Laboratory of Physiology, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
Submitted 2 September 2003 ; accepted in final form 14 October 2003
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ABSTRACT |
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distal colon; electrogenic Na+ absorption; short-circuit current; whole cell patch clamp; -ENaC
Since the amiloride-sensitive epithelial Na+ channel (ENaC) composed of three homologous -,
-, and
-subunits (
-,
-, and
-ENaC) was first cloned from rat distal colonic epithelium (7, 8, 30, 31), it has been hypothesized that the subunits may contribute to the molecular nature of the amiloride-sensitive epithelial Na+ channel naturally expressed in the mammalian distal colon. Extensive molecular studies have supported this hypothesis. Northern and Western analyses have shown that
-subunit is constitutively expressed, and expression of
- and
-ENaC subunits is upregulated by aldosterone (1, 14, 18, 21). In situ hybridization and immunocytochemical studies have also provided evidence that the subunits are present mostly in surface epithelial cells (12, 21, 44, 46, 50), with the restricted expression being in line with the functional observations mentioned above (13, 28, 32). Furthermore, transepithelial amiloride-sensitive potential difference in the rectum of mice bearing Liddle's mutation on the
-subunit of ENaC is shown to be larger than that of normal mice (40), suggesting a role of the ENaC subunit in the electrogenic Na+ absorption.
Despite these extensive molecular studies, electrophysiological properties of amiloride-sensitive Na+ currents naturally expressed in the colonic surface epithelial cells still remain largely unknown even at the whole cell current level, most probably because of the technical difficulties. Thus it is not known whether the native channel currents would fulfill the hallmark electrophysiological properties of heterologously expressed -ENaC currents, including a high selectivity for Na+ over K+ (PNa/PK > 10), a Li+ conductance 1.5- to 2.0-fold greater than the Na+ conductance, a high affinity for amiloride (Ki of 0.10.5 µM), a unitary single-channel conductance of about 5 and 8 pS for Na+ and Li+, respectively, and a slow kinetics of gating (8, 25). Given a functional diversity of amiloride-sensitive Na+-permeable channels with pore properties that are not consistent with those expected from expressed
-rENaC currents in various native Na+-transporting epithelia (20), molecular study alone may have a limitation in identifying the proteins composing a native ENaC channel in the mammalian distal colon. Even though the subunits of ENaC may be involved, different oligomeric structures of the three proteins might form conducting pores with altered functional properties. In fact, heterologous expression studies using the Xenopus oocytes expression system suggest that subunit combinations may result in channels with diverse biophysical properties (4, 7, 8, 19, 34, 54).
Thus the aim of the present work was to examine whether electrophysiological properties of macroscopic currents of native amiloride-sensitive Na+ channels expressed in surface cells of the distal colon are similar to those reported for heterologously expressed -rENaC. Using the short-circuit current (Isc) measurement technique, we first show the presence of amiloride-sensitive electrogenic Na+ currents (Isc-amil) in mucosa prepared from the rectal colon, but not from proximal parts of the distal colon, of rats fed a normal salt diet. We then used RT-PCR to confirm the presence of transcripts of
-,
-, and
-rENaC subunits in the mucosal preparation of rectal colon. Using the conventional whole cell patch-clamp technique, we finally show that electrophysiological properties of an amiloride-sensitive whole cell current in surface cells in cryps freshly isolated from the rectal mucosa resemble those reported for macroscopic currents of heterologously coexpressed
-rENaC subunits in terms of amiloride sensitivity, ion selectivity, and extracellular Na+ concentration dependency of the currents. The present work provides electrophysiological characterization of native amiloride-sensitive whole cell Na+ currents, which are most likely responsible for electrogenic Na+ transport in surface cells of rat rectal colon, and functional evidence for the correlation between the physiological (functional) and molecular expression of ENaC subunits in surface cells of the mammalian distal colon.
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MATERIALS AND METHODS |
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Each segment was cut open longitudinally and flushed with the standard NaCl-rich solution to remove fecal pellets. Epithelium (mucosa) was separated from underlying submucosa and muscle layers by using a glass slide to gently scrape along the length of the colonic segment, as described elsewhere (53). Stripped mucosa was mounted in a modified Ussing chamber with a tissue holder (EasyMount Chamber; Physiologic Instruments, San Diego, CA) having an aperture surface area of 0.5 cm2 and was bathed bilaterally in a solution (pH 7.4) consisting of (in mM) 115 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 25 NaHCO3, 10 HEPES (pH 7.4, adjusted with NaOH), and 10 D-glucose. In experiments in which effects of removal of Na+ on amiloride-sensitive Isc were examined, the basolateral and apical solutions containing 115 mM N-methyl-D-glucamine (NMDG)-Cl and 10 mM HEPES (pH 7.4, adjusted with NMDG) instead of NaCl and HEPES (pH 7.4, adjusted with NaOH) in a control solution (pH 7.4) consisting of (in mM) 115 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 25 KHCO3, and 10 HEPES (pH 7.4) were used. The bathing solution was continuously gassed with a mixture of 95% O2-5% CO2 and had a pH of 7.4. In a large number of experiments, the luminal and basolateral sides of the epithelium were perfused with the bathing solution (at 37°C) continuously at a rate of 1 ml/min (chamber volume of 5 ml). When drugs were applied to either side of the bathing solution, 5 µl of stock solutions were added to give the desired final concentration, immediately followed by continuous perfusion (1 ml/min) with the solution containing the appropriate concentration of drugs. When drugs were removed, 2.5 ml of the solution were withdrawn from the apical or basolateral bath and replaced successively with an equal volume of the standard solution containing no drugs. This procedure was repeated 20 times in parallel to continuous perfusion with a drug-free solution. We also performed experiments under a nonperfused condition and found that the two methods basically yielded similar results. Furthermore, basal bioelectrical properties were not affected by the presence or absence of indomethacin (10 µM), which suppresses prostaglandin production, in the solutions bathing serosal surfaces (data not shown). Thus the data under these different experimental conditions were pooled and are reported together. Tetrodotoxin was not included in the solution. All preparations were allowed to equilibrate for 4090 min after being mounted on the chambers before the measurements were taken, since Isc usually displayed an initial transient phase that started at high values, followed by a lower plateau phase, as reported elsewhere (43).
