Amiloride-sensitive epithelial Na+ channel currents in surface cells of rat rectal colon

A. Inagaki, S. Yamaguchi, and T. Ishikawa

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Surface cells of the mammalian distal colon are shown to molecularly express the amiloride-sensitive epithelial Na+ channel composed of three homologous subunits ({alpha}-, {beta}-, and {gamma}-ENaC). However, because basic electrophysiological properties of amiloride-sensitive Na+ channels expressed in these cells are largely unknown at the cellular level, functional evidence for the involvement of the subunits in the native channels is incomplete. Using electrophysiological techniques, we have now characterized functional properties of native ENaC in surface cells of rectal colon (RC) of rats fed a normal Na+ diet. Ussing chamber experiments showed that apical amiloride inhibited a basal short-circuit current in mucosal preparation of RC with an apparent half-inhibition constant (Ki) value of 0.20 µM. RT-PCR analysis confirmed the presence of transcripts of {alpha}-, {beta}-, and {gamma}-rENaC in rectal mucosa. Whole cell patch-clamp experiments in surface cells of intact crypts acutely isolated from rectal mucosa identified an inward cationic current, which was inhibited by amiloride with a Ki value of 0.12 µM at a membrane potential of –64 mV, the inhibition being weakly voltage dependent. Conductance ratios of the currents were Li+ (1.8) > Na+ (1) >> K+ ({approx}0), respectively. Amiloride-sensitive current amplitude was almost the same at 15 or 150 mM extracellular Na+, suggesting a high Na+ affinity for current activation. These results are consistent with the hypothesis that a heterooligomer composed of {alpha}-, {beta}-, and {gamma}-ENaC may be the molecular basis of the native channels, which are responsible for amiloride-sensitive electrogenic Na+ absorption in rat rectal colon.

distal colon; electrogenic Na+ absorption; short-circuit current; whole cell patch clamp; {alpha}{beta}{gamma}-ENaC


ELECTROGENIC NA+ ABSORPTION in the mammalian distal colon plays an important role in electrolyte and water balance of the body under physiological and pathophysiological conditions (3). A large number of studies, using transepithelial current and ion flux measurement techniques, have provided evidence that the electrogenic Na+ absorption involves the two-step process: 1) electrogenic Na+ influx across the apical membrane along an electrochemical potential and 2) Na+ efflux mediated by Na+-K+-ATPase across the basolateral membrane against an electrochemical potential. In this process, the amiloride-sensitive epithelial Na+ channel(s) located in the apical membrane of the epithelial cells constitutes the Na+ influx pathway, serves the rate-limiting step for the overall Na+ absorption, and is regulated by mineralocorticoids, such as aldosterone (3). Furthermore, the electrogenic Na+ transport in the distal colon is shown to be mostly confined to the surface epithelium and upper parts of crypts (13, 28, 32).

Since the amiloride-sensitive epithelial Na+ channel (ENaC) composed of three homologous {alpha}-, {beta}-, and {gamma}-subunits ({alpha}-, {beta}-, and {gamma}-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 {alpha}-subunit is constitutively expressed, and expression of {beta}- and {gamma}-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 {beta}-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 {alpha}{beta}{gamma}-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 0.1–0.5 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 {alpha}{beta}{gamma}-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 {alpha}{beta}{gamma}-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 {alpha}-, {beta}-, and {gamma}-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 {alpha}{beta}{gamma}-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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transepithelial current measurements. All experiments were performed in accordance with a protocol approved by the Laboratory Animal Care and Use Committee of Graduate School of Veterinary Medicine, Hokkaido University. Male Sprague-Dawley rats (200–400 g) were fed with a standard rat diet (Labo MR Standard; Na content 2.1g/kg, K content 7.8 g/kg; Nosan, Yokohama, Japan) and tap water. Animals were killed by cervical dislocation, and the distal colon was immediately removed and rinsed with the standard NaCl solution (pH 7.4) containing (in mM): 145 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 10 D-glucose; pH was adjusted with NaOH. Two segments of the colon, termed rectal colon (RC) and distal colon (DC) in the present study, correspond to late and early segments of distal colon, respectively, as reported elsewhere (14, 17). In brief, the pelvic bones were cut open, and specimens of RC were excised from the very last part of the colon, located between the lymph node (typically situated 3 cm apart from the anus) at the pelvic brim and the anus. The DC was also removed and defined as the ~7-cm-long segment proximal to the lymph node. The RC was divided into two segments equal in length, termed RC-1 and RC-2 (near by anus), respectively. The DC was also split into four segments. termed DC-1, DC-2, DC-3, and DC-4 (next to RC-1), respectively (see also Fig. 1B). To minimize possible variations in electrogenic Na+ transport in the RC and DC due to a circadian cyclicity (3, 48), the segments were usually prepared in the morning (generally between 8:00 AM and 10:00 AM).



