IK channels are involved in the regulatory volume decrease in human epithelial cells

Jun Wang, Shigeru Morishima, and Yasunobu Okada

Department of Cell Physiology, National Institute for Physiological Sciences; and Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation, Okazaki 444-8585, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Parallel activation of Ca2+-dependent K+ channels and volume-sensitive Cl- channels is known to be responsible for KCl efflux during regulatory volume decrease (RVD) in human epithelial Intestine 407 cells. The present study was performed to identify the K+ channel type. RT-PCR demonstrated mRNA expression of Ca2+-activated, intermediate conductance K+ (IK), but not small conductance K+ (SK1) or large conductance K+ (BK) channels in this cell line. Whole cell recordings showed that ionomycin or hypotonic stress activated inwardly rectifying K+ currents that were reversibly blocked by IK channel blockers [clotrimazole (CLT) and charybdotoxin] but not by SK and BK channel blockers (apamin and iberiotoxin). Inside-out recordings revealed the existence of CLT-sensitive single K+-channel activity, which exhibited an intermediate unitary conductance (30 pS at -100 mV). The channel was activated by cytosolic Ca2+ in inside-out patches and by a hypotonic challenge in cell-attached patches. The RVD was suppressed by CLT, but not by apamin or iberiotoxin. Thus we conclude that the IK channel is involved in the RVD process in these human epithelial cells.

Ca2+-activated K+ channel; patch clamp; clotrimazole; osmotic swelling


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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CELL VOLUME REGULATION is an essential function for animal cells because osmotic perturbation is coupled to a variety of physiological and pathological processes, such as cell proliferation, cell differentiation, and apoptosis (23, 37). Under hypoosmotic conditions, a regulatory volume decrease (RVD) is accomplished by efflux of K+, Cl-, and organic osmolytes, which results in the extrusion of osmotically obliged water in a variety of cell types (15, 35, 36).

Osmotic cell swelling has been reported to be associated with the activation of different types of K+ channels, including Ca2+-activated K+ channels (5, 21, 27, 39, 50, 52, 57, 58), stretch-activated K+ channels (8, 31, 42, 54), Ca2+- and stretch-activated K+ channels (20, 38), voltage-gated K+ channels (1, 6, 9, 43, 46), MinK channels (3, 28), and two-pore (2P) domain TASK channels (33). Summarizing previous observations, Pasantes-Morales and Morales-Mulia (40) recently suggested that the RVD in most types of epithelial cells involves Ca2+-dependent K+ channels, whereas that in nonepithelial cells involves Ca2+-independent K+ channels.

Ca2+-activated K+ channels are ubiquitously distributed in mammalian cells, and these channels play important roles in many different cell functions. On the basis of their electrophysiological characteristics, three major classes of Ca2+-activated K+ channels have been described (24): voltage-dependent, large-conductance K+ channels (BK or hSlo for its alpha -subunit); voltage-independent, small-conductance K+ channels (SK); and inwardly rectifying, intermediate-conductance K+ channels (IK). It is well known that the BK, IK, and SK channel proteins are products of three different genes (10, 56). Although there is ample evidence that the RVD process involves Ca2+-activated K+ channels in Intestine 407 cells (13, 14), it is not known which type of Ca2+-activated K+ channel protein is involved. Thus, in the present study, we had the aim of determining the molecular identity of this volume-regulatory K+ channel.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Cell culture. A human epithelial cell line, Intestine 407, was cultured in monolayer in Fischer's medium supplemented with 10% newborn bovine serum, as described previously (13). For patch-clamp and volume measurements, cells were detached from the plastic substrate and suspended, as described previously (22). After cell culture was suspended with agitation, the cells were placed in a chamber (0.3 ml) and, after they had attached to the glass bottom, perfused with bath solution at about 3 ml/min by gravity feed from reservoirs. Cells used for some cell-attached recordings were first loaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) by preincubation with 50 µM BAPTA-AM for 15 min at 37°C.