The tissues were continuously short-circuited to monitor Isc (µA/cm2) by using a voltage-clamping amplifier (CEZ-9100; Nihon Kohden, Tokyo, Japan). Transepithelial electrical potential difference (Vte) was measured by a pair of pipette-shaped voltage-sensing electrodes made of sintered Ag-AgCl pellet (Physiologic Instruments) filled with a solution of 3% (wt/vol) agarose in 3 M KCl solution, with the transepithelial current being passed across the tissue through a pair of pipette-shaped electrodes made of Ag wire (Physiologic Instruments) filled with a solution of 3% (wt/vol) agarose in 3M KCl solution. Isc is referred to as positive for current flowing across the epithelium from the mucosal side to the serosal side. Typically, the transepithelial resistance (Rte) was estimated from the current change in response to square voltage pulses (±2 or +0.5 mV, 1-s duration) imposed across the mucosa at 5- or 2-min intervals. The resistance of the bathing fluid between the voltge-sensing electrodes was measured and compensated by the amplifier before each experiment.
To measure basolateral Na+ pump activity, short-circuited epithelia bathed with the standard NaCl-rich solution were first exposed to apical amiloride (10 µM) to block the Na+ channels in this membrane, which was then permeabilized using nystatin (200 µg/ml). Apical nystatin caused a slowly developing increase in current, which was reduced by addition of 1 mM ouabain to the basolateral solution (see also Fig. 1C). We assumed that the ouabain-sensitive current would be proportional to the transport activity of the Na+-K+ pumps present in the basolateral membrane, as described elsewhere (41).
Patch-clamp analyses. The preparation of colonic crypts was similar to that described elsewhere (6, 11, 29). In brief, portions of mucosa obtained from RC were rinsed with a standard NaCl-rich bath solution (see below) three times and put into a 50-ml flask with 5 ml of a solution (pH 7.4 adjusted with NaOH) containing (in mM) 145 NaCl, 5 KCl, 10 HEPES, 10 D-glucose, and 10 EDTA. Mucosal portions were consecutively incubated at 37°C for 12 min, every 4 min during which they were agitated gently for 30 s to release surface and crypt epithelium. Isolated crypts were collected by centrifugation (40 g for 30 s), resuspended in the standard NaCl-rich solution containing amiloride (10 µM), and stored at 4°C until use.
Isolated crypts were transferred onto a poly-L-lysine (0.01%)-coated glass coverslip in a recording chamber mounted on the stage of an inverted microscope (IX50, Olympus). Bathing solutions were continuously perfused into the chamber by gravity feed from reservoirs. All experiments were performed at room temperature (2023°C). Current recordings were made only from surface epithelial cells in isolated crypts, using the standard whole cell configuration of the patch-clamp technique (22). Care was taken to clearly define the surface cells by their location under the microscope. The patch-clamp pipettes, which were pulled from glass capillaries (LG16, Dagan, Minneapolis, MN) with the use of a vertical puller (model PP-830; Narishige, Tokyo, Japan), had resistances of 58 M when filled with a standard Cs-glutamate-rich solution, described below.
An Axopatch-1D patch-clamp amplifier (Axon Instruments, Union City, CA) was used to measure whole cell currents. The reference electrode was a Ag-AgCl electrode that was connected to the bath via an agar bridge [1% (wt/vol)] filled with a NaCl-rich bathing solution. The amplifier was driven by pCLAMP 6.0 software to allow the delivery of voltage-step and ramp protocols with concomitant digitization of the whole cell currents. The whole cell currents were filtered through an internal four-pole Bessel filter at 500 Hz, sampled at 2 kHz, and stored directly into the computer's hard disk through a Digidata 1200 interface (Axon Instruments). Subsequent data analysis was performed by using programs supplied with pCLAMP 6.0 software.
Current-voltage (I-V) relationships were studied by using 10-mV voltage pulses, each of 400 ms in duration, delivered at voltages ranging between 60 mV and +30 mV, and voltage pulses were separated by 7 s, during which the cell potential was held at 40 mV. As an alternative to voltage steps, voltage ramps were also applied in the whole cell experiments. Typically, the command voltage was varied from 60 mV to +50 mV over a duration of 800 ms every 10 s. Amiloride-sensitive currents were estimated under each condition by subtraction of currents measured under identical conditions except for the addition of 10 µM amiloride.
To analyze titration curves for amiloride inhibition of the macroscopic Li+ current, the ratio I/Io measured in the presence of amiloride (I) and in its absence (Io), normalized to the value in the presence of 10 µM amiloride, was described by the following equation
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The capacitance transient current was compensated by using the Axopatch-1D amplifier in most experiments. The cell capacitance was 10.1 ± 0.7 pF (n = 178). The values are similar to those reported for surface cells of rat colonic crypts, where there is no evidence for cell-to-cell coupling (26). The series resistance (Rs) in these studies, which was 22.1 ± 0.4 M (n = 178), was not compensated. The pipette potential was corrected for the liquid junction potentials between the pipette solution and the external solution, and between the external solution and the agar bridge, as described elsewhere (2, 35).
