Secretory activation of basolateral membrane Cl
channels in guinea pig distal colonic crypts
Yingjun
Li,
Susan Troutman
Halm, and
Dan R.
Halm
Department of Physiology and Biophysics, Wright State
University, Dayton, Ohio 45435
 |
ABSTRACT |
Cell-attached recordings
revealed Cl
channel activity in basolateral membrane of
guinea pig distal colonic crypts isolated from basement membrane.
Outwardly rectified currents (gpClor) were
apparent with a single-channel conductance (
) of 29 pS at resting
membrane electrical potential; another outward rectifier with
of 24 pS was also observed (~25% of gpClor). At a
holding potential of
80 mV
was 18 pS for both
gpClor currents, and at +80 mV
was 67 and
40 pS, respectively. Identity as Cl
channels was
confirmed in excised patches by changing bath ion composition. From
reversal potentials, relative permeability of K+ over
Cl
(PK/PCl)
was 0.07 ± 0.03, with relative permeability of Na+
over Cl
(PNa/PCl) = 0.08 ± 0.04. A second type of Cl
channel was seen
with linear current-voltage (I-V) relations (gpClL), having subtypes with
of 21, 13, and 8 pS. Epinephrine or forskolin increased the number of open
gpClor and gpClL. Open
probabilities (Po) of
gpClor, gpClL21, and
gpClL13 were voltage dependent in cell-attached
patches, higher at more positive potentials. Kinetics of
gpClor were more rapid with epinephrine
activation than with forskolin activation. Epinephrine increased
Po at the resting membrane potential for
gpClL13. Secretagogue activation of these
Cl
channels may contribute to stimulation of electrogenic
K+ secretion across colonic epithelium by increasing
basolateral membrane Cl
conductance that permits
Cl
exit after uptake via
Na+-K+-2Cl
cotransport.
potassium ion secretion; chloride secretion; epinephrine; prostaglandin E2; forskolin
 |
INTRODUCTION |
EPITHELIAL ION SECRETION
DRIVES fluid secretion by producing transepithelial osmotic
gradients (8, 18, 19). Electrogenic Cl
secretion is a common type of transport in these fluid secretory epithelia. The cellular mechanism for this secretion includes Cl
channels in the apical membrane that are activated by
various secretagogues through the action of intracellular signaling
pathways. Colonic epithelia of mammals also produce electrogenic
K+ secretion via a cellular mechanism similar to that for
Cl
secretion except that apical Cl
conductance is not activated (17, 19, 23, 50, 51). Secretagogues producing electrogenic K+ secretion with
little or no accompanying steady-state Cl
secretion
include epinephrine (17, 19, 50, 68), prostaglandin E2 (PGE2) (23, 50), aldosterone
(21, 51), and cholinergic agonists such as carbachol
(6, 11). Sensitivity of this K+ secretion to
bumetanide (17, 50, 51, 68) supports a requirement for
basolateral membrane Na+-K+-2Cl
cotransporters. Because Cl
entering together with
Na+ and K+ does not exit into the lumen, a
second basolateral Cl
transport process is necessary to
allow maintenance of intracellular Cl
concentration
during steady-state K+ secretion (24, 27).
Inhibition of K+ secretion by DIDS further supports that
another basolateral Cl
transport pathway is involved
(17). Basolateral membrane Cl
channels have
been proposed as this Cl
exit step that would contribute
to the observed positive charge flow across the epithelium from blood
side to lumen (17). Activation of basolateral membrane
Cl
channels as well as apical membrane K+
channels presumably would occur during secretagogue stimulation to
initiate and sustain steady-state electrogenic K+ secretion.
Basolateral membrane Cl
conductance
(g
) serves multiple functions in
epithelial cells. Electrolyte absorption in the thick ascending limb of
Henle's loop (52) and in the intestine of teleost fish
(25) uses a cellular mechanism in which Cl
enters across the apical membrane via
Na+-K+-2Cl
cotransporters and
exits, in part, through basolateral membrane Cl
channels.
The cochlea and vestibular labyrinth of the inner ear secrete
K+ by a mechanism (67) similar to that
proposed for the colonic epithelium (17). In addition,
g
is activated during regulatory volume
decrease in colonic crypts as well as other cell types (12, 47,
57, 62). Similarities may exist for the control of
Cl
channels during transepithelial ion flow and cell
volume regulation, but whether the same channels serve both types of
function in epithelia has not been determined.