RT-PCR. Poly(A)+ RNA was extracted from Intestine 407 cells by using the Direct mRNA purification kit with magnetic porous glass (MPG; CPG, Lincoln Park, NJ). Briefly, the cells were detached from culture flasks and homogenized in a buffer containing LiDs and ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Poly(A)+ RNA was extracted with MPG-bound oligo(dT)25s. The isolated poly(A)+ RNA was reverse transcribed by using the SuperScript preamplification system (Invitrogen, Carlsbad, CA). The resultant first strand cDNA was used for PCR. Primers were designed and synthesized according to the published sequences of cDNA encoding human SK1 (hSK1), human IK (hIK) (18), and human BK alpha -subunit (hSlo) (51), as summarized in Table 1. As a positive control, we also amplified mRNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the primers listed in Table 1.

                              
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Table 1.   Primer sequence

PCR was performed in a total volume of 50 µl of a buffer solution containing (in mM) 20 Tris · HCl (pH 8.4), 50 KCl, 1.5 MgCl2, 0.2 dNTP, and 250 units Ampli Taq Gold (Perkin-Elmer, Norwalk, NC). The thermal cycle protocol used was 94°C for 1 min, 55°C for 2 min, and 72°C for 3 min for 40 cycles with a programmable thermal cycler (GeneAmp PCR System 9600; Perkin Elmer Life Sciences, Boston, MA). A negative control experiment was performed using primers and RNA that had not been reverse transcribed. The products of RT-PCR were electrophoresed and sized separately on a 2% agarose gel. Positive bands were cut out of the gel, and the cDNA was extracted and purified using the GENE CLEAN kit (Bio101, Carlsbad, CA), after which it was immediately subjected to sequencing with the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit and an ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA).

Cell volume measurements. Cell volume was measured at room temperature (22-26°C) or 37°C by an electronic sizing technique with a Coulter-type cell size analyzer (CDA-500; Sysmex, Kobe, Japan), as previously described (13). The mean volume of the cell population was calculated from the cell volume distribution after the machine was calibrated with latex beads of known volume.

Isotonic (310 mosmol/kgH2O) or hypotonic (200 mosmol/kgH2O) solution consisted of (in mM) 95 NaCl, 4.5 KCl, 1 MgCl2, 1 CaCl2, 110 or 0 mannitol, and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)/NaOH (pH 7.3).

Patch-clamp experiments. Whole cell and single-channel recordings were performed at room temperature. Pipettes were pulled from borosilicate glass capillaries with a micropipette puller (P-2000; Sutter Instruments, Novato, CA). The electrode had a resistance of 1.8-2.5 MOmega for whole cell recordings and of around 7 MOmega for single-channel recordings when filled with pipette solution. Data were acquired using an EPC-9 amplifier and Pulse software (HEKA Electroniks, Lambrecht, Germany). Current signals were low-pass filtered at 2.9 and 1.0 kHz using a four-pole Bessel filter and digitized at 10 and 4 kHz, in whole cell and single-channel recordings, respectively. Sampled data were analyzed by an original software application called PulseMate and Origin 6.1 (Origin Lab, Northampton, MA). In most experiments, a grounded Ag-AgCl pellet electrode was placed in the perfusion solution. When Cl--free bath solution was used, a 3 M KCl-agar bridge was used.

In whole cell recordings, series resistance (<6 MOmega ) was compensated (to 60-70%) to minimize voltage errors. The time course of whole cell current activation and recovery was monitored by repetitively applying (every 15) ramp pulses (1-s duration) from -100 to +100 mV from a holding potential of -40 mV. To obtain whole cell I-V relations, step pulses (1-s duration, 5-s interval) were applied from a prepotential of -100 mV (for 400 ms) to test potentials (1-s duration) of -100 to +100 mV (in 20-mV increments). To minimize Ca2+- and/or swelling-activated anion currents, whole cell recordings were carried out using low-Cl- intracellular solution and Cl--free external solution containing a Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The Cl--free isotonic (320 mosmol/kgH2O) or hypotonic (280 mosmol/kgH2O) bath solution contained (in mM) 4.2 K-gluconate, 140 Na-gluconate, 1 MgSO4, 2 CaSO4, 40 or 0 mannitol, and 10 HEPES/NaOH (pH 7.4), as well as 0.05 NPPB. The low-Cl- intracellular (pipette) solution (300 mosmol/kgH2O) contained (in mM) 147 K-gluconate, 1 MgCl2, 2 Mg-ATP, 0.08 EGTA, 0.03 CaCl2, 10 mannitol, and 10 HEPES/KOH (pH 7.3). The free Ca2+ concentration ([Ca2+]) was 0.036 µM. In some experiments, 5 mM BAPTA was added to the low-Cl- pipette solution from which EGTA and CaCl2 were removed. To test the K+ selectivity of the channel observed, K-gluconate in the bath solution was replaced with Na-gluconate on an equimolar basis.