The composition of the standard pipette and bath solutions was as follows. The pipette solution (pH 7.4) contained (in mM) 120 Cs-glutamate, 10 CsCl, 1 MgCl2, 10 HEPES, and 10 EGTA, with pH adjusted with CsOH at 7.4. The cells were initially immersed in a bath solution (pH 7.4) containing (in mM) 145 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 D-glucose, and 10 HEPES. In most experiments, the bath solution was changed to the one (pH 7.4) containing (in mM) 145 Na-glutamate, 1 MgCl2, and 10 HEPES. The pH was adjusted with NaOH (5 mM). In some experiments, the bath solution was changed to one (pH 7.4) containing (in mM) 145 Li-glutamate, K-glutamate, or NMDG-glutamate, 1 MgCl2, and 10 HEPES. The pH of the solution was adjusted with LiOH, KOH, or NMDG, respectively. All chemicals employed were of reagent grade. Amiloride, bumetanide, d-aldosterone, indomethacin, nystatin, HEPES, and EGTA were obtained from Sigma (St. Louis, MO); theophylline was from Wako Chemicals (Osaka, Japan); and poly-L-lysine was from Nacalai Tesque (Kyoto, Japan). Stock solutions of amiloride (10 mM) and theophylline (25 mM) were prepared in distilled water and the bathing solution, respectively. Stock solutions of d-aldosterone (10 µM) and indomethacin (10 mM) were dissolved in methanol and ethanol, respectively. Stock solutions of other drugs were dissolved in dimethyl sulfoxide (DMSO). Nystatin stock solution (200 mg/ml) was made and sonicated for 6 min just before use.
The results are reported as means ± SE of several experiments (n). Statistical significance was evaluated by using the two-tailed paired or unpaired Student's t-test. Differences between means were considered to be statistically significance at a value of P < 0.05.
RT-PCR analysis. Total RNA was extracted from RC mucosa, prepared as described above, using Trizol reagent (Life Technologies, Grand Island, NY) following the producer's instructions. First-strand cDNA was generated from total RNA by using SuperScript II RT (Life Technologies) according to the producer's instructions. The specific oligonucleotide primers for the RT-PCR for -,
-, and
-rENaC were as follows:
-rENaC, 5'ACA ACA CCA CCA TCC ACG 3' (sense), 5' GCC ACC ATC ATC CAT AAA G 3' (antisense);
-rENaC 5' CCT ACA AGG AGC TGC TAG TGT G 3' (sense), 5' GAA GTG CCT TCT CTG TCA TG 3' (antisense); and
-rENaC, 5' CTC GTC TTC TCT TTC TAC AC 3' (sense), 5' GCA GAA TAG CTC ATG TTG 3' (antisense); which were derived from the published sequences of the rat epithelial Na+ channel (GenBank accession no. X70497
[GenBank]
, X77932
[GenBank]
, and X77933
[GenBank]
, respectively). The size of the expected fragment was 914 bp (
-rENaC), 786 bp (
-rENaC), and 542 bp (
-rENaC), respectively. The PCR reaction was performed with Taq DNA polymerase (Promega, Madison, WI). PCR products were gel-excised, purified, cloned into the pGEM-T Easy vector (Promega), and sequenced. The PCR conditions were as follows: denaturation at 94°C for 30 s; annealing at 58°C (
), 53°C (
), or 50°C (
) for 30 s; and extension at 72°C for 1.5 (
) or 1 min (
or
) for 35 cycles. As a control,
-actin-cDNA was amplified by using the primers 5'-GAC TAC CTC ATG AAG ATC CT-3' (sense) and 5'-CCA CAT CTG CTG GAA GGT GG-3' (antisense), and a 510-bp product was obtained.
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RESULTS |
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In contrast to RC segments, four segments of the distal colon (DC1DC4) exhibited much lower Isc-amil (Fig. 1B). However, negligible Isc-amil in these DC segments appeared not to be related to an erroneous damage of the preparations for the following reasons. First, ouabain-sensitive Na+-pump current in DC segments (DC3 and DC4: 89.0 ± 35.4 µA/cm2, n = 6) was not significantly different from that in RC segments (RC1 and RC2: 87.7 ± 29.4 µA/cm2, n = 6; P = 0.98) (Fig. 1C). Second, basolateral bumetanide (100 µM)-blockable, theophylline (2.5 mM)-induced Isc in distal colon (61.5 ± 17.3 µA/cm2, n = 6; 58.0 ± 16.3 µA/cm2, n = 7; 37.1 ± 6.0 µA/cm2, n = 10; and 29.4 ± 5.4 µA/cm2, n = 12, for DC1, DC2, DC3, and DC4, respectively) were rather larger than those for rectal colon (13.5 ± 6.8 µA/cm2, n = 4, and 10.5 ± 6.7 µA/cm2, n = 6, for RC1 and RC2, respectively).
We also confirmed that Isc-amil in both RC and DC segments of rats fed a normal Na+ diet could be enhanced by in vitro incubation with 10 nM aldosterone, as reported previously (14), so that after 5- to 8-h incubation of RC (RC1 and RC2) and DC segments (DC2, DC3, and DC4) with aldosterone, Isc-amil reached maximum values of 67.9 ± 11.1 µA/cm2 (n = 13) and 31.9 ± 10.0 µA/cm2 (n = 5), which were 323.7 ± 50.2% (n = 13) and 650.7 ± 185.4% (n = 5), respectively, of the Isc-amil initial values of 29.6 ± 8.1 µA/cm2 (n = 13) and 7.3 ± 3.0 µA/cm2 (n = 5). In control experiments, where RC segments were incubated with methanol alone, a solvent of aldosterone, the initial Isc-amil of 43.2 ± 15.5 µA/cm2 (n = 6) decreased to 9.1 ± 2.2 µA/cm2 (n = 6) (41.6 ± 15.7% of the initial values).