Numerous classes of Cl
channels have been identified that
are involved in transepithelial flow and cell volume control as well as
contributing to conductances in specific cells to support synaptic
signaling and modulation of excitability (15, 33, 47, 60, 62,
69). Some of these Cl
channel types are pertinent
to epithelial function, and three have a defined molecular identity:
CFTR (33, 60), the CLC family (33, 69), and
the Ca2+-activated Cl
channel family CLCA
(16). Volume-regulated Cl
channels
(Clvol) and an outwardly rectified Cl
channel
(Clor) are of uncertain molecular identity but are present in intestinal epithelia (3, 5, 12, 47, 57, 62). CFTR is a
Cl
channel with voltage-independent single-channel
conductance (
) of ~9 pS activated by cellular protein kinases
(33, 60). Members of the CLC family have voltage-dependent
currents variously inwardly rectified and outwardly rectified with
from ~1 pS to >40 pS (33, 52, 69). CLCA is activated by
Ca2+ with
of 15-30 pS (16). Cell
swelling activates Clvol, which has outwardly rectified
currents with
of uncertain size (12, 47, 61, 62). The
outwardly rectified
- and depolarization-enhanced open probability
(Po) of Clor (20, 26, 43,
61) appears to be distinct from that of Clvol
(47, 61, 62). Blockers have been described for these
channel types, but most are relatively nonspecific so that defining
channel type by blocker sensitivity is problematic at present
(33, 47, 48, 58, 61, 62).
Several of these Cl
channel types have been localized to
colonic epithelia. CFTR mRNA is expressed primarily in the lower two-thirds of colonic crypts (63). A basolateral
localization of CFTR would be counter to the commonly accepted function
as a secretory Cl
channel, but its presence at low levels
in the basolateral membrane of sweat gland duct cells may support
Cl
absorption from the primary sweat (10,
34). CLCA-1 mRNA is expressed in colon, particularly crypt
goblet cells (16). In the CLC family CLC-2, CLC-3, CLC-4,
CLC-6, and CLC-7 are broadly distributed (33). CLC-2
appears in the basolateral membrane of surface and crypt epithelium in
rat colon and at an intracellular location in human colonic crypts
(40); in guinea pig distal colon, CLC-2 was localized only
to the basolateral membrane of the surface epithelium (7).
An intracellular location also is expected for CLC-3, -4, -5, -6, and
-7 (33). A splice variant, CLC-3B, is expressed
predominantly in epithelia and alters plasma membrane Cl
conductance (46). CLC-4 colocalizes with CFTR to the
apical membrane of rat ileal crypt cells (44); an
intracellular localization near the basolateral membrane was apparent
for CLC-5 in rat colon (65). CLC-K has been localized to
the basolateral membrane in the thick ascending limb of Henle's loop
and collecting duct of kidney as well as cochlea and vestibular
labyrinth of inner ear (33, 52, 55, 66). Similarly,
basolateral Cl
channels in colonic crypt cells would
serve to permit exit of Cl
from the cell into the
interstitial space and thus support electrogenic K+
secretion. This study provides results indicating that K+
secretagogues activated basolateral membrane Cl
channels
in colonic crypt cells. The observed stimulation of several
Cl
channel activities, through an increased number of
open channels and an increased channel Po, would
lead to greater g
consistent with an
involvement in Cl
exit during electrogenic K+ secretion.
 |
METHODS |
Male guinea pigs (400- to 700-g body wt) received standard
guinea pig chow and water ad libitum. Guinea pigs were killed by decapitation in accordance with a protocol approved by the Wright State
University Institutional Laboratory Animal Care and Use Committee.
Distal colon was removed and defined as the ~20-cm-long segment
ending roughly 5 cm from the rectum. Colonic segments were cut open
along the mesenteric line and flushed with ice-cold Ringer solution to
remove fecal pellets. Epithelium was separated from underlying
submucosa and muscle layers by using a glass slide to gently scrape
along the length of the colonic segment. The plane of dissection
occurred at the base of the crypts such that only components of the
mucosa immediately adherent to the epithelium remained. These isolated
colonic mucosal sheets were used for measurement of transepithelial
current and conductance (23) as well as for further
isolation of intact crypts, allowing patch-clamp recording of
basolateral membrane currents (39).