Single-channel recordings were made in the cell-attached mode or the excised, inside-out mode. When patches contained only one active channel, single-channel analysis was performed for events, the closed level of which was flat and stable. Amplitude histograms were plotted from data samples of 30- to 60-s duration from at least eight patches and were fitted by two Gaussian curves using a Peak Fitting module in Origin 6.1. Single-channel conductance was determined by the difference of these two Gaussian's center values, and open probability (Po) was by the area under the curve of each Gaussian. For inside-out recordings, the intracellular side was perfused with Cl--free bath solution (285 mosmol/kgH2O) consisting of (in mM) 144 K-gluconate, 1 MgSO4, 2 EGTA, 10 HEPES/NaOH (pH 7.4), and CaSO4 at concentrations calculated by CaBuf software (provided by Dr. G. Droogmans, KUL, Belgium) to give [Ca2+] of 0.01, 0.1, 1, and 100 µM. To test the K+ selectivity of the channel, K-gluconate in the bath solution was replaced with equimolar Na-gluconate. The pipette was filled with extracellular solution (285 mosmol/kgH2O) composed of (in mM) 144 K-gluconate, 2 CaCl2, 1 MgCl2, and 10 HEPES/KOH (pH 7.4). At the end of experiments, patches were perfused with 5 µM clotrimazole (CLT) to confirm that the current was a CLT-sensitive one. For on-cell patches, high-K+ bath solution was used to nullify the membrane potential. High-K+, Cl--free isotonic (290 mosmol/kgH2O) or hypotonic (230 mosmol/kgH2O) bath solution contained (in mM) 110 K-gluconate, 4 Na-gluconate, 1 MgSO4, 2 CaSO4, 60 or 0 mannitol, and 10 HEPES/KOH (pH 7.4). The pipette solution (285 mosmol/kgH2O) contained (in mM) 144 K-gluconate, 2 CaCl2, 1 MgCl2, and 10 HEPES/KOH (pH 7.4).

Chemicals. EGTA, BAPTA, BAPTA-AM, and Na-gluconate were purchased from Wako Pure Chemical Industries (Osaka, Japan), and clofilium was from Research Biochemicals (Natick, MA). All other reagents were obtained from Sigma-Aldrich Japan (Tokyo, Japan). Stock solutions of 5 mM CLT, 50 mM NPPB, and 1 mM ionomycin were prepared in dimethyl sulfoxide (DMSO). Tetraethylammonium (TEA), charybdotoxin (ChTX), iberiotoxin, and apamin were directly added to the appropriate solution before use.

Statistical analysis. Data are presented as means ± SE of n observations. Statistical differences in data were evaluated by one-dimensional ANOVA and Scheffé's post hoc multiple comparison tests. Data were considered to be significant at P < 0.01.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Molecular expression of IK channels. RT-PCR was performed on RNA isolated from Intestine 407 cells to examine the expression of five types of Ca2+-activated K+ channels. As shown in Fig. 1 (lanes 2 and 4), DNA fragments of expected size at 241 and 457 bp were amplified by hIK-specific primers (Table 1, pairs 1 and 2) from reverse transcribed cDNA. The nucleotide sequence of these PCR products was completely identical to the corresponding sequence in the hIK channel (18). However, no PCR product was amplified when reverse transcriptase was omitted from the reaction (Fig. 1, lanes 3 and 5).