Figure 1D, inset, shows a representative experiment in which the effect of addition of different concentrations of amiloride (1 nM to 10 µM) to the apical bath solution was examined on the basal Isc in RC segments and demonstrates that amiloride indeed inhibited the Isc in a dose-dependent manner. Based on the concentration-response curve for the inhibition by apical amiloride, the IC50 value was estimated to be 198 ± 48 nM (n = 7) (Fig. 1D). Benzamil, an amiloride analog, also reduced Isc in RC segments with an IC50 value of 51.1 ± 8.4 nM (n = 5) (Fig. 1D). We also confirmed that the amiloride sensitivity observed in the untreated RC segments was similar to that for the segments pretreated with aldosterone in vitro. The IC50 value for the amiloride-inhibition of Isc in the aldosterone-pretreated segments was estimated to be 291 ± 43 nM (n = 12), which was not significantly different from the corresponding values for the nontreated RC segments (P > 0.20). These results together suggest the presence of a functional amiloride-sensitive Na+ channel in the RC segments even without pretreatment with aldosterone. Thus we decided to characterize native amiloride-sensitive Na+ channels in RC segments under basal conditions (i.e., normal conditions).
RT-PCR analysis. We next performed RT-PCR analysis to examine whether the RC segments would express mRNA encoding -,
-, and
-rENaC subunits. In these experiments, we extracted RNA from mucosa of RC segments directly after completion of the electrophysiological measurements. Figure 2 shows an example of such experiments and indicates that epithelial cells (e.g., surface and crypt cells) and/or nonepithelial cells (e.g., nerve endings, cells of the immune system, etc.) in rectal mucosa express transcripts of the three subunits. Similar results were obtained in three independent experiments.
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Amiloride-sensitive whole cell conductance. It is well established that the amiloride-sensitive electrogenic Na+ transport is confined to the surface cells and upper parts of crypts of distal colon under various experimental conditions (13, 28, 32). We thus hypothesized that amiloride-sensitive channels responsible for Isc-amil observed in RC should be localized in the surface epithelial cells. Subsequently, we attempted to record whole cell currents from surface cells of intact crypts isolated from rectal mucosa. Because previous patch-clamp studies have shown that colonic surface epithelial cells exhibit both K+ and Cl channel currents (6, 11, 13), the currents were minimized with the use of a Cs-glutamate-rich solution in the pipette. Under these experimental conditions, the success rates for obtaining gigaohm membrane seals and for making successful whole cell current recordings were 18 and 4% of the total trial, respectively. Despite these technical difficulties of patching, we could record an amiloride-sensitive whole cell conductance, which was observed in 22% of cells tested. The following results were obtained in these selected cells that expressed an amiloride-sensitive current. Figure 3A shows tracings of the whole cell currents evoked by voltage steps from a holding potential of 49 mV between 69 and +21 mV and demonstrates that addition of amiloride (10 µM) to the standard NaCl-rich bath solution reduced the currents. By subtracting the whole cell current records observed after the addition of the inhibitor from those observed before its addition, we obtained the traces of components and the corresponding I-V relationship for the amiloride-sensitive current (Fig. 3B). In the voltage range tested, the amiloride-sensitive currents rapidly reached steady state after each voltage pulse and did not reveal any voltage-dependent activation or inactivation. We could not further characterize the currents associated with a membrane hyperpolarization more negative than 70 mV because the cells often exhibited a hyperpolarization-activated current, which may not be carried by monovalent cation (Inagaki A and Ishikawa T, unpublished observation). Amiloride-sensitive I-V relationships obtained from 35 cells are summarized in Fig. 3C. To examine whether the amiloride-sensitive currents were mediated by Na+ ions, we next compared whole cell I-V relationship for the amiloride-sensitive whole cell current with that for the whole cell Na+ current estimated by NMDG+ substitution for Na+. As shown in Fig. 3D, there was little, if any, difference between the amiloride-sensitive currents and the Na+ currents in the same cell at different membrane potentials, indicating that the amiloride-sensitive current was solely carried by Na+ ions.
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We also found that stability of the amiloride-sensitive whole cell currents was variable under the present experimental conditions and that in some experiments amiloride-sensitive whole cell currents ran down. Figure 4A shows an example of time course of rundown of amiloride-sensitive whole cell currents. In this experiment, amiloride-sensitive current amplitude at 63 mV was 212.3 pA at 2 min after whole cell dialysis, which then decreased to 50.5 pA 21 min later. Figure 4B summarizes data obtained from 8 experiments, in which the time course of change in amiloride-sensitive whole cell currents was systematically monitored for longer than 10 min, and demonstrates the variation in the stability of the currents. In 7 of 14 other experiments, we also observed rundown of the currents so that amiloride-sensitive current amplitude at 63 mV decreased to 66.2 ± 4.7% (n = 7) of the intial level within 410 min after whole cell dialysis. Although we did not pursue the mechanism of the variation in the stability of the currents in the present study, the results may suggest that some diffusible cytosolic molecules, which were not added to the pipette solution, might play an important role in maintaining amiloride-sensitive channel activity in these cells. A rundown of ENaC activity in the standard whole cell configuration has been also reported in acutely dissociated renal collecting duct tubules (39) and in heterologous expression systems (24). Thus, to minimize the contribution of the rundown to the experimental results, we performed the following experiments using a voltage-ramp pulse protocol that enabled us to obtain control and experimental data in an apparent steady-state condition (i.e., in a short period), which was also ensured by checking reversibility whenever currents were decreased upon an experimental maneuver.
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We next examined the amiloride sensitivity of the whole cell currents by using Li+ as a charge carrier (see Ion selectivity of the amiloride-sensitive whole cell current). Figure 5A shows that external amiloride inhibited inward currents in a dose-dependent manner. Because the amiloride block of the currents mediated by naturally expressed ENaC and by the cloned ENaC is known to be weakly voltage dependent (25, 36, 42, 49), we further analyzed the voltage dependence of the block. As also summarized in Fig. 5B, the Ki for the amiloride effect at 64 or 4 mV was estimated to be 0.12 ± 0.03 or 1.63 ± 1.29 µM (n = 5), respectively. When Ki values obtained at different membrane potentials were fitted with the Woodhull equation (see MATERIALS AND METHODS) (Fig. 5B, inset), a mean slope parameter z for the voltage dependency of amiloride block was estimated to be 0.24 ± 0.03 (n = 5) at 20°C. Therefore, the electrical distance was 24% of the transmembrane electric field, suggesting that the amiloride-binding site is located within the outer entrance of the ion conductive pathway of native ENaC.