Four mucosal sheets from each animal were mounted in Ussing chambers
with an aperture of 0.64 cm2. These sheets were supported
on the serosal face by Nuclepore filters (Whatman) with a thickness of
~10 µm and a pore diameter of 5 µm. Bathing solutions (10 ml)
were circulated by gas lift through water-jacketed reservoirs that were
maintained at 38°C. Standard Ringer solution contained (in mM) 145 Na+, 5 K+, 2 Ca2+, 1.2 Mg2+, 125 Cl
, 25 HCO
, 4 H(3
X)PO
, and 10 D-glucose. Solutions were continually gassed with 95%
O2 and 5% CO2, which maintained solution pH at
7.4. Transepithelial electrical potential difference
(Vt) was measured by two calomel electrodes
connected to the chambers by Ringer-agar bridges. Chambers were
connected to automatic voltage clamps (Physiologic Instruments, San
Diego, CA) that permitted continuous measurement of short-circuit current (Isc) and compensation for solution
resistance. Current was passed across the tissue through two Ag-AgCl
electrodes connected by Ringer-agar bridges. Isc
is described as positive for current flowing across the epithelium from
the mucosal side to the serosal side. Transepithelial conductance
(Gt) was measured by recording currents
resulting from bipolar square voltage pulses (±5 mV, 3-s duration)
imposed across the mucosa at 1-min intervals.
Portions of mucosa were mounted with cyanoacrylate glue onto Lucite
holders with apertures 1 cm wide and 4 cm long to permit isolation of
intact crypts. Mucosal portions on holders were incubated at 38°C in
HEPES-buffered solution, with indomethacin (1 µM) to reduce
spontaneous fluid and mucus secretion (22, 23, 50). Standard HEPES-buffered Ringer solution contained (in mM) 142 Na+, 5 K+, 2 Ca2+, 1.2 Mg2+, 143 Cl
, 4 H(3
X)PO
, 10 HEPES, and 10 D-glucose.
Solutions were continually aerated with room air. Isolation of crypts
from the mucosa followed general procedures developed previously
(39). Solutions for separating epithelium from underlying
connective tissue contained (in mM) 192 Na+, 5 K+, 97 Cl
, 4 H(3
X)PO
, 10 HEPES, 10 D-glucose, and
either 30 mM citrate or EDTA. Isolation solution containing EDTA also
had 0.1% bovine serum albumin. Best results were obtained when the
EDTA solution was prepared on the day of the isolation, as noted
previously (2). Mucosal portions were consecutively
incubated in 30 mM citrate Ringer with indomethacin (1 µM) for
15-30 min and 30 mM EDTA Ringer for 15-20 min at 38°C. Holders then were agitated in HEPES-buffered Ringer with indomethacin (1 µM) and dithiothreitol (1 mM) to release surface and crypt epithelium. Inclusion of dithiothreitol reduced clumping of epithelium within extruded mucus. Isolated crypts were stored on ice or in a
refrigerator until use. Patch-clamp recording on these crypts began
~2 h after removal of the colon from the animal and were suitable for
patch-clamp experiments up to ~36 h.
Isolated crypts were transferred onto a poly-lysine-coated plastic
coverslip in the electrical recording chamber mounted on the stage of
an inverted microscope (Diaphot, Nikon). Bathing solutions were
perfused into the chamber by a peristaltic pump (Gilson, Middleton,
WI), at room temperature. Pipettes were fabricated from 7052 glass
(WPI, Sarasota, FL) with a two-stage puller (Narishige, Tokyo, Japan),
coated with Q-dope (GC Electronics, Rockford, IL), and fire-polished.
Pipettes filled with either high-Na+ or high-K+
solution (Table 1) had resistances of
5-10 M
and were connected to the head stage of an EPC-7
patch-clamp amplifier (List-Medical) via a 150 mM KCl-agar salt bridge
inside a holder containing a Ag/AgCl electrode (14). The
reference electrode was a Ag/AgCl pellet connected to the bath through
a 150 mM KCl-agar salt bridge. Currents were recorded on videotape with
3-kHz filtering using a pulse code-modulated VCR (Vetter Instruments,
Rebersburg, PA). Seals were made on the central tubular portion of
isolated crypts bathed in standard HEPES-buffered Ringer solution.
Seals of >1 G
were obtained in about one of five attempts. Before
excision of patches the bath solution was changed to one containing
EGTA (Table 1) to maintain low free Ca2+ that would mimic
maximal intracellular conditions (~10 µM) but be low enough to
avoid activation of nonselective cation channels (5).