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Fig. 1.   RT-PCR analysis of Ca2+-activated K+ channel mRNA expression in Intestine 407 cells. Lane 1: markers (100 bp DNA ladder); lane 2: hIK (human IK, intermediate-conductance K+ channel) PCR product resulting from RT-PCR with reverse transcriptase (primer pair 1: predicted size 241 bp); lane 3: negative control hIK PCR product resulting from RT-PCR without reverse transcriptase (primer pair 1); lane 4: hIK PCR product resulting from RT-PCR with reverse transcriptase (primer pair 2: predicted size 457 bp); lane 5: negative control hIK PCR product resulting from RT-PCR without reverse transcriptase (primer pair 2); lane 6: SK1 (SK, small-conductance K+ channel) PCR product (primer pair 1); lane 7: hSlo (human large-conductance K+ alpha -subunit channel) PCR product (primer pair 1); and lane 8: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR product (predicted size 452 bp).

In contrast, as shown in Fig. 1 (lanes 6 and 7), no specific DNA products were amplified by primers (Table 1, pair 1) specific for SK1 and hSlo. The same results were obtained using another set of primers (Table 1, pair 2). However, GAPDH mRNA was consistently detected (Fig. 1, lane 8).

Functional expression of IK channels. When Intestine 407 cells were dialyzed with low-Cl- pipette solution and exposed to Cl--free bath solution containing a Cl- channel blocker, NPPB (50 µM), addition of ionomycin (1 µM) increased whole cell currents, as observed previously (17). As shown in Fig. 2A, the ionomycin-activated current exhibited slight inward rectification. Time-dependent activation was observed at large positive potentials (upper inset). When the extracellular K+ concentration ([K+]o) was increased, the reversal potential (Erev) shifted in the positive direction. The Erev shift per 10-fold increase in [K+]o was 53 mV (lower inset), indicating high selectivity of K+ (PNa/PK < 0.01). A wide spectrum K+ channel blocker (24, 56), TEA (20 mM), partially suppressed the currents (Fig. 2Ba). The current was largely abolished by 200 nM CLT (Fig. 2Bb), which is a blocker specific to IK (53). The ionomycin-activated current was also sensitive to 20 nM ChTX (Fig. 2Bc), which is known to block not only BK channels (32) but also IK channels (11, 45). The Erev values for TEA-, CLT-, and ChTX-sensitive currents were -80.6 ± 5.3, -82.8 ± 4.2, and -83.6 ± 3.0 mV (n = 6), respectively, and these values are close to that of the equilibrium potential for K+ (-90 mV). In contrast, the ionomycin-activated current was insensitive to 100 nM apamin (Fig. 2Bd) and 100 nM iberiotoxin (data not shown, n = 3), blockers that are specific to SK and BK (24, 56), respectively.


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Fig. 2.   Ionomycin-induced whole cell K+ channel currents in Intestine 407 cells and their sensitivity to K+ channel blockers. Arrowheads indicate zero current level. A: current-voltage relations. Data represent the mean values of 8 experiments. SE values are presented as vertical bars, where the values exceed the symbol size. Upper inset: representative traces of the current response to step pulses from -100 to 100 mV in 20-mV increments. Lower inset: relation between the reversal potential (Erev) and the logarithm of the extracellular K+ concentration ([K+]o). The slope value given in the figure was determined by regression fitting. B: representative traces of the current response to step pulses 3-5 min after application of 20 mM tetraethylammonium (TEA) (a), 200 nM clotrimazole (CLT) (b), 20 nM charybdotaxin (ChTX) (c), and 100 nM apamin (d). Data represent 8 similar experiments.