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Ion selectivity of the amiloride-sensitive whole cell current. With the standard Cs-glutamate-rich pipette solution and Na+-rich bath solution, amiloride (10 µM)-sensitive whole cell currents were not usually reversed in the voltage range tested (see also Fig. 3, B and D), suggesting that Na+ permeability of the channel was much greater than Cs+ permeability. When a zero-current potential of the currents was simply estimated by extrapolation of values between +27 and +47 mV, a mean value of 50.3 ± 5.2 mV (n = 26) was obtained. Assuming that amiloride-sensitive currents are solely carried by Na+ and Cs+ and a zero intracellular Na+ concentration, the relative permeability of Na+ to Cs+ (PNa/PCs) was estimated to be 11.0 ± 2.4 (n = 26). This may be an underestimated value because some residual Na+ intracellular concentration may be present because of incomplete intracellular dialysis.
To characterize the ion selectivity of the amiloride-sensitive currents mediated by native ENaC expressed in surface cells, we next examined the relative permeabilities for various cations of the whole cell currents. Figure 6A, inset, shows representative whole cell current tracings recorded from a surface cell. In this experiment, amiloride (10 µM)-sensitive whole cell currents were measured in the presence of Na+, K+, or Li+ as the major cation in the bathing solution. When Li+ was used as the main charge carrier, it produced larger amiloride-sensitive currents than did Na+. When the amiloride-sensitive Na+ and Li+ currents were measured in the same cells, the corresponding current amplitudes at 63 mV were 84.0 ± 23.9 pA and 167.0 ± 58.5 pA (n = 6), respectively, and the ratio of the amiloride-sensitive Li+ to Na+ current at this membrane potential was calculated to be 1.8 ± 0.1 (n = 6) (Fig. 6A). In contrast to these cations, however, replacement of Na+ with K+ failed to support the amiloride-sensitive inward current (Fig. 6A). From these experiments, we estimated the ion selectivity sequence of the current to be Li+ (1.8) > Na+ (1) >> K+ (0). The sequence was also confirmed in current-clamp experiments, where the shift of a zero-current voltage by external amiloride (10 µM) was monitored in the presence of Na+, K+, or Li+ as the major cation in the bathing solution (Fig. 6B).
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In addition to the amiloride-sensitive Na+ conductance, we also observed an amiloride (10 µM)-insensitive Na+ conductance. Figure 6C shows an example of tracings of whole cell current recorded from a surface cell of an intact crypt freshly isolated from rat rectal colonic mucosa. Addition of amiloride (10 µM) to the bath had no effects on the current, whereas when Na+ in the bath solution was totally replaced with the impermeant NMDG+ cation, inward current was largely reduced. In 15 cells, amiloride-insensitive, inward whole cell conductances measured in a voltage range between 63 (or 66) and 3 (or 6) mV before and after the Na+ replacement were 3.9 ± 0.6 and 1.7 ± 0.2 nS, respectively. When Li+ or K+ was substituted for Na+, the conductance was not significantly changed so that the ratio of the amiloride-insensitive Li+ or K+ conductance to Na+ conductance was 0.9 ± 0.1 (n = 4; P = 0.11) or 1.1 ± 0.1 (n = 5; P = 0.35), respectively (Fig. 6D). Under the current-clamp condition, the NMDG+ substitution for Na+ caused a shift of zero-current potential from 2.7 ± 1.0 mV to 28.7 ± 3.0 mV (n = 14). When extracellular Na+ was totally replaced with Li+ or K+, zero-current potential was not significantly changed so that the zero-current potential was 1.0 ± 1.3 (n = 3; P = 0.88) or 1.3 ± 1.7 mV (n = 3; P = 0.13), respectively. These results suggest that the amiloride-insensitive Na+ current is mediated by a nonselective cation conductance. However, physiological significance and molecular identity of the currents are unclear at this stage.
Extracellular Na+ dependency of the amiloride-sensitive whole cell conductance. To further characterize the native amiloride-sensitive currents expressed in surface epithelial cells, we examined extracellular Na+ concentration ([Na+]o) dependence of the macroscopic currents. In these experiments, amiloride-sensitive instantaneous I-V relationships were determined by applying voltage ramps in the presence of different [Na+]o. As shown in Fig. 7A, when [Na+]o was reduced from 150 mM to 3 mM, the amiloride-sensitive Na+ current at 63 mV reversibly decreased to 12.2% of the corresponding value at 150 mM, but a change of [Na+]o from 150 mM to 15 mM did not decrease the amiloride-sensitive Na+ current (Fig. 7, A and B). When the current amplitude at 63 mV was normalized to the corresponding value at 150 mM [Na+]o, plotting the normalized inward Na+ current amplitude as a function of [Na+]o revealed a saturating relation (Fig. 7C) so that the normalized amiloride-sensitive Na+ current amplitude at 63 mV was 0.16 ± 0.02 (n = 3), 0.96 ± 0.08 (n = 3), 1.08 ± 0.08 (n = 2), 0.98 (n = 1), and 1.00 ± 0 (n = 7) at 3, 15, 30, 75, and 150 mM, respectively. When the relation was fitted to the Michaelis-Menten equation, an apparent Km value was estimated to be 7 mM. One experiment in which amiloride-sensitive current at 150 mM was much smaller than at lower Na+ concentration (15, 30, or 75 mM) was excluded from the analysis.