Lower levels of bath free Ca2+ (~100 nM and <10 nM) were
produced by adding only 0.1 mM Ca2+ or no Ca2+,
respectively, to these bath solutions. Solution osmolarity was 292 mosM
(290-294 mosM), except for the 300 Cl bath.
Drugs were added in small volumes from concentrated stock solutions.
PGE2, indomethacin, and NS-398 were obtained from Cayman Chemical (Ann Arbor, MI) and epinephrine from Elkins-Sinn (Cherry Hill,
NJ). All other chemicals were obtained from Sigma (St. Louis, MO).
PGE2 was prepared in an ethanol stock solution that added 0.1% ethanol at 10 µM of PGE2; additions of 1% ethanol
alone did not alter transepithelial measures of K+ or
Cl
secretion (23).
Data analysis.
Concentration responses of Isc and
Gt to forskolin were fit to
Henri-Michaelis-Menten binding curves with a nonlinear least-squares procedure. Two independent binding curves were required
(23), I = IA/[1 + (EC
/C)] + IB/[1 + (EC
/C)] or G = GA/[1 + (EC
/C)] + GB/[1 + (EC
/C)], such that total Isc or Gt was a
combination of these two components (IA and
IB; GA and
GB) at each concentration (C). Results are
reported as means ± SE. Statistical comparisons were made with a
two-tailed Student's t-test for paired responses, with
significant difference accepted at P < 0.05.
Patch-clamp current data were transferred via DigiData-1200 interface
to a computer for analysis with pCLAMP6 software (Axon Instruments,
Foster City, CA). Currents were filtered at 700 Hz. Junction potentials
(1, 45) at pipette tip and bath reference bridge were
calculated to correct holding potentials
(Vhold). Relative ion permeabilities were
calculated with the Goldman-Hodgkin-Katz potential equation together
with the measured reversal potential and solution ion composition.
Po was calculated from all-points histograms of
current amplitude. Area (A) under each current peak was
determined by a Gaussian fit. Po was obtained
from the relation: Po = (
iAi)/
Ai/N
with i indicating each peak starting at 0 for baseline and
increasing to N, the number of active channels. A I-V relation was constructed from the lowest current peaks
to ensure that the lowest peak at each Vhold
indicated the closed state. Records of sufficient length (5-10
min) were obtained for each condition to allow a reliable measure of
N from the number of observed peaks (29). In
records containing only one channel further kinetic analysis was
performed by producing histograms of open and closed durations from an
events list. For this analysis, current records were sampled at a rate
of 20 µs/point and then filtered at 1 kHz to minimize noise but also
maximize bandwidth. Log binning was used to improve fitting and display
of exponential curves (32); maximal likelihood estimates
were used to obtain time constants from open and closed durations.
 |
RESULTS |
Isolated crypts had basolateral membranes that were accessible to
sealing with patch pipettes; seals were obtained on the middle section
of the crypt cylinder. Identity of cells as either columnar or goblet
(22) was not readily discernable. Reversal potentials of
ionic currents while cell attached aided identification of the channel
types producing those currents (39). For crypt epithelial
cells, currents from Cl
and K+ channels
recorded with high-Na+ pipette solution were expected to
reverse at positive and negative Vhold,
respectively, as determined by the ion concentration gradients (24, 27) together with a cell membrane electrical
potential difference (PD) (Vcell) of about
65
mV (42). Thus, at resting Vcell (Vhold = 0 mV), Cl
currents would be inward (net outward
Cl
flow) and K+ currents would be outward. In
addition, nonselective cation channel currents would reverse at large
positive Vhold corresponding to a
Vcell of 0 mV. Using high-K+ pipette
solution (Table 1) has the advantage of shifting the reversal potential
for K+ channels toward a Vcell of 0 mV. Reversal of K+ channel currents near a
Vhold of +40 mV (39) indicates that resting Vcell was about
40 mV in these
isolated crypts. The shift in reversal potential for K+
channels (to Vcell = 0 mV) also provided
greater separation from the expected Cl
channel reversal potential.
Spontaneous single-channel currents consistent with Cl
channel activity were observed while cell attached (Fig.