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Fig. 1. A: effects of amiloride (10 µM) on short-circuit current (Isc), transepithelial voltage (Vte), and transepithelial resistance (Rte) of the rectal colonic mucosa. A positive deflection of Isc indicates cation movement from apical to basolateral bathing medium or anion movement from basolateral to apical medium. Values for Vte were referred to the serosal side of the mucosa. Rte was determined by applying short (1 s) voltage pulses (0.5 mV) every 2 min, and Vte was calculated according to Ohm's law (Vte = Isc x Rte). Where indicated, amiloride (10 µM) was added to the apical solution. B: segmental heterogeneity of amiloride (10 µM)-sensitive short-circuit currents (Isc-amil) in rectal (RC) and distal colon (DC) of rats fed a normal Na+ diet. The RC, located between the lymph node (typically situated ~3 cm from the anus) at the pelvic brim and the anus, was divided into 2 segments (RC1 and RC2) equal in length. The DC, defined as the ~7-cm-long segment proximal to the lymph node, was split into 4 segments (DC1–DC4). Values are means ± SE of 6–27 experiments. C: comparison of basolateral Na+-pump activity in RC and DC segments. Bar graphs show Isc-amil and ouabain-sensitive basolateral Na+-pump currents (Isc-oua) in RC and DC segments. Values are means ± SE of 6 experiments. Top: representative experiments in which Isc-oua were examined in RC1 (left) and DC3 segments (right) prepared from the same animal. A, amiloride (10–5 M); N, nystatin (200 µg/ml); O, ouabain (10–3 M). D: dose-response curves for inhibition of Isc by amiloride or its analog, benzamil in rectal colonic segments of rats fed a normal Na+ diet. The effect of different concentrations of amiloride or benzamil (I/I0) was normalized to a value in the presence of 10–5 M of each drug. Values are means ± SE of 7 or 5 experiments. Inset: representative experiment in which the effect of different concentrations of amiloride were examined on basal Isc.

 

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 40–90 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 (20–23°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 5–8 M{Omega} 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

(1)
where Ki is the inhibitory constant of the blocker, A is the concentration of the blocker, and nH is a pseudo Hill coefficient. In the case of a voltage-dependent block, Ki(V) is the voltage-dependent inhibitory constant, which has been expressed by Woodhull (52) as a Boltzmann relationship with respect to the voltage

(2)
where Ki(0) is the inhibitory constant at 0 mV, Z is a slope parameter, and F,V, R, and T have their conventional meanings. Z is equal to the product of the actual valence of the blocking ion z and the fraction of the membrane potential (or electrical distance) delta ({delta}) acting on the ion.

