1 Centre National de la Recherche Scientifique, Unité de Recherche en Physiologie Cellulaire, Université de Bretagne Occidentale, 29200 Brest, France; 2 Laboratoire de Biologie et Physiologie Animales, Université des Antilles et de la Guyane, Campus Fouillote, 97159 Pointe à Pitre, Guadeloupe; and 3 Cellular Physiology Research Unit, University College Cork, Cork, Ireland
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ABSTRACT |
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Single channel patch-clamp techniques were used to
demonstrate the presence of outwardly rectifying chloride channels in the basolateral membrane of crypt cells from mouse distal colon. These
channels were rarely observed in the cell-attached mode and, in the
inside-out configuration, only became active after a delay and
depolarizing voltage steps. Single channel conductance was 23.4 pS
between 100 and
40 mV and increased to 90.2 pS between 40 and 100 mV. The channel permeability sequence for anions was: I
> SCN
> Br
> Cl
> NO3
> F
SO42
gluconate. In inside-out
patches, the channel open probability was voltage dependent but
insensitive to intracellular Ca2+ concentration. In
cell-attached mode, forskolin, histamine, carbachol, A-23187, and
activators of protein kinase C all failed to activate the channel, and
activity could not be evoked in inside-out patches by exposure to the
purified catalytic subunit of cAMP-dependent protein kinase A. The
channel was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, 9-anthracenecarboxylic acid, and DIDS. Stimulation of G proteins with
guanosine 5'-O-(3-thiotriphosphate) decreased the channel open probability and conductance, whereas subsequent addition of
guanosine 5'-O-(2-thiodiphosphate) reactivated the channel.
ionic channels; colonic crypts; basolateral membrane; mouse; G protein
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INTRODUCTION |
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THE TRANSMEMBRANE MOVEMENT of ions in the cells of the colonic epithelium is involved in two distinct processes: the transepithelial movement of fluid and the regulation of cell volume. The first process is responsible for the overall fluidity of the colonic luminal content, which is determined by the balance between fluid secretion and absorption occurring in the crypt and surface epithelial cells. The second process is based on the uptake or efflux of osmotically active compounds and is responsible for the rapid compensation of cell volume changes resulting from fluctuating entry or exit of ions and osmotically obliged water and from variations of the osmotic pressure in the luminal compartment of the colon.
Current understanding of the mechanisms underlying transepithelial
fluid movements through the colon is based on observations of the
opposing processes of Cl secretion within the crypts and
Na+ and Cl
uptake by the
surface epithelial cells. Several types of Cl
channels
have been characterized on the apical membrane of isolated rat
enterocytes (9, 14) and human colon
carcinoma cells (20) and on the surface cells of intact
human mucosa (9, 39). Most of these channels
belong to the family of outwardly rectifying Cl
channels
(ORCC) and, along with the so-called cystic fibrosis transmembrane
conductance regulator Cl
channel, are thought to share a
major role in Cl
secretion. In the basolateral membrane,
a Cl
channel with a linear current-voltage
(I-V) relationship and a slope conductance of 29 pS has been reported in isolated crypts from rat distal colon
(9), and ORCC have been observed in isolated crypts of
mouse jejunum (4) and guinea pig enterocytes
(36), but the role of these basolateral Cl
channels has not been clearly identified.
Similarly, various studies on intestinal epithelial cells, e.g.,
isolated enterocytes (32) or intact crypt cells
(8, 35, 37), have implicated
Cl conductances in regulatory volume decrease (RVD)
following hypotonic shock. In this experimental situation, the data of
Worrell et al. (52) and Kubo and Okada (24)
implicated a role of ORCCs in the volume regulatory process.
On the basis of this, it appears that members of the large family of
ORCCs may be involved in both volume regulation and in ion secretion or
absorption. A Cl channel located on the basolateral
membrane of crypt cells could provide a pathway for the exit of
Cl
ions during RVD and/or be involved in the basolateral
Cl
exit step during NaCl absorption. However, there is a
marked lack of information concerning conductive Cl
movements through the basolateral membrane of colonic cells.
