effect on outward ClCa currents in IMCD-K2 cells. The inhibition developed slowly. DTT (2 mM) inhibited the current by 35% at 100 mV in 46 min (Student's t-test, paired data, P = 0.01). A similar result was obtained in X. laevis oocytes (not shown).
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Tamoxifen is an effective blocker for swelling-activated Cl channels (4) and CLCA (17), but it did not block ClCa currents after 36 min in either IMCD-K2 cells (Fig. 5B) or X. laevis oocytes (not shown).
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DISCUSSION |
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ClCa currents have previously been studied in IMCD-3 and IMCD-K2 cell lines derived from IMCD. ClCa currents in IMCD-3 cells (40) are different from those in IMCD-K2 cells. In the absence of any agents that would raise intracellular Ca2+, the majority of IMCD-3 cells (64%) possessed a large, spontaneously active, outwardly rectifying, and time/voltage-independent Cl conductance (45). This suggested that the ClCa channels were open at resting Ca2+ levels. Changes in [Ca2+]i produced by chelating cytosolic Ca2+ by pre-loading the cells with BAPTA-AM or by elevating cytosolic Ca2+ with ionomycin or ATP altered current amplitude but did not alter the kinetics of the current (45). Because the biophysical properties are similar to those described for mouse CLCA1 functionally expressed in HEK-293 cells, it was suggested that the ClCa currents in IMCD-3 cells might be displayed by the CLCA family (10).
In contrast, ClCa currents in IMCD-K2 cells
(27) showed time and voltage
dependence. After the induction of slow ramps in
[Ca2+]i produced by exposing BAPTA-loaded
IMCD-K2 cells to ionomycin, whole cell currents exhibited pronounced outward
rectification with time-dependent activation or inactivation
(3). We have extended these
findings here. We applied whole cell recording to IMCD-K2 cells and observed
the Ca2+ activation by directly changing
[Ca2+]i. Lower Ca2+
(<800 nM) activated outwardly rectifying Cl currents,
whereas high Ca2+ elicited nearly linear
Cl currents. The currents activated at low
Ca2+ showed time-dependent activation and deactivation.
Ca2+ activation and Ca2+ affinity
of the ClCa channel were voltage dependent. The Ca2+
activation of the channel was dose dependent, and the EC50 was
0.35 µM at +100 mV and
0.95 µM at 100 mV. These data
indicate that ClCa currents in IMCD-K2 cells resemble those in secretory
epithelia and X. laevis oocytes
(2,
33).
One may be tempted to speculate that the differences in ClCa currents between IMCD-3 and IMCD-K2 cells may be related to the fact that IMCD-3 cells are derived from the terminal segment of the duct (40) and IMCD-K2 cells are derived from the initial segment (27). However, to our knowledge, there are no data on Ca2+-activated Cl channels in intact IMCD. Therefore, one must consider the possibility that the ClCa currents in these cell lines may not reflect the situation in the intact tissue. To determine whether ClCa currents differ between initial and terminal segments of the IMCD, electrophysiological recording of native cells from different segments of IMCD may clarify this point.
Comparison of Properties of ClCa Currents Between X. Laevis Oocytes and IMCD-K2 Cells
We have extensively studied ClCa channels in X. laevis oocytes (28, 38, 39). Comparing the properties of ClCa currents in oocytes with those in IMCD-K2 cells, we found that both channels resemble each other biophysically and pharmacologically, as summarized in Table 1. ClCa current activation in both cell types was controlled strictly by [Ca2+]i, and the Ca2+ activation showed voltage dependency. At low [Ca2+] <1 µM, the currents activated slowly on depolarization and deactivated on hyperpolarization and the steady-state I-V plot was strongly outwardly rectifying. At higher [Ca2+], the currents did not rectify and were time independent. This difference in behavior at different [Ca2+] was due to the different affinity of the channel for Ca2+. Both ClCa channels showed a higher affinity for Ca2+ at positive Vm but a lower affinity at negative Vm. The affinity difference is about two- to threefold between 100 and +100 mV.
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The similarity of the biophysical properties of ClCa channels for both IMCD-K2 and oocytes suggests a similar channel pore structure. The pharmacological properties support the hypothesis. We studied in detail the voltage-dependent block of ClCa channels by various anion blockers for Cl channels with excised patches from oocytes. All drugs studied blocked the channel from the outside in a voltage-dependent manner. The order of block potency was NFA > A9C > DIDS > DPC at +100 mV. Our analysis suggests that the channel is an elliptical cone with the largest opening facing the extracellular space (38). In this study, the four drugs applied in the bath also mainly blocked the outward ClCa currents in whole cell recordings of IMCD-K2 cells. The block also showed voltage dependence. The order of potency was similar to that for oocytes, although DIDS was more potent than A9C for IMCD-K2 cells (Table 1).