When single-channel recordings were performed in inside-out patches under symmetrical K+ conditions, unitary events were found to be induced by increases in the intracellular Ca2+ concentration (Fig. 3A). The amplitude histogram was well fitted by two Gaussian curves. The Po value increased from 0 to 0.17 ± 0.04 and 0.45 ± 0.03 (n = 8) at -100 mV, when [Ca2+] was increased from 0.01 to 0.1 and 1 µM, respectively. This channel activity showed no obvious voltage dependence. The Po values at -100, -40, +40, and +100 mV were 0.46 ± 0.06, 0.41 ± 0.05, 0.42 ± 0.04, and 0.44 ± 0.03 (n = 8), respectively, at 100 µM [Ca2+]. As shown in Fig. 3B, the single-channel activity at 100 µM [Ca2+] was sensitive to 200 nM CLT in a reversible manner. The current-voltage curve exhibited slight inward rectification (Fig. 3C). The mean unitary conductance was 30 ± 2 pS at -100 mV and 18 ± 4 pS (n = 6) at +100 mV. When the intracellular K+ concentration was decreased from 144 to 4.2 mM, the Erev value shifted from 0 to +69 mV, indicating K+ selectivity. Both BK-like larger conductance and SK-like smaller conductance events were never observed in inside-out patches in the tested [Ca2+] range.


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Fig. 3.   Ca2+-activated single K+ channel currents in inside-out patches excised from Intestine 407 cells and their sensitivity to CLT. Arrowheads indicate the closed-state level. A: effects of Ca2+ concentration on the single-channel activity recorded at -100 mV. B: effect of CLT on the single-channel activity recorded at -100 mV and [Ca2+] = 100 µM. The middle and bottom records were obtained around 3 min after application and washout, respectively, of CLT. C: current-voltage curves for single-channel currents recorded at [Ca2+] = 100 µM under symmetrical K+ conditions ([K+]o = 144 mM) and asymmetrical K+ conditions ([K+]o = 4.2 mM). Data represent the mean of 6 experiments (vertical bar: SE).

Combining the results of the whole cell and single-channel recordings, we conclude that Intestine 407 cells functionally express CLT-sensitive, Ca2+-dependent IK channels.

Swelling-induced activation of IK channels. A hypotonic challenge reversibly induced increases in cell size and membrane currents in Intestine 407 cells under whole cell clamp in which low-Cl- pipette solution and Cl--free bath solution containing 50 µM NPPB were used (Fig. 4A). The profile of the swelling-induced current (Fig. 4Aa) and the I-V relation (Fig. 4Ac) were similar to those of ionomycin-induced current (Fig. 2A). The swelling-induced current was very sensitive to CLT (200 nM; Fig. 4Ab). The Erev for CLT-sensitive current was -84.3 ± 7.2 mV (Fig. 4Ac). When intracellular Ca2+ was chelated with 5 mM BAPTA introduced to the pipette solution, a hypotonic challenge induced cell swelling but activated whole cell currents very little, as shown in Fig. 4B.


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Fig. 4.   Swelling-induced whole cell K+ currents in Intestine 407 cells and their sensitivity to CLT and cytosolic Ca2+ chelation. Arrowheads represent zero current level. A: representative record showing currents before, during, and after exposure to hypotonic solution in the absence or presence of CLT (200 nM) during application of ramp pulses (from -100 to +100 mV) and step pulses of -100 to +100 mV in 20-mV increments. Expanded traces of the record under isotonic and hypotonic conditions are shown in a and b, and the current-voltage relationships before (black-triangle) and during exposure to hypotonic solution in the absence (open circle ) and presence () of CLT are shown in c. Each symbol represents the mean instantaneous current of 7 observations ± SE (vertical bar). B: representative record showing currents during application of ramp pulses and step pulses before, during, and after exposure to hypotonic solution under conditions in which cytosolic Ca2+ was chelated with 5 mM BAPTA added to the pipette solution. Data represent 8 similar experiments.

Single-channel events were observed only rarely in cell-attached patches on Intestine 407 cells exposed to isotonic solution (Fig. 5). Osmotic swelling induced by hypotonic stress was always associated with a prominent increase in single-channel activity with an intermediate unitary current of 3.1 ± 0.2 pA (n = 10) at a pipette potential of +100 mV (intracellular potential of around -100 mV), under high K+ conditions (Fig. 5A). Unitary events with SK-like smaller or BK-like larger single-channel conductance were never observed. The Po value of intermediate type single-channel events increased from around 0 to 0.43 ± 0.04 (n = 10) after induction of osmotic swelling. For BAPTA-loaded swollen cells, however, swelling-induced activation of intermediate-conductance single-channel events were far less prominent (Fig. 5B), and the Po value was 0.004 ± 0.001 (n = 10) under hypotonic conditions. Also, swelling-induced activation of single-channel events was largely prevented by 200 nM CLT when it was added to the pipette solution (Fig. 5C). The Po value was 0.010 ± 0.005 (n = 8) during osmotic swelling in the presence of 200 nM CLT.