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{Omega} (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 {alpha}-, {beta}-, and {gamma}-rENaC were as follows: {alpha}-rENaC, 5'ACA ACA CCA CCA TCC ACG 3' (sense), 5' GCC ACC ATC ATC CAT AAA G 3' (antisense); {beta}-rENaC 5' CCT ACA AGG AGC TGC TAG TGT G 3' (sense), 5' GAA GTG CCT TCT CTG TCA TG 3' (antisense); and {gamma}-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 ({alpha}-rENaC), 786 bp ({beta}-rENaC), and 542 bp ({gamma}-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 ({alpha}), 53°C ({beta}), or 50°C ({gamma}) for 30 s; and extension at 72°C for 1.5 ({alpha}) or 1 min ({beta} or {gamma}) for 35 cycles. As a control, {beta}-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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Basal bioelectric properties of rectal colonic epithelium of rats fed a normal Na+ diet. It has been shown that the rat distal colon exhibits a marked amiloride-sensitive electrogenic Na+ transport generally when acute or chronic hyperaldosteronism is induced by dietary Na+ depletion or administration of exogenous aldosterone to animals (3). However, we have unexpectedly found that mucosa prepared from rectal colon of rats fed a normal Na+ diet display amiloride-sensitive shortcircuit current (Isc-amil). Figure 1A shows an example of Isc measurement experiments in mucosa prepared from rectal colon (RC2) of rats fed a normal Na+ diet. In this experiment, basal Isc, Vte, and Rte were 90.6 µA/cm2, –6.1 mV (lumen negative), and 67.6 {Omega}·cm2, respectively. Addition of amiloride (10 µM) to the apical solution decreased Isc to 23.8 µA/cm2, changed Vte to –1.8 mV, and increased Rte to 73.5 {Omega}·cm2. Isc-amil in RC segments was most likely carried by Na+ ions, because omission of Na+ from the apical solution blunted Isc-amil, the effect being reversible (n = 3; data not shown). Basal bioelectrical properties of rectal colonic segments (RC1 and RC2) are summarized in Table 1.


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Table 1. Basal bioelectric properties of rectal colon

 

In contrast to RC segments, four segments of the distal colon (DC1–DC4) 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 {alpha}-, {beta}-, and {gamma}-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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Fig. 2. RT-PCR analysis of {alpha}-, {beta}-, and {gamma}-subunits of rat epithelial Na+ channel (rENaC) in mucosa prepared from rectal colon of rats fed a normal Na+ diet. The mucosa had exhibited an Isc-amil of 71.8 µA/cm2. PCR products were resolved by 1% agarose gel electrophoresis, and amplicons ({alpha}-rENaC, 914 bp; {beta}-rENaC, 786 bp; and {gamma}-rENaC, 542 bp) were detected by staining with ethidium bromide. As a control, {beta}-actin cDNA (510 bp) was amplified. No DNA fragment was amplified with the template without RT treatment. Sequence comparison of the PCR products with those for {alpha}-(GenBank accession no. X70497 [GenBank] ), {beta}-(no. X77932 [GenBank] ), and {gamma}-rENaC (no. X77933 [GenBank] ) showed 100%, 99.7% (2 nucleotide differences), and 100% identity, respectively. M, size markers (100-bp DNA ladder).

 

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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Fig. 3. A: traces of whole cell currents obtained from a surface cell of an intact crypt of rat rectal colon in the absence (control, top) or presence (middle) of external amiloride (10 µM). Traces of amiloride-sensitive currents (bottom) were obtained from traces shown at top and middle. The cell was held at –49 mV and stepped for 400 ms to potentials ranging between –69 and +21 mV in 10-mV intervals. The pipette was filled with the standard Cs-glutamate-rich solution, and the bath contained the standard NaCl-rich solution. Inset: protocol. B: instantaneous current-voltage (I-V) relationships of whole cell currents obtained from the same cell as in A. Currents were elicited in the absence (control) and presence of external amiloride (10 µM) by applying ramp command voltages, each of 800-ms duration, from –69 to +41 mV. A trace of the I-V relationship of amiloride-sensitive current (bottom) was obtained from traces shown at top. C: summary of I-V relationships of amiloride-sensitive currents. Values are means ± SE of 35 experiments. The bath contained the standard Na-glutamate-rich solution. D: comparison of amiloride (10 µM)-sensitive currents and Na+ currents in the same surface cell. Instantaneous I-V relationsip was measured when the bath contained a Na-glutamate-rich solution having 10 µM amiloride or an NMDG-glutamate-rich solution (NMDG+). Control I-V relation was measured before addition of amiloride to the bath solution. Effect of amiloride was completely reversible. Control I-V relation before replacement of Na+ with NMDG+ was thus almost the same as that for amiloride (not shown).