1) with both high-Na+ and
high-K+ pipette solutions. Currents from outwardly
rectified Cl
channels (Fig. 1A) occasionally
were seen together with inwardly rectified guinea pig K+
channels (gpKir; Ref. 39),
supporting a resting Vcell of about
40 mV in these recordings. Other currents consistent with Cl
channels also were seen, reversing at Vhold of
about +5 mV. Several sizes of current events were apparent (Fig. 1,
B and C), generally smaller than the outwardly
rectified Cl
currents. Nonselective cation channels
rarely were observed in guinea pig crypts, as noted previously
(39).

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Fig. 1.
Cl channel currents in crypt cells. Current
traces are shown of cell-attached patches from basolateral membrane of
isolated colonic crypts. Pipette solution was high-K+
containing 140 mM K+ (Table 1). Dashed lines indicate
closed state. A: an outwardly rectified Cl
channel (gpClor) is shown together with an
inwardly rectifying K+ channel
(gpKir). Currents from
gpKir reverse near +40 mV, and those for
gpClor reverse near +5 mV; trace at +80 mV was
chosen to show only gpClor, and traces at
negative holding potential (Vhold) were chosen
to show only gpKir. B: current
traces are shown for a voltage-independent (linear- )
Cl channel of 12 pS. C: current traces are
shown for 2 linear- Cl channels of 6 and 17 pS.
|
|
Outwardly rectified Cl
channels were seen with two sizes
of outward current in guinea pig crypts
(gpClor); inward currents were similar in size
(Fig. 2A). Activity of
gpClor was observed in 16 of 231 patches (7%).
Cl
currents (Fig. 2B) with linear
I-V relations (gpClL) were observed,
congregated into three groups (Fig. 2C) on the basis of
single-channel
: 17-25 pS (17 of 231; 7%), 11-15 pS (15 of 231; 6%) and 5-9 pS (10 of 231; 4%). Although these distinctions are somewhat arbitrary, the three groups were clearly separable as indicated by the small variability within each group. The
presence of multiple Cl
channel classes also was
supported by appearance of two or more types of
gpClL in some patches (Fig. 1C), so
that differing cell composition alone could not account for the
distinct conductances. Cl
channels were observed in 47 of
231 patches (20%), with more than one Cl
channel type
occasionally present in a single patch (Fig. 1C). Positive
reversal potentials for gpClor and
gpClL (Fig. 2, A and B)
support outwardly directed Cl
flow through these channels
at spontaneous Vcell, consistent with the
predicted requirements for conductive Cl
flow across the
basolateral membrane during electrogenic K+ secretion.

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Fig. 2.
Cl channel conductive properties. A:
current-voltage (I-V) relations are shown (means ± SE)
for gpClor activity observed in cell-attached
patches of crypt basolateral membrane. Two distinct groups were
observed, with larger outward currents ( ,
n = 9) and with smaller outward currents
( , n = 3). B: I-V
relations are shown for linear- Cl channel
(gpClL) activity, exhibiting both inward and
outward currents, observed in cell-attached patches of
crypt basolateral membrane. Three distinct sizes of activity were
observed ( , n = 7; ,
n = 10; , n = 9).
C: single-channel conductance (chord conductance; ) is
shown, from I-V relations in A and B.
Mean for gpClL21,
gpClL13, and gpClL8 was
21.4 ± 1.0, 13.2 ± 0.4, and 8.3 ± 0.5 pS,
respectively; each group was significantly different from the other 2 (P < 0.05).
|
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Cl
channels were seen together with
gpKir (Fig. 1A) and were
distinguished by currents exhibiting distinct reversal potentials and
rectification. In five patches, gpClor was
observed together with gpKir. On the basis of
the proportion of patches exhibiting each of these channel types
(39), four patches would have been expected to contain
both gpClor and gpKir,
assuming uniform channel distribution among patched cells. Preferential
localization, of the channel types being compared, to separate cell
types would reduce the expected number of joint occurrences. Each of
the linear-
Cl
channels also was observed with
gpKir (expected number of patches): seven
(4) for gpClL21, seven
(4) for gpClL13, and three
(3) for gpClL8. Appearance of
these channels together with gpKir further
supported identification as Cl selective, because nonselective cation
currents would have reversed near +40 mV similar to
gpKir. The various Cl
channels,
as distinguished by conductance, also were observed together. Presence
of linear-
Cl
channels together with
gpClor was observed (expected number of
patches): four (1) for gpClL21,
four (1) for gpClL13, and zero
(1) for gpClL8. The presence
together of multiple linear-
Cl
channel types also was
observed (expected number of patches): three (1) for
gpClL21 with
gpClL13, three (1) for
gpClL21 with gpClL8,
and two (1) for gpClL13 with
gpClL8. The results suggest a clustering of
these basolateral channel types.