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Fig. 5.   Swelling-induced single K+ channel events in cell-attached patches on Intestine 407 cells and their sensitivity to cytosolic Ca2+ chelation and extracellular CLT. Arrowheads indicate the closed-state level. A: representative record showing currents at the pipette potential of +100 mV before and after exposure to hypotonic solution. Bottom panels a and b represent expanded traces. B: representative record of currents before and during exposure to hypotonic solution from an on-cell patch of a cell preincubated with 50 µM BAPTA-AM. C: representative record of currents before and during exposure to hypotonic solution with CLT (200 nM) present in the pipette solution.

Both these whole cell and single-channel data indicate that activation of CLT-sensitive, Ca2+-dependent, IK channels is associated with osmotic swelling in Intestine 407 cells.

Involvement of IK channel in the RVD process. After Intestine 407 cells were exposed to hypotonic solution at room temperature, the mean cell volume promptly increased and then gradually recovered (Fig. 6), as observed previously (13). The RVD was inhibited by the application of 200 nM CLT (Fig. 6A). However, application of SK and BK channel blockers, apamin (100 nM) and iberiotoxin (100 nM), failed to significantly affect the RVD (Fig. 6A). Also, the RVD was not inhibited by another K+ channel blocker, clofilium (100 µM; Fig. 6B), which is known to block both KCNQ (MinK) (2) and KCNK5 (TASK) channels (33). CLT sensitivity and clofilium insensitivity of the RVD that were essentially the same were observed at 37°C (data not shown, n = 6 each). In light of these data, we conclude that the K+ channel type involved in the RVD of Intestine 407 cells is the CLT-sensitive IK channel.


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Fig. 6.   Effects of K+ channel blockers on volume regulation after osmotic swelling in Intestine 407 cells. Blockers were added 2-3 min before and during application of hypotonic solution. Each symbol represents the mean ± SE (vertical bar) of 7-11 observations. A: regulatory volume decrease (RVD) in the absence of CLT (control, open circle ), presence of CLT (200 nM, ), presence of apamin (100 nM, black-triangle), or presence of iberiotoxin (100 nM, black-down-triangle ). Data points at the given time were compared among groups by ANOVA, and statistical difference was analyzed by Scheffé's post hoc test. * P < 0.01 vs. control. B: RVD in the absence (control, open circle ) or presence of clofilium (100 µM, ).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A number of types of K+ channels have been demonstrated to be involved in K+ efflux during volume regulation after osmotic swelling (40). In human epithelial Intestine 407 cells, we previously concluded that the volume-regulatory K+ channel is classified into a Ca2+-activated one, based on the following observations: the RVD was inhibited by chelation of cytosolic Ca2+ and by application of a K+ channel blocker Ba2+ (13), osmotic cell swelling brought about activation of Ca2+-dependent K+ conductance under voltage-clamp (13), and swelling-induced K+ conductance activation was associated with an increase in the cytosolic free Ca2+ concentration measured with Ca2+-selective microelectrodes (14). The present study provided five lines of evidence that the identity of this channel is an hIK channel (16, 18): 1) RT-PCR demonstrated exclusive expression of hIK mRNA (Fig. 1), 2) whole cell K+ currents activated by ionomycin exhibited weak voltage dependence and were sensitive to the IK channel blockers CLT and ChTX (Fig. 2), 3) single K+-channel events induced by an increase in cytosolic Ca2+ exhibited slight inward rectification with an intermediate unitary conductance and were sensitive to CLT (Fig. 3), 4) swelling-induced whole cell and single-channel currents were both inhibited by the presence of CLT and by cytosolic Ca2+ chelation (Figs. 4 and 5), and 5) the RVD was inhibited by CLT (Fig. 6A).