The present study used the patch-clamp technique for recording single
ion channel activity, with the aim of identifying and characterizing
the Cl channels present under steady-state conditions in
the basolateral membrane of enterocytes in intact mouse distal colon
crypts. We report the presence of a Cl
channel
characterized by a strong outward rectification, sensitive to DIDS,
5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), 9-anthracenecarboxylic acid (9-AC), and GTP. Preliminary data have been presented in abstract
form (34).
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METHODS |
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Isolation of colonic crypts. Isolated crypts were prepared by a modification of the procedure described by Siemer and Gögelein (47). Female mice weighing 20-30 g were anesthetized (ketamine, 10-20 mg/ml) and killed by cervical dislocation. The distal colon was taken above the pelvic brim, dissected, and rinsed in ice-cold NaCl solution containing (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES-Tris buffer, 10 glucose, and 1 dithiothreitol (DTT), pH 7.4. The intact colon was everted and filled with a Ca2+-free solution containing (in mM) 96 NaCl, 1.5 KCl, 10 HEPES-Tris, 27 Na-EDTA, 55 sorbitol, 44 sucrose, and 1 DTT, pH 7.4, and incubated for 20 min, then transferred into a high-Ca2+ NaCl solution containing (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES-Tris, and 10 glucose, pH 7.4. The crypts were released into solution by gentle shaking, washed twice by centrifugation at 600 g for 2 min, and stored on ice until use. Before patch-clamp experiments, the crypts were fixed to the glass bottom of an experimental chamber (volume 0.4 ml) coated with poly-L-lysine (0.01% wt/vol). Patch-clamp experiments were performed at room temperature (20-22°C).
Experimental solutions and drugs.
The composition of solutions used in patch pipettes and bathing
solutions is given in Table 1. The
Ca2+ concentration used in the bathing and the pipette
solutions was adjusted using EGTA to pCa 3 in cell-attached
configuration, and it was adjusted to pCa 8 in the bathing solutions in
the excised inside-out configuration. All solutions were equilibrated
in air, filtered through 0.2-µm Millipore cellulose disks, and had a
final osmolality of 310 mosmol/kgH2O. Osmolality
was determined by vapor pressure osmometry (Wescor). A set of
reservoirs connected to perfusion pipettes was used to test the effects
of different solutions on channel activity in excised patches. Solution
changes were performed within a few seconds by manual switching between
reservoirs. The permeability of the ORCC to anions other than
Cl was assessed using appropriate potassium salts
(KX
), where X
= SCN
,
I
, Br
, F
,
NO3
, SO42
, or gluconate (see Table
1). NPPB was obtained from Research Biochemicals International. All
other chemicals were from Sigma.
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Current recordings.
Characterization of the single channel current was performed in both
the cell-attached and excised inside-out patch configurations. Single
channel currents were recorded by the method of Hamill et al.
(17) using a RK400 patch clamp amplifier (Biologic, Claix, France) filtered at 0.3 or 1 kHz, digitized (48 kHz), and stored on a
digital audio tape (DTR 1204, Biologic). For analysis, the data were
played back, transferred to a computer, and analyzed by the PAT
computer program (Dempster, Strathclyde Electrophysiology Software).
Patch pipettes (tip resistance ranging between 10 and 15 M) were
prepared from borosilicate glass capillaries (GC 150F, Clark), pulled,
and polished on a programmable puller (DMZ, Werner Zeitz Augsburg,
Germany). Three to five gigaohm seals were obtained by calibrated
suction using a syringe connected to the patch pipette. Under these
conditions, the success rate of obtaining gigaohm seals was 59%. The
sign of the clamped voltage
(Vp) refers to the pipette
solution with respect to the bath, and outward currents (positive
charges flowing across the patch membrane into the pipette) are shown
as an upward deflection in the current traces. In the excised
configuration, the imposed membrane potential
(Vm) is referred to as
Vp. I-V curves were
constructed by plotting the mean current amplitude for each clamped potential.
Liquid junction potentials.
The liquid junction potential (LJP) was defined as the potential of the
bath solution with respect to the pipette solution (2),
and the Vm was calculated as
Vm = Vp + LJP, where Vp is the reading
provided by the patch-clamp amplifier. When bath solutions of different
composition were successively applied to the patch membrane, the
corresponding changes in LJPs were corrected using the Henderson
equation (JPCalc computer program)
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Data analysis.