Ion selectivity.
Activity of gpClor often persisted after
excision into an inside-out (I/O) configuration (8 of 12 patches;
67%), which permitted ion selectivity to be determined more precisely.
Increasing or decreasing bathing solution Cl
concentration (Table 1) shifted the reversal potential as expected for
a Cl
-selective channel (Fig.
3A). Relative ion permeability
was calculated for Cl
with respect to K+ and
Na+: relative permeability of K+ over
Cl
(PK/PCl) = 0.07 ± 0.03 (n = 7) and relative permeability of
Na+ over Cl
(PNa/PCl) = 0.08 ± 0.04 (n = 4). Ion selectivity of
gpClL was more difficult to determine precisely
because these channels generally inactivated on excision; but ion
substitution in a few cases (n = 3) supported
preference for Cl
over K+ (data not shown).
In addition, excision into symmetrical Cl
concentrations
could be expected to produce inward rectification for
gpClL; however, activity did not persist in
enough cases to resolve the excised I-V relations
completely.

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Fig. 3.
Ion selectivity of gpClor . A:
after excision into inside-out (I/O) configuration, bath solution
(Table 1) was changed among NaCl ( , ;
n = 7, 2), KCl ( ; n = 7), K-gluconate ( ; n = 5), Na-gluconate
( ; n = 2), 300 KCl ( ;
n = 2), and 300 NaCl ( ;
n = 2). Pipette solution was high-K+
(filled symbols) or high-Na+ (open symbols). Bath solutions
containing gluconate would have lower Ca2+ activities than
those with EGTA alone, because of Ca2+ binding by
gluconate, but gpClor activity was not
noticeably altered by reduction in Ca2+ activity (data not
shown). B: single-channel conductance (chord conductance) is
shown from I-V relations in A.
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Increasing Cl
concentration at the cytoplasmic face of
the patch increased
for gpClor at negative
Vm (Fig. 3B), as expected for
conductive Cl
exit. The dependence of
on
intracellular Cl
activity (Vm =
80 mV) conformed to a Henri-Michaelis-Menten binding curve with
max of 59 pS and K1/2 of 87 mM.
This apparent Cl
affinity for conduction was
approximately threefold higher than for T84Clor
(20). The presence of Na+ in the pipette
rather than K+ (Fig. 3B) yielded lower
at
negative Vm when the intracellular Cl
concentration was 160 mM, but not at 50 mM
Cl
. Comparison of cell-attached currents with those after
excision into a low Cl
concentration that mimics
intracellular values (Fig. 4A)
suggests that the small-
form of gpClor
predominated in the excised condition, even though the larger form was
more common when cell attached (75%). For the large-
form of
gpClor, cell-attached
at negative
Vm was similar to the excised
in 50 mM
Cl
but at positive Vm
cell-attached
was similar to excised
in 160 mM Cl
(Fig. 4B), further suggesting that
may be controlled by
cytosolic components.

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Fig. 4.
Conductive properties of gpClor after
excision. A: I-V relations are shown for I/O
condition with K-gluconate bath ( ) together with those
for cell-attached condition (Fig. 2A) after correction
( , large gpClor;
, small gpClor) for
apparent cell membrane potential difference
(Vcell) of 40 mV (39),
Vm = Vhold + Vcell. Apparent intracellular Cl
concentration for cell-attached condition was 36 mM, obtained with
reversal potentials of corrected currents. B: single-channel
conductance (chord conductance) is shown from I-V relations
in A together with conductance from Fig. 3B
( , symmetrical KCl).
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Secretagogue activation of Cl
channels.