 

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 4–10 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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Fig. 4. A: example of time course of rundown of amiloride-sensitive whole cell currents (Iamil) obtained from a surface cell of rectal colon. Pipette and bath solutions were Cs-glutamate and Na-glutamate rich, respectively. Instantaneous amiloride-sensitive I-V relationships at times after whole cell dialysis are shown. B: summary of 8 different experiments in which the time course of Iamil at –63 mV was determined. Lines connect data obtained from the same patch. Current amplitudes at –63 mV were normalized to the corresponding values of initial Iamil (at 0 min) (–208.4 ± 45.6 pA, n = 8, at –63 mV), which were measured within 2 min after whole cell dialysis.

 

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 {delta} 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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Fig. 5. Amiloride sensitivity of whole cell Li+ currents in rat rectal colonic surface cells. A: traces represent the effect of different concentrations of amiloride obtained from a rectal colonic surface cell of an intact crypt. Currents were elicited by 800-ms voltage ramps from –64 to +46 mV in the presence of 150 mM Li+ in the bath solution. The pipette was filled with a Cs-glutamate-rich solution. B: dose-inhibition relationship for the amiloride effect at –64 mV ({bullet}) and –4 mV ({blacktriangleup}) of whole cell Li+ currents in rectal colonic surface cells. The effect of different concentrations of amiloride (I/I0) was normalized to the value in the presence of 10–5 M amiloride. Data were fitted with Eq. 1 (see MATERIALS AND METHODS). For comparison, I/I0 was renormalized to an estimated maximum value. Values are means ± SE of 5 experiments. Inset: effect of membrane potential on Ki values for amiloride inhibition. The Ki values were normalized to the values obtained at –64 mV. Data derived from 5 experiments were fitted with Eq. 2 (see MATERIALS AND METHODS).

 

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+ ({approx}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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Fig. 6. Ion selectivity of Iamil in rectal colonic surface cells. A: Iamil (10 µM amiloride) current amplitudes normalized to values at –63 mV in the presence of 150 mM Na+ in the bath solution are plotted against membrane potentials. The bath solution contained 150 mM Na+ ({bullet}), Li+ ({blacktriangleup}), or K+ ({circ}). Currents were elicited by 800-ms voltage ramps. The pipette was filled with a Cs-glutamate-rich solution. Values are means ± SE of 5–26 experiments. Inset: Iamil (10 µM amiloride) recorded from the same rectal surface cell in the presence of 150 mM Na+, Li+, or K+ in the bath solution. B: effect of amiloride (10 µM) on the zero-current potential of rectal colonic surface cells. Bar graphs show the summary of amiloride-induced changes in zero-current voltage in the presence of 150 mM Na+, Li+, or K+ in the bath solution. Zero-current voltage was measured in current-clamp mode. Values are means ± SE of 4–23 experiments. C: amiloride-insensitive whole cell cationic currents in rat rectal surface cells. Traces are examples of instantaneous I-V relationships of whole cell currents elicited by 800-ms voltage ramps. The bath contained a Na-glutamate-rich solution without (control) or with 10 µM amiloride or an NMDG-glutamate-rich solution. The pipette solution was the standard Cs-glutamate-rich solution. D: ion selectivity of amiloride-insensitive whole cell currents in rectal colonic surface cells. Amiloride (10 µM)-insensitive whole cell current amplitudes normalized to values at –63 mV in the presence of 150 mM Na+ in the bath solution are plotted against membrane potentials. The bath solution contained 150 mM of Na+ ({bullet}, n = 15), Li+ ({blacktriangleup}, n = 4), K+ ({circ}, n = 5), or NMDG+ ({diamondsuit}, n = 15). The pipette was filled with the standard Cs-glutamate-rich solution. Data are means ± SE of 4–15 experiments. Inset: amiloride (10 µM)-insensitive whole cell current amplitudes plotted against membrane potentials. Data are from the same experiments shown in D.