Open probability (Po) was determined as the
fraction of digitized points above a threshold set midway between the
closed and open peaks of current-amplitude histograms. In these
conditions, Po was defined as the ratio of the
total time spent in the open state to the total time of the complete
record. Po was determined from stable recordings
immediately after identification of the channels. Except for
NPo calculation, analysis was confined to patches containing a single channel. When more than one channel open
state was observed, N was determined as the maximum number of channels observed. The NPo value was
determined from the amplitude histograms for each record. Patches often
contained more than one channel (the average number of ORCCs observed
simultaneously was 1.96 ± 0.08, n = 120). To
determine this, the Vm was clamped to 70 mV for
10-20 min, and when channel activity was detected this potential
was maintained for 5-10 more minutes to check that all channels
present under the patch were activated. When multiple channels were
present, they always activated before this time. The
I-V curve was first obtained to confirm that the
channel was the ORCC. Where only a single channel was found, this was
confirmed by holding the Vm at 70 mV for several
minutes before performing any kinetic analysis at either +50 or 50 mV
with data collected first at +50 mV. Moreover, at the end of each
experiment, and especially when only one channel was observed, the
Vm was always clamped to a high positive voltage
to check that no additional channels were present. For all of the data
relevant to single channel open state, only the data in which this
protocol was fully accomplished were considered. The probability of
simultaneous opening of 1-5 channels was also calculated as
described by Colquhoun and Hawkes (6). Calculations were
made on continuous data, filtered at 1 KHz, that began with a
closed-open transition and ended with an open-closed transition. From
these records, we calculated the mean open time (MOT), mean closed time
(MCT), and the number of open states per second (NOS). Conventional
50% threshold analyses yielded distribution of dwell times that were
fit by multiexponential or power functions consistent with multiple
open and closed states. Gaps were defined as closed intervals of
relatively long duration. To get the best fit, the gaps were defined as
closed intervals >100 ms. Bursts consisted of all channel activity
between gaps. Within the bursts we calculated the burst MOT, the burst
MCT, and the NOS. The I-V plots obtained were fit
with a second-order polynomial equation, and values for reversal
potentials (Er) were obtained from the fitted curves.
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RESULTS |
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The dissociation protocol used in the present experiments eliminated connective tissue, and cells with basolateral membranes free of basal lamina were routinely obtained, resulting in a 59% success rate for obtaining gigaohm seals. Observation of the isolated preparations indicated that the crypts remained viable for at least 4 h after dissociation, after which some cell rounding occurred. Patch-clamp experiments were carried out before changes in cell shape became visible.
Channel activation in intact cells.
Spontaneous Cl channel activity was only rarely found in
the cell-attached mode. Of 735 successful seals, only 7 cell-attached patches showed spontaneous single channel activity consistent with
Cl
-selective channels. Figure
1A gives an example of these
recordings obtained with 145 mM KCl in the pipette and with isotonic
Ringer solution in the bath at a range of applied potentials in
cell-attached patches. The single channel I-V
relationship is presented in Fig. 1B. Under these
conditions, the single channel current exhibited outward rectification
and reversed polarity at the resting Vm (
40 mV
as measured with microelectrodes; Dr. J. P. Pennec, personal communication). This is very close to the Nernst potential for Cl
(Ecl =
36 mV) calculated in
these cells with 35 mM intracellular Cl
concentration
(51).
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Kinetic analysis of the channel in intact cells.
Figure 1B shows that NPo was a direct
function of the imposed potential (Vp) with
the highest values for positive potentials. In addition, as illustrated
in Fig. 1A, the number of channels simultaneously active
(N) was an increasing function of Vm.
Cl channels in excised inside-out patches.
Spontaneous channel activity always disappeared immediately after
excision of active cell patches. After excision in the inside-out configuration from quiescent cell-attached patches, an activity consistent with Cl
channels was observed in 120 out of
735 seals. However, such activity usually occurred only after
10-20 min and application of depolarizing voltage steps of +70 mV
(
Vp). Table 2
summarizes all mean values ± SE of the Er
and slope conductances at Er
(gEr), between
100 and
40 mV
(g
), between +40 and +100 mV
(g+), calculated from single channel currents in
the excised inside-out configuration, with different bathing and
pipette-filling solutions. Figure
2A shows an example of the
current records obtained with Kint solution (pCa 8)
in the bath and 145 mM KCl (pCa 3) in the pipette at a range of imposed
potentials. As shown in Fig. 2B, the
I-V relationship showed the strong outward
rectification characteristic of ORCC. The channel slope conductance was
23.4 ± 1.2 pS (n = 25) between
100 and
40 mV
and increased to 90.2 ± 1.7 pS (n = 25) between
+40 and +100 mV. Under these conditions, the channel activity reversed
at a Vm of 2.5 ± 0.2 mV (n = 25). Table 2 shows that the ORCC was distributed along the crypts
from the base to the apex. For all of the following experiments,
recordings were performed at the crypt base. Seals were generally
easier to achieve and crypt structure integrity was better conserved in
this region.