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DISCUSSION |
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The present findings in DC segments are consistent with the previous ones showing that, unlike in other species such as rabbit, human, and pig (10, 16, 23, 51), the rat distal colon shows a marked amiloride-sensitive electrogenic Na+ transport when acute or chronic hyperaldosteronism is induced by dietary Na+ depletion or administration of exogenous aldosterone to animals (3), or when the isolated epithelia are incubated with exogenous aldosterone in vitro (14). On the other hand, the presence of a large basal Isc-amil in the RC segments seems to be inconsistent with previous studies showing a minimal basal Isc in RC segments of rats fed a normal Na+ diet (14, 43). The reason for the inconsistency is unclear at this stage because the animals were not particularly stressed in the present study. A slight change in endogenous factors such as a resting level of aldosterone in animals used might result in variations in basal Isc-amil in RC segments, because the rat rectal colon has been shown to be much more sensitive to aldosterone than a more proximal segment of distal colon (14, 17). Nonetheless, it is clear that Isc-amil observed in RC segments under the present experimental conditions are similar to those described previously for the mammalian distal colon [in terms of amiloride (and benzamil) inhibition and aldosterone stimulation], and it is thus reasonable to assume that the apical membrane of the rectal colonic epithelial cells contain functional amiloride-sensitive channels.
The present whole cell patch-clamp study has provided direct evidence that surface cells of the RC segments indeed exhibit amiloride-sensitive whole cell currents, which are most likely responsible for Isc-amil, with the following properties: 1) the currents were highly sensitive to extracellular amiloride with a Ki of 0.12 µM; 2) amiloride block of the currents was weakly voltage-dependent, with the amiloride-binding site being estimated to be 24% of the transmembrane electric field; 3) the sequence of conductance ratios was Li+ > Na+ >> K+ NMDG+; and 4) amiloride-sensitive currents increased with increasing [Na+]o with a low apparent Km (clearly <15 mM). These properties are similar to those reported for heterologously expressed
-rENaC currents in Xenopus oocytes (8) and in mammalian epithelial cells (25) and for a native amiloride-sensitive Na+ current, which is believed to be mediated by
-rENaC (20), recorded from acutely isolated cortical collecting duct tubules (CCT) of rats fed a low-Na+ diet (15). Furthermore, it is interesting to note that amiloride-sensitive Na+ current density in the native surface cells (
10 pA/pF at 64 mV) (see also MATERIALS AND METHODS and Fig. 3C) is also comparable with those reported in renal collecting duct tubules of rats fed a low-Na+ diet or infused with aldosterone (
200300 pA at 60 mV with a cell capacitance of 4050 pF) (37) and in Xenopus oocytes heterologously expressing
-rENaC [approximately several hundreds of nA at 60 mV (8) with a capacitance of 100 nF (47)]. In accordance with these functional similarities, RT-PCR analysis confirmed the presence of the transcripts of three subunits in mucosa prepared from rectal colon, although it remains to be determined whether the corresponding proteins are expressed in the plasma (apical) membrane of surface cells.
Previous studies have shown that the channel complex formed by ,
-, or
-subunits can produce small but significant amiloride-sensitive currents in the Xenopus oocyte expression system (4, 7, 8, 19, 34, 54), although all three subunits may be required to form functional ENaC in mammalian epithelial expression systems (25, 45). However, the native amiloride-sensitive currents in surface cells are clearly distinguished from expressed
-rENaC currents by their monovalent cation selectivity sequence (based on macroscopic conductance) and amiloride sensitivity: expressed
-channels exhibit currents with a ILi/INa ratio of 0.8 and a Ki of 4.4 µM (34). [Na+]o dependency (an apparent Km seems to be much less than 15 mM) of the native currents appears to be slightly different from that (an apparent Km of 35 mM) reported for
-rENaC currents expressed in Xenopus oocytes, although there are shared common properties (i.e., amiloride sensitivity and a ILi/INa ratio) between the native and expressed
-rENaC currents (34). Unfortunately, biophysical properties of the native currents cannot be compared with those of homomeric channels formed by
-rENaC in Xenopus oocytes, because their detailed information is not available, probably due to a small expressed current. The native currents seem to obviously differ from amiloride-sensitive currents reported for LM (TK) cells transfected with
-rENaC though, with the currents being mediated by nonselective cation channels activated by membrane stretch (27).
Assuming that the native channels were formed by the three subunits, then an interesting finding in the present study would be that the native whole cell currents (proportional to the product of single-channel conductance and open probability of the active channels) saturated at a lower Na+ concentration (i.e., 15 mM) than did the single-channel currents reported for artificially expressed -rENaC and for a highly selective Na+ channel in rat CCT (i.e., an apparent Km ranging between 20 and 50 mM) (25, 38, 39). Because [Na+]o dependence of the native currents was determined in the conventional whole cell patch-clamp configuration, which keeps intracellular ionic environment almost constant, it is unlikely that changes in [Na+]o would affect the currents mainly by altering the cytosolic environment. Thus the native channel activity might be regulated by [Na+]o. In this point of view, it is noteworthy that a discrepancy of the Km values between macroscopic and single-channel currents in rat CCT has been interpreted as indicating a mechanism called "self-inhibition," which is defined as a decrease in overall open probability of active channels by [Na+]o via a direct interaction of [Na+]o and the channel (39). Such a mechanism has been also demonstrated for the human
-ENaC in a heterologous expression system (9). Furthermore, there is indeed an evidence for self-inhibition in rabbit descending colon (33). Nevertheless, the [Na+]o dependency of the native amiloride-sensitive macroscopic currents observed in the present study may be, at least, advantageous for effective absorption of Na+, because the surface cells of the rectal colon form the last absorptive epithelium in the gut and can be exposed to a low concentration of Na+ in the luminal content in physiological and pathophysiological conditions. Further experiments are required to determine whether such a self-inhibition by [Na+]o is involved in the regulation of native amiloride-sensitive Na+ channel activity in colonic surface cells.
In conclusion, we have characterized electrophysiological properties of macroscopic currents mediated by ENaC naturally expressed in rectal colonic surface cells and shown a similarity to the macroscopic currents reported for heterologously expressed -rENaC. Further characterization of native ENaC channels at the single-channel level is indeed required to prove that the biophysical properties completely match those reported for heterologously expressed
-rENaC and for ENaC naturally expressed in rat CCT. In addition, cellular and subcellular localization of the subunits protein must be determined to extend the present findings on functional properties to the molecular level in rectal colonic surface epithelial cells.