Maxi-K+ or BK channels, which exhibit striking voltage dependence (24), have been found to be activated during osmotic swelling in a number of other cell types (4, 7, 19, 44, 49). In human osteoblast-like C1 cells, BK channels, together with IK channels, were shown to be involved in the RVD (58). In Intestine 407 cells, however, a BK channel blocker, iberiotoxin, failed to inhibit the RVD and Ca2+-activated whole cell K+ currents. Also, swelling- or Ca2+-activated K+ channel events exhibited weak voltage dependence and never exhibited Maxi unitary conductance. Moreover, mRNA of the BK channel alpha -subunit hSlo was never detected by RT-PCR. Involvement of apamin-sensitive SK channels in the RVD of Intestine 407 cells could also be excluded by the observation that there was no RT-PCR signal for SK1 and that the RVD and the Ca2+-activated whole cell K+ currents were not sensitive to apamin.

Recently, Niemeyer et al. (33) provided clear evidence for the involvement of the TASK-2 type of 2P domain K+ channels in the RVD of Ehrlich ascites tumor cells. It is known that 2P domain K+ channels are independent of cytosolic Ca2+ and are insensitive to Ba2+ (41). Thus, in Intestine 407 cells, TASK-2 channels may not play a role in the RVD, which was found to be dependent on cytosolic Ca2+ (13, 14) and sensitive to Ba2+ (13). In the present study, in fact, the RVD of Intestine 407 cells was found to be totally insensitive to clofilium (Fig. 6B), which is known to block TASK-2 (33). Clofilium insensitivity may also rule out the involvement of the MinK channel, which is sensitive to this class III antiarrhythmic drug (2).

The involvement of IK channels in the RVD process has been reported in mouse erythroid cells (32), human T lymphocytes (21), and human tracheal cells (25). Vázquez et al. (55) reported that CFTR expression is a prerequisite to swelling-induced IK channel activation in tracheal cells. However, the present study provided an example of the involvement of IK channel activation in Intestine 407 cells, which do not express CFTR (12). In Intestine 407 cells, IK channels could be activated by a cytosolic Ca2+ rise due to swelling, as well as due to stimulation of phospholipase C-linked P2Y2 receptor by ATP released from swollen cells (37).

The RVD mechanism is of essential importance to some cell types, such as enterocytes, in which the swelling-inducing osmotic gradient across the cell membrane is produced by active solute uptake (29, 34, 47). K+ channel activation may play an important role in volume regulation during Na+-coupled absorption of organic solutes in small intestine, as Ba2+-sensitive K+ conductance activation was observed in Necturus small intestinal enterocytes during exposure to galactose (25, 26). Single-channel recordings demonstrated that L-alanine application activated Ca2+-dependent K+ channels with an intermediate unitary conductance in Necturus enterocytes (48). In guinea pig jejunum enterocytes, the RVD that takes place after osmotic swelling due to Na+-coupled solute absorption was found to be inhibited by ChTX (29, 30). Our results suggest that IK channels play, at least in part, a volume-regulatory role in small intestinal epithelial cells during Na2+-dependent organic solute absorption in vivo.


    ACKNOWLEDGEMENTS

We are grateful to R.Z. Sabirov for discussion, to K. Shigemoto, S. Tanaka, and E.L. Lee for technical assistance, and to T. Okayasu for secretarial assistance.


    FOOTNOTES

Address for reprint requests and other correspondence: Y. Okada, Dept. of Cell Physiology, National Institute for Physiological Sciences, Myodaiji-cho, Okazaki 444-8585, Japan (E-mail: okada{at}nips.ac.jp).

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.

First published September 11, 2002;10.1152/ajpcell.00132.2002

Received 20 March 2002; accepted in final form 4 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Baraban, SC, Bellingham MC, Berger AJ, and Schwartzkroin PA. Osmolarity modulates K+ channel function on rat hippocampal interneurons but not CA1 pyramidal neurons. J Physiol 498: 679-689, 1997[Abstract].

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