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Ion selectivity of the ORCC.
Table 2 and Fig. 3 show the effects of
substituting NaCl or K-gluconate for KCl in the bath or in the pipette.
Replacement of KCl by NaCl had no significant (P < 0.05) effect on the I-V relationship (Table 2),
whereas in the presence of K-gluconate in the pipette, the reversal
potential shifted to 34.3 ± 1.9 mV (Fig. 3, n = 6). The anionic vs. cationic selectivity was determined by changing
from Kint to half-strength KCl solution
(Kint1/2), with only half of the concentration of KCl on
the cytosolic side of the patch. This maneuver significantly
(P < 0.01) shifted the I-V curve
to the left and the reversal potential was 14.0 ± 1.5 (n = 9) mV, consistent again with
ECl (
17.9 mV). The relative permeability
(PCl/Pcations) derived
from the Goldman-Huxley-Katz relation was 14.6 ± 4.7 (n = 9). The measured conductances of the channel
(g+, g
, and gEr) are
also significantly reduced (P < 0.01) in the presence of Kint1/2.
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Relative anion permeability.
The relative permeability of various anions compared with
Cl was assessed from measurements of current reversal
potentials in ion substitution experiments. KCl 145 mM, initially
present in the bathing solution, was replaced by KCl 72.5 mM + KX
72.5 mM (X
being the anion to be
tested), and the Cl
concentration in the pipette was kept
constant at 145 mM. The shift of the reversal potential was expressed
with respect to the KCl solution after correction for the calculated
junction potential. Ion selectivity was calculated from
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Kinetic analysis of the channel in excised inside-out patches.
Figure 2 shows that Po was an increasing linear
function of Vm. Moreover, in spite of the
relatively low frequency of channel occurrences, several identical
channels (up to five) were simultaneously present in active membrane
patches. For example, a total of 206 multiple ORCCs were observed in
120 recordings. In addition, Fig. 1A clearly shows that for
a given membrane patch, the number of channels simultaneously active
was proportional to the Vm with the highest
values seen at positive potentials. However, the observed increase in
open states (also observed in the cell-attached configuration) most
likely results from the voltage dependence of
Po. The probability of simultaneous opening of
1-5 channels was also calculated as described by Colquhoun and
Hawkes (6). This probability was in good agreement with
that predicted for independent channels by a binomial distribution with
a single channel Po of 0.57. Dwell time analysis
was performed on patches containing only one Cl channel,
and the kinetic analysis was made at a holding potential of +50 or
50
mV. At these potentials, the channel consistently displayed burst/gap
behavior. The ORCC kinetics were characterized by very low
Po at negative potentials
(Po= 0.23 ± 0.07, n = 11) compared with positive potentials (Po= 0.57 ± 0.04, n = 19), corresponding to longer MCT
(30.7 ± 8.4 ms, n = 11, vs. 7.6 ± 0.9 ms,
n = 19). Within bursts, the channel kinetics were
characterized by a lower Po at negative
potentials (0.32 ± 0.01, n = 413 bursts) compared with positive potentials (0.65 ± 0.01, n = 195 bursts), as shown in Table 4. Open time
durations were fitted best by the sum of two exponential distributions
at +50 mV (Table 4) and by a single exponential distribution at
50
mV. Closed time distribution was always fitted by the sum of three
exponentials.
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Effect of channel activators or blockers in intact cells.
Several attempts were made to induce channel activity in the
cell-attached configuration with the aid of agents known to increase intracellular pH, Ca2+, or cAMP or to directly activate
protein kinases A or C. All trials were made either by incubating the
crypts in Ringer solutions containing the drugs at 30, 60, or 90 min
before patch clamping (n = 15-20 for each drug
concentration) or by addition to the bath after obtaining the gigaohm
seal (n = 15-20 for each drug concentration).