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ACKNOWLEDGMENTS |
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This work was supported in part by Grants-in-Aid for Scientific Research C from the Ministry of Education, Science, Sports and Culture of Japan, a grant from the Akiyama Foundation, and a grant from the Suzuken Memorial Foundation.
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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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Barry PH and Lynch JW. Liquid junction potentials and small cell effects in patch-clamp analysis. J Membr Biol 121: 101117, 1991.[ISI][Medline]
3. Binder HJ and Sandle GI. Electrolyte transport in the mammalian colon. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by Johnson LR, Alpers DH, Christensen J, Jacobson ED, and Walsh JH. New York: Raven, 1994, p. 21332171.
4. Bonny O, Chraïbi A, Loffing J, Jaeger NF, Gründer S, Horisberger JD, and Rossier BC. Functional expression of a pseudohypoaldosteronism type I mutated epithelial Na+ channel lacking the pore-forming region of its alpha subunit. J Clin Invest 104: 967974, 1999.
5. Bridges RJ, Cragoe EJ, Frizzell RA, and Benos DJ. Inhibition of colonic Na+ transport by amiloride analogues. Am J Physiol Cell Physiol 256: C67C74, 1989.
6. Butterfield I, Warhurst G, Jones MN, and Sandle GI. Characterization of apical potassium channels induced in rat distal colon during potassium adaptation. J Physiol 501: 537547, 1997.[Abstract]
7. Canessa CM, Horisberger JD, and Rossier BC. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 361: 467470, 1993.[CrossRef][ISI][Medline]
8. Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, and Rossier BC. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367: 463467, 1994.[CrossRef][ISI][Medline]
9. Chraïbi A and Horisberger JD. Na self inhibition of human epithelial Na channel: temperature dependence and effect of extracellular proteases. J Gen Physiol 120: 133145, 2002.
10. Cremaschi D, Ferguson DR, Henin S, James PS, Meyer G, and Smith MW. Post-natal development of amiloride sensitive sodium transport in pig distal colon. J Physiol 292: 481494, 1979.[Abstract]
11. Diener M, Rummel W, Mestres P, and Lindemann B. Single chloride channels in colon mucosa and isolated colonic enterocytes of the rat. J Membr Biol 108: 2130, 1989.[ISI][Medline]
12. Duc C, Farman N, Canessa CM, Bonvalet JP, and Rossier BC. Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry. J Cell Biol: 19071921, 1994.
13. Ecke D, Bleich M, Schwartz B, Fraser G, and Greger R. The ion conductances of colonic crypts from dexamethasone-treated rats. Pflügers Arch 431: 419426, 1996.[ISI][Medline]
14. Epple HJ, Amasheh S, Mankertz J, Goltz M, Schulzke JD, and Fromm M. Early aldosterone effect in distal colon by transcriptional regulation of ENaC subunits. Am J Physiol Gastrointest Liver Physiol 278: G718G724, 2000.
15. Frindt G and Palmer LG. Regulation of Na channels in the rat cortical collecting tubule: effects of cAMP and methyl donors. Am J Physiol Renal Fluid Electrolyte Physiol 271: F1086F1092, 1996.
16. Frizzell RA, Koch MJ, and Schultz SG. Ion transport by rabbit colon. I. Active and passive components. J Membr Biol 27: 297316, 1976.[ISI][Medline]
17. Fromm M, Schulzke JD, and Hegel U. Control of electrogenic Na+ absorption in rat late distal colon by nanomolar aldosterone added in vitro. Am J Physiol Endocrinol Metab 264: E68E73, 1993.
18. Fuller PJ, Brennan FE, and Burgess JS. Acute differential regulation by corticosteroids of epithelial sodium channel subunit and Nedd4 mRNA levels in the distal colon. Pflügers Arch 441: 94101, 2000.[CrossRef][ISI][Medline]
19. Fyfe GK and Canessa CM. Subunit composition determines the single channel kinetics of the epithelial sodium channel. J Gen Physiol 112: 423432, 1998.
20. Garty H and Palmer LG. Epithelial sodium channels: function, structure, and regulation. Physiol Rev 77: 359396, 1997.
21. Greig ER, Baker EH, Mathialahan T, Boot-Handford RP, and Sandle GI. Segmental variability of ENaC subunit expression in rat colon during dietary sodium depletion. Pflügers Arch 444: 476483, 2002.[CrossRef][ISI][Medline]
22. Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391: 85100, 1981.[ISI][Medline]
23. Hawker PC, Mashiter KE, and Turnberg LA. Mechanisms of transport of Na, Cl, and K in the human colon. Gastroenterology 74: 12411247, 1978.[ISI][Medline]
24. Ishikawa T, Jiang C, Stutts MJ, Marunaka Y, and Rotin D. Regulation of the epithelial Na+ channel by cytosolic ATP. J Biol Chem 278: 3827638286, 2003.
25. Ishikawa T, Marunaka Y, and Rotin D. Electrophysiological characterization of the rat epithelial Na+ channel (rENaC) expressed in MDCK cells. Effects of Na+ and Ca2+. J Gen Physiol 111: 825846, 1998.
26. Jacobi C, Leipziger J, Nitschke R, Ricken S, and Greger R. No evidence for cell-to-cell coupling in rat colonic crypts: studies with Lucifer Yellow and with photobleaching. Pflügers Arch 436: 8389, 1998.[CrossRef][ISI][Medline]
27. Kizer N, Guo XL, and Hruska K. Reconstitution of stretch-activated cation channels by expression of the alpha-subunit of the epithelial sodium channel cloned from osteoblasts. Proc Natl Acad Sci USA 94: 10131018, 1997.