 

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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Fig. 7. Extracellular Na+ dependence of Iamil in rat rectal colonic surface cells. A and B: tracings of amiloride-sensitive instantaneous I-V relationships obtained from the same cells in the presence of different extracellular Na+ concentrations. Currents were elicited by voltage ramps in varying extracellular Na+ concentrations from 150 to 3 mM (A) or to 15 mM (B) by equimolar replacement with NMDG+. The pipette was filled with the standard Cs-glutamate-rich solution. C: plot of the amiloride-sensitive inward current amplitude at –63 mV as a function of extracellular Na+ concentration. Currents were normalized to the value at 150 mM Na+ (Iamil/Iamil 150 mM). Data are means ± SE of 1–7 experiments. The dotted line is a nonlinear least-squares fit of the Michaelis-Menten equation.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present Isc measurements in vitro show that the mucosa of rectal colon of rats fed a normal Na+ diet displayed basal amiloride-sensitive Isc, which was not obvious in the distal colon. Several lines of evidence support the view that the segmental heterogeneity of Isc-amil is most likely attributable to a different basal activity of apically located amiloride-sensitive Na+ channels. First, the channels responsible for the Isc in rectal colon appeared to be highly sensitive to amiloride (Ki = 198 nM) and benzamil (Ki = 51 nM), with the pharmacological profile being in good agreement with those for an enhanced Isc in rectal and distal parts of colonic mucosa by incubation with aldosterone in vitro and with those reported for the distal colon obtained in mineralocorticoid-treated rats (5). Second, Na+-K+-pump current was not significantly different between RC and DC segments, implying that the activity of the pump is also unlikely to limit Isc-amil in DC segments. Third, a cAMP-related agent, theophylline, induced rather higher Isc (likely mediated by bumetanide-sensitive electrogenic Cl secretion) in DC segments than in RC segments, and Isc-amil in DC segments could be also enhanced by exogenous aldosterone in vitro, with the findings excluding the possibility of erroneous damage of mucosal preparations of DC segments.

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+ {approx} 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 {alpha}{beta}{gamma}-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 {alpha}{beta}{gamma}-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 (~200–300 pA at –60 mV with a cell capacitance of 40–50 pF) (37) and in Xenopus oocytes heterologously expressing {alpha}{beta}{gamma}-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 {alpha}, {alpha}{beta}-, or {alpha}{gamma}-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 {alpha}{beta}-rENaC currents by their monovalent cation selectivity sequence (based on macroscopic conductance) and amiloride sensitivity: expressed {alpha}{beta}-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 {alpha}{gamma}-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 {alpha}{gamma}-rENaC currents (34). Unfortunately, biophysical properties of the native currents cannot be compared with those of homomeric channels formed by {alpha}-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 {alpha}-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 {alpha}{beta}{gamma}-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 {alpha}{beta}{gamma}-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 {alpha}{beta}{gamma}-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 {alpha}{beta}{gamma}-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.


    ACKNOWLEDGMENTS
 
GRANTS

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.


    FOOTNOTES
 

Address for reprint requests and other correspondence: T. Ishikawa, Laboratory of Physiology, Dept. of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido Univ., Sapporo 060-0818, Japan (E-mail: torui{at}vetmed.hokudai.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.


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