Forskolin (10 or 20 µM), histamine (10 µM), carbachol (50 µM),
and A-23187 (1 or 100 µM) failed to induce any channel activity
consistent with a Cl conductance. In the same patches,
ORCC could be activated by voltage after transition to the excised
inside-out configuration. Activators of protein kinase C (phorbol
12-myristate 13-acetate, 1, 2, and 4 µM; phorbol 12,13-dibutyrate, 1, 2, and 3 µM) also failed to activate ORCC in cell-attached patches.
Effect of channel activators or blockers in excised patches.
Figure 4 shows the effects of 50 µM
NPPB on the channel activity. Addition of the blocker to the bathing
solution led within seconds to a 95% inhibition of ORCC activity. The
blockade was characterized by a reduction (94.0 ± 1.9%) in
NPo from 0.77 ± 0.12 (n = 10) to 0.03 ± 0.01 (n = 10) in parallel with a
decrease in the unitary current (control = 2.58 ± 0.08 pA
and NPPB = 1.45 ± 0.19 pA; n = 10). This
unitary current decrease results from the flickery block induced by
NPPB. Subsequent washout of NPPB from the bathing solution slowly led
to 51.7 ± 10.4% recovery of the channel
NPo.
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Absence of channel activation by phosphorylation in excised
patches.
Previous studies have shown that protein kinase A stimulates ORCC in
different types of epithelial cells (3, 28,
44). However, in the mouse colonic crypt cells in the
excised inside-out configuration, all attempts (n = 10)
to activate the channel with the catalytic subunit of bovine or porcine
cAMP-dependent protein kinase (50 or 100 nM) in the presence of its
cofactor Mg2+-ATP, failed to show any effect. This implies
that the Cl channel is not regulated by protein kinase A
phosphorylation or that such regulation requires some additional factor
lost on excision.
G protein regulation.
To determine whether G proteins regulate ORCC activity, we examined the
effects of guanosine 5'-O-(3-thiotriphosphate) (GTPS) and
guanosine 5'-O-(2-thiodiphosphate) (GDP
S) on the
Po and amplitude of the single channel current
in inside-out patches. GTP
S (5, 10, 15, or 20 µM) added to the
solution bathing the cytoplasmic side of the patch reduced
Po and single channel activity by ~50% (Fig.
6, A and C) with no
apparent significant concentration dependence (at least in the range of
5-20 µM) observed. This lack of concentration dependency could
reflect the fact that 5 µM GTP
S already induced a maximal effect
on the channel Po and unitary current. Because the observed decrease in the channel conductance induced by GTP
S could result from a flickery block, the unitary current in the presence
of this nucleotide was determined for different filter frequencies (500 Hz and 1, 2, and 5 kHz). No increase in the measured current was
observed with increasing filtration frequency, suggesting that a
flickery block was not involved, although the possibility that the
frequency of block exceeded the fastest filter rate used (5 kHz) cannot
be definitively excluded.
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DISCUSSION |
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The purpose of the present study was to identify and characterize
the Cl channels present in the basolateral membrane of
enterocytes in intact mouse distal colonic crypts. Compared with other
methods using isolated enterocytes, the technique of dissociation of
intact crypts has the obvious advantage of maintaining a clear
differentiation between the apical and basolateral poles of the cells,
along with the base-to-apex topology of the crypt.
Only one type of Cl channel was found, and this was an
ORCC. The ORCC demonstrates an intermediary conductance (20-90
pS), strong outward rectification, and inhibition by DIDS, NPPB, 9-AC, and GTP. Outwardly rectifying anion channels have been described in a
wide variety of epithelial cells and also in nonepithelial cell
membranes. In most of these reports, the occurrence of such ORCCs was
found to be extremely low in the cell-attached configuration, consistent with our findings of spontaneous activity in only ~1% of
the patches under steady-state conditions. An exception to this is the
intermediate conductance ORCC from chicken colon epithelial cells
described by Fischer et al. (12). Although this channel was reported to show significant spontaneous activity, the study involved isolated cells, so it was not possible to determine whether the channel was located in the apical or basolateral membrane. In
intact rat colon epithelium, Diener et al. (9) described an ORCC (13.5-40 pS) on the apical membrane that was spontaneously active in cell-attached patches and that remained active after excision
of the patch. Some reports have suggested that ORCC activity can be
induced in intact cells by cAMP (13, 16,
18, 20). However, this is not a general
feature of ORCC, since, as in the present study, there are several
reports of cAMP failing to influence ORCC activity (9,
49).