28. Köckerling A, Sorgenfrei D, and Fromm M. Electrogenic Na+ absorption of rat distal colon is confined to surface epithelium: a voltage-scanning study. Am J Physiol Cell Physiol 264: C1285C1293, 1993.
29. Li Y and Halm DR. Secretory modulation of basolateral membrane inwardly rectified K+ channel in guinea pig distal colonic crypts. Am J Physiol Cell Physiol 282: C719C735, 2002.
30. Lingueglia E, Renard S, Waldmann R, Voilley N, Champigny G, Plass H, Lazdunski M, and Barbry P. Different homologous subunits of the amiloride-sensitive Na+ channel are differently regulated by aldosterone. J Biol Chem 269: 1373613739, 1994.
31. Lingueglia E, Voilley N, Waldmann R, Lazdunski M, and Barbry P. Expression cloning of an epithelial amiloride-sensitive Na+ channel. A new channel type with homologies to Caenorhabditis elegans degenerins. FEBS Lett 318: 9599, 1993.[CrossRef][ISI][Medline]
32. Lomax RB, McNicholas CM, Lombes M, and Sandle GI. Aldosterone-induced apical Na+ and K+ conductances are located predominantly in surface cells in rat distal colon. Am J Physiol Gastrointest Liver Physiol 266: G71G82, 1994.
33. Luger A and Turnheim K. Modification of cation permeability of rabbit descending colon by sulphydryl reagents. J Physiol 317: 4966, 1981.[Abstract]
34. McNicholas CM and Canessa CM. Diversity of channels generated by different combinations of epithelial sodium channel subunits. J Gen Physiol 109: 681692, 1997.
35. Neher E. Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol 207: 123131, 1992.[ISI][Medline]
36. Palmer LG. Voltage-dependent block by amiloride and other monovalent cations of apical Na channels in the toad urinary bladder. J Membr Biol 80: 153165, 1984.[ISI][Medline]
37. Palmer LG, Antonian L, and Frindt G. Regulation of the Na-K pump of the rat cortical collecting tubule by aldosterone. J Gen Physiol 102: 4357, 1993.[Abstract]
38. Palmer LG and Frindt G. Amiloride-sensitive Na channels from the apical membrane of the rat cortical collecting tubule. Proc Natl Acad Sci USA 83: 27672770, 1986.[Abstract]
39. Palmer LG, Sackin H, and Frindt G. Regulation of Na+ channels by luminal Na+ in rat cortical collecting tubule. J Physiol 509: 151162, 1998.
40. Pradervand S, Wang Q, Burnier M, Beermann F, Horisberger JD, Hummler E, and Rossier BC. A mouse model for Liddle's syndrome. J Am Soc Nephrol 10: 25272533, 1999.
41. Ramminger SJ, Inglis SK, Olver RE, and Wilson SM. Hormonal modulation of Na+ transport in rat fetal distal lung epithelial cells. J Physiol 544: 567577, 2002.
42. Schild L, Schneeberger E, Gautschi I, and Firsov D. Identification of amino acid residues in the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation. J Gen Physiol 109: 1526, 1997.
43. Schulzke JD, Fromm M, Hegel U, and Riecken EO. Ion transport and enteric nervous system (ENS) in rat rectal colon: mechanical stretch causes electrogenic Cl-secretion via plexus Meissner and amiloride-sensitive electrogenic Na-absorption is not affected by intramural neurons. Pflügers Arch 414: 216221, 1989.[ISI][Medline]
44. Smith PR, Bradford AL, Dantzer V, Benos DJ, and Skadhauge E. Immunocytochemical localization of amiloride-sensitive sodium channels in the lower intestine of the hen. Cell Tissue Res 272: 129136, 1993.[ISI][Medline]
45. Snyder PM. Liddle's syndrome mutations disrupt cAMP-mediated translocation of the epithelial Na+ channel to the cell surface. J Clin Invest 105: 4553, 2000.
46. Staub O, Yeger H, Plant PJ, Kim H, Ernst SA, and Rotin D. Immunolocalization of the ubiquitin-protein ligase Nedd4 in tissues expressing the epithelial Na+ channel (ENaC). Am J Physiol Cell Physiol 272: C1871C1880, 1997.
47. Stühmer W. Electrophysiological recording from Xenopus oocytes. Methods Enzymol 207: 319339, 1992.[ISI][Medline]
48. Wang Q, Horisberger JD, Maillard M, Brunner HR, Rossier BC, and Burnier M. Salt- and angiotensin II-dependent variations in amiloride-sensitive rectal potential difference in mice. Clin Exp Pharmacol Physiol 27: 6066, 2000.[CrossRef][ISI][Medline]
49. Warncke J and Lindemann B. Voltage dependence of Na channel blockage by amiloride: relaxation effects in admittance spectra. J Membr Biol 86: 255265, 1985.[ISI][Medline]
50. Watanabe S, Matsushita K, Stokes JB, and McCray PB Jr. Developmental regulation of epithelial sodium channel subunit mRNA expression in rat colon and lung. Am J Physiol Gastrointest Liver Physiol 275: G1227G1235, 1998.
51. Wills NK, Alles WP, Sandle GI, and Binder HJ. Apical membrane properties and amiloride binding kinetics of the human descending colon. Am J Physiol Gastrointest Liver Physiol 247: G749G757, 1984.
52. Woodhull AM. Ionic blockage of sodium channels in nerve. J Gen Physiol 61: 687708, 1973.
53. Yajima T, Suzuki T, and Suzuki Y. Synergism between calcium-mediated and cyclic AMP-mediated activation of chloride secretion in isolated guinea pig distal colon. Jpn J Physiol 38: 427443, 1988.[ISI][Medline]
54. Zhang P, Fyfe GK, Grichtchenko II, and Canessa CM. Inhibition of alphabeta epithelial sodium channels by external protons indicates that the second hydrophobic domain contains structural elements for closing the pore. Biophys J 77: 30433051, 1999.