Molecular Identification of Potential Genes for ClCa Currents in IMCD-K2 Cells
So far, two molecular candidates for ClCa channels have been identified, bestrophins and CLCA. Bestrophin is the product of the gene isolated from a person with Best vitelliform macular dystrophy (VMD-2) by positional cloning (35, 37). Northern blot analysis has shown that bestrophin mRNA is strongly distributed in retina, brain, and spinal cord, and RT-PCR has indicated that bestrophin also existed in kidney. Bestrophin localizes to the basolateral plasma membrane of the retinal pigment epithelium (RPE) (34) and contributes to the slow light peak of the electrooculogram (8). The function of bestrophins has been enigmatic until recently, Sun et al. (46) reported that human bestrophins are Ca2+-sensitive Cl channels. Under whole cell recording, heterologously expressed human bestrophin-1 in HEK-293 cells showed voltage-independent Cl currents at 40 nM Ca2+. The heterologously expressed human bestrophin-1 has very high sensitivity to Ca2+. The EC50 is 0.1 µM at +100 mV, and the currents are time independent at all [Ca2+] (Qu Z, unpublished observations). These properties are different from those of ClCa currents in IMCD-K2 cells.
Although a putative family of Ca2+-sensitive Cl channels (CLCA) has been cloned (1, 7, 10, 1417, 43), this remains controversial. The properties of the heterologously expressed CLCA channels (10, 14, 15, 25) differ from those of native ClCa currents in various cells, but the possibility remains that a subunit is needed for CLCA channels to express currents like those for native ClCa. Mouse CLCA1 was cloned from a mouse lung library using homology cloning (10). The analysis of the tissue distribution of mCLCA1 showed strong expression in mouse epithelial tissues, including kidney. The properties of mouse CLCA1 have been studied by heterologous expression in HEK-293 cells (10). Mouse CLCA1 currents were activated by intracellular Ca2+ in whole cell recording. The currents were outwardly rectifying, but time independent at 2 µM [Ca2+]i. Obviously, the properties of mouse CLCA1 currents are not identical to those in IMCD-K2 cells.
Although our RT-PCR data indicated that the bestrophin and CLCA gene families were expressed in IMCD-K2 cells (data not shown), bestrophin and CLCA channels appear to be different from the ClCa currents we have studied in IMCD-K2 cells.
Physiological Functions of ClCa Conductance in IMCD-K2 Cells
Although the mouse and X. laevis are greatly separated from each other on the phylogenetic tree, the properties of ClCa channels in both species are very similar, indicating that the channel structure may be highly conserved; the IMCD-K2 cell line was originally established from the initial IMCD of mice (27). Reconstituted mouse IMCD-K2 epithelia electrogenically absorb Na+ (12, 27) through nucleotide-gated cation channels and display transepithelial anion secretion (27). Anion secretion in IMCD-K2 cells involves uphill accumulation of Cl/HCO3 across the basolateral membrane, followed by downhill movement across the apical membrane through Cl channels. Boese et al. (3) presented evidence that ClCa conductance was present at the apical membrane of reconstituted IMCD-K2 epithelial layers and participated in transepithelial anion secretion when ATP and ionomycin were applied to the layers. It is likely that a variety of hormones and paracrines can activate apical ClCa currents and affect IMCD anion secretion via increasing [Ca2+]i.
Wild-type CFTR was also expressed in all segments of rat kidney nephron, including IMCD (36). Cl currents mediated by CFTR were also revealed in IMCD and IMCD-K2 cells (3, 22, 48). Because the renal deficit in cystic fibrosis is not profound compared with that in the pancreas or small intestine, alternative mechanisms must exist to compensate for the loss of CFTR function (11, 44, 49). The possibility exists that ClCa in mouse IMCD may compensate for defective CFTR. It has been hypothesized that the severity of the deficit due to defective CFTR in different organs correlates with the expression of an apical-epithelial ClCa conductance and that this ClCa conductance can functionally compensate for the loss of CFTR activity in humans as in the transgenic cystic fibrosis mouse (5, 11, 49).
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DISCLOSURES |
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FOOTNOTES |
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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.
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REFERENCES |
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