Another consistent property of ORCC from a variety of tissues is so-called excision activation (25, 48). At room temperature, ORCCs are usually quiescent after excision, and activation requires application of depolarizing membrane voltages (70-100 mV). In contrast, at 37°C the channels may activate after excision without application of membrane voltage. In the present study, channel activation at 37°C could not be studied because the isolated crypts rapidly lost their morphological integrity. At room temperature, the protocol for successful channel activation in this study (15- to 20-min waiting time and 70-mV depolarization steps) is very similar to that reported in the literature for other ORCCs (11, 13, 16, 28, 44). Under these conditions, channel activity was recorded in 16.5% of all seals. The observation of activation after excision has led to the suggestion that ORCCs might be tonically inhibited as long as the cell membrane is in contact with the cytoplasm and that the cell might contain a cytosolic inhibitor that diffuses away after patch membrane excision (11, 23, 26).
As with the effect of cAMP on ORCC activity in intact cells, channel activation by exposure to purified cAMP-dependent protein kinase catalytic subunit has been reported, but with varying success (3, 28, 44). As an example, in HT-29 cells, the number of patches presenting channel activity increased from 3 to 42% after exposure of the excised inside-out patches to protein kinase A + ATP (20). On the contrary, no activation was seen in T84 cell patches (48). Similarly, no activation by the protein kinase A catalytic subunit was observed in excised patches in the present study, although it was subsequently shown by voltage activation that the patches did contain ORCC.
The permeability order for the mouse colon basolateral ORCC described
here was found to be I > SCN
> Br
> Cl
> NO3
> F
SO42
gluconate,
corresponding to the Eisenmann I sequence (53). Similar
sequences have been reported for other Cl
channels from
enterocytes, including ORCCs (13, 24,
36). However, a higher relative permeability to
nitrate than Cl
has been reported for several other ORCCs
(1, 27), including those in intestinal cells
(15, 38), and this characteristic appears to
be a common feature for this type of Cl
channel. Like
respiratory cells (25) or rabbit parietal cells (41), a higher permeability of Cl
over
nitrate was observed for the ORCC described in these crypt intestinal
cells. The higher permeability for larger ions indicates that small
ionic size does not favor flow through the channel. This may indicate
that an important factor for permeation is the energy necessary to
dehydrate the anion, because large anions have lower hydration energies
and move more easily from the aqueous phase to the cationic sites
located inside the channel (16). Partial substitution of
Cl
by other anions modified single channel conductance as
well as the Er. A similar anionic selectivity is
obtained when determined from the shift in Er or
from the conductance measured at Er. However, in
contrast to observations for other ORCCs (13,
15, 24, 27), the selectivity
sequence determined from conductance measured between
40 and
100 mV
is different from the sequence derived from relative permeabilities.
In addition, the data show that stimulation of protein kinase C failed to activate the basolateral ORCC in mouse colon crypt cells, although this has been reported to be successful in the ORCC of airway epithelial cells (29). The absence of response to histamine, carbachol, and A-23187, all of which are known to cause increases in intracellular Ca2+ levels, demonstrates that Ca2+ is not a trigger for ORCC activation. This is further supported by the fact that changing the pCa value in the bath solution in the excised inside-out configuration did not modify single channel activity. A similar insensitivity to intracellular Ca2+ levels has been previously demonstrated for other ORCCs (4, 13, 14, 24, 36).
In contrast to the above, GTPS had pronounced effects on the
activity of the mouse colon basolateral ORCC, decreasing single channel
Po and conductance by 50%. In addition, this
could be readily reversed by addition of GDP
S. The indication is
that G proteins likely play a key role in the regulation of the
basolateral ORCC of crypt cells. Inhibitory effects of G proteins on
other ORCC have been previously described (19,
45), as well as on high-conductance (21,
31) and low-conductance Cl
channels
(42, 43). On the other hand, activation was
seen in inwardly rectifying Cl
channels (22,
50), as well as for high-conductance Cl
channels (33, 46) or for Cl
channels of very low conductance (30). As yet, the precise mechanisms underlying these stimulatory or inhibitory effects of G
proteins remain unclear. It was suggested (45) that the inhibition of ORCC activity could occur through an activation of
phospholipase A2 and subsequent production of arachidonic acid or from
the inhibition of adenylate cyclase and the subsequent decreased pool
of intracellular cAMP. It would seem that the latter mechanism is most
unlikely for the mouse colon basolateral ORCC, since we never observed
any activation of ORCC by cAMP-dependent protein kinase. Alternatively,
the activity of the ORCC may be regulated by a
phosphorylation/dephosphorylation process that is influenced by a G
protein-dependent activation of phosphatases (40,
45). In the present study, activation of G proteins
decreases the unitary current of the ORCC as well as its
Po. In a number of studies, the inhibitory
effect of the activation of G proteins on Cl
channels is
via a decrease of the Po without affecting the
single channel conductance (21, 31,
43, 45). To the best of our knowledge, only
one report of an effect of modulating G protein activity on channel
conductance has been published. In this case, the inhibition of the
unitary conductance by pertussis toxin, an agent known to
inactivate Gi proteins, increased the
Po of an immunopurified ORCC incorporated in
planar bilayer but also conferred linearity to the
I-V relationship of this channel
(19). The authors suggested that the rectification of the
channel partially involved its interaction with the G protein. However,
they also showed that the addition of GTP by itself had no effect on
the channel conductance. The direct effect of GTP and GDP on the ORCC activity described in the present study seems to be a unique feature. G
protein regulation of both Po and unitary
current provides a more efficient regulation of the channel activity
than an effect on Po alone. Clearly, the
regulation of the activity of the mouse colon basolateral ORCC and the
possible role of G proteins in such regulation is worthy of further investigation.
As to the possible physiological role(s) of this basolateral ORCC in
the mouse colon, it is clear that such channels could be involved in
the transport of Cl through the basolateral membrane of
colonic crypts during the process of NaCl absorption. However, the
stimulation of NaCl absorption, for example by somatostatin or
by increased absorption of short-chain fatty acids (7,
10), is known to induce cell swelling and the induction of
the subsequent process of regulatory volume decrease. Moreover, in
intestinal cell lines, activation of ORCC was detected during cell
volume regulation following hypotonic shock (24, 52). In some preliminary experiments, we attempted to
examine changes in channel activity following exposure to hypotonic
medium in the native mouse colon basolateral ORCC using the
cell-attached configuration. However, these proved to be unsuccessful
because of the fragility of the seal due to the large variations of
membrane tension and to unavoidable movements of the intact crypt in
the perfusion flow.
In conclusion, we have been able to demonstrate for the first time the
presence of a Cl channel characterized by a strong
outward rectification and sensitive to DIDS, NPPB, 9-AC, and GTP on the
basolateral membrane of crypt cells from mouse distal colon. The
presence of this ORCC could be demonstrated in both cell-attached and
excised inside-out patch-clamp configurations. The existence of such a
basolateral Cl
channel is frequently postulated in the
models for NaCl absorption in colonic cells, and our findings are
therefore consistent with such a physiological role. Furthermore, the
possible involvement of this basolateral ORCC in volume regulation
processes following hypotonic shock should be considered, particularly
as we have previously demonstrated a key role of a Cl
exit pathway sensitive to NPPB and 9-AC during RVD in these cells (35). Clearly, further investigations are needed to
precisely define the physiological role, or roles, of this channel in
the basolateral membrane.
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ACKNOWLEDGEMENTS |
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We thank Dr. Trevor Shuttleworth, Dr. Alexandre Ghazi, Dr. Ted Begenesich, and Jill Thompson for helpful discussion and for critiquing the manuscripts.
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FOOTNOTES |
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Address for reprint requests and other correspondence: O. Mignen, Univ. of Rochester Medical Center, Dept. of Pharmacology and Physiology, 601 Elmwood Ave., Rochester, NY 14642.
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. §1734 solely to indicate this fact.
Received 2 December 1999; accepted in final form 2 March 2000.
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