Distinct groups of secretagogues stimulate various amounts of
electrogenic K+ and Cl
secretion across
distal colonic epithelia (19, 23). Epinephrine or
PGE2 stimulates electrogenic K+ secretion, and
at higher concentrations PGE2 also stimulates Cl
secretion (23, 50). In addition,
cholinergic agonists such as carbachol (CCh) stimulate K+
secretion when added alone but also stimulate Cl
secretion when added together with other secretagogues such as PGE2 (6, 11). Forskolin, which increases
intracellular cAMP through activation of adenylyl cyclase (4,
64), stimulated a negative Isc across
guinea pig distal colonic mucosa (Fig. 5, A and
B), consistent with cation
secretion. Identification of this forskolin-stimulated
Isc as electrogenic K+ secretion was
supported (Fig. 5) by sensitivity to bumetanide as well as a
transepithelial equivalent electromotive force similar to that with
other K+ secretagogues, such as aldosterone, epinephrine,
and PGE2 (21, 23, 50). Also, Ba2+
(10 mM) added to the mucosal solution or DIDS (100 µM) added to the
serosal solution inhibited forskolin-stimulated
Isc and Gt (data not
shown), similar to the action on epinephrine-stimulated electrogenic
K+ secretion (17). Forskolin stimulated (Fig.
5, C and D) negative Isc
consistent with K+ secretion at low concentrations and
additionally at higher concentrations positive
Isc consistent with Cl
secretion
(50), which mimicked the response to PGE2
(23, 50) and cAMP (17).

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Fig. 5.
Forskolin stimulation of K+ secretion.
Short-circuit current (Isc) and transepithelial
conductance (Gt) were recorded from guinea pig
distal colonic mucosa. Spontaneous rates of ion secretion due to
apparent autocrine stimulation (23) were suppressed by
repeated (3×) replacement of bathing solutions and addition of
cyclooxygenase inhibitors indomethacin (2 µM) and NS-398 (2 µM).
Amiloride (100 µM) added to mucosal solution inhibited electrogenic
Na+ absorption. Forskolin (0.3 µM) addition to mucosal
and serosal solutions (0 min) stimulated both
Isc (A) and Gt
(B). The equivalent electromotive force (EMF = Isc/ Gt) was 24
mV, similar to other K+ secretagogues (21, 23,
50). Subsequent (35.8 min) serosal addition of bumetanide (100 µM) inhibited both Isc and
Gt. Cumulative forskolin concentration responses
(n = 7) for Isc (C)
and Gt (D) exhibited 2 saturable
components. Solid lines indicate fits for a 2-component model (see
METHODS), with high-affinity EC50 values of 64 (Isc) and 74 (Gt) nM and
low-affinity EC50 values of 4.0 (Isc) and 2.3 (Gt) µM.
Dashed lines indicate individual fit components; EMF
(Imax/Gmax) for high-affinity
response was 23 mV and for lower-affinity response was +24 mV.
Forskolin-stimulated values after bumetanide (100 µM) addition are
also shown ( ).
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Stimulation of Cl channel activity was measured as increases in number
(N) and Po. The conductance
(g) contributed by each channel type can be calculated from
these values together with single-channel conductance,
g = NPo
. Spontaneous activity
of gpClor occurred in 10 of 16 patches with
detectable gpClor (62%). Linear-
Cl
channels occurred spontaneously in 25 of 36 patches
with discernable gpClL (69%). These incidence
rates overestimate spontaneous activity by ignoring channels that
remained resistant to activation because of experimental conditions.
This relatively high level of apparently spontaneous Cl
channel activity in the basolateral membrane is consistent with stimulation of K+ secretion by low concentrations of
PGE2 and other possible endogenously produced lipid
mediators (23).
Epinephrine (5 µM) activated gpClor
(increased N) in 4 of 57 quiescent cell-attached patches
(7%), and all were the larger-
form. Onset was abrupt, with rapid
opening and closing kinetics (Fig. 6).
PGE2 (100 nM) failed to activate
gpClor in 18 quiescent patches (a rate similar
to epinephrine would have predicted 1 activated
gpClor). Addition of forskolin (1 µM) to the
bath during cell-attached recording (Fig.
7A) led to
gpClor activation (3 of 58 quiescent patches;
5%). Subsequent addition (Fig. 7B) of epinephrine (5 µM)
activated another gpClor, PGE2 (10 µM) addition led to a single gpClor, and CCh
(10 µM) produced erratic current amplitudes together with rapid
kinetics. Currents at the beginning of bursts were near the pre-CCh
size but declined during the burst. These CCh-attenuated currents at
positive Vhold had a
of roughly 60% of the
preceding conditions (data not shown). In two other cell-attached
patches with active gpClor, CCh (10 µM)
addition in combination with PGE2 (10 µM) also converted
channel kinetics from flickering closures to short openings and
closings, but
was not decreased (data not shown). Reversal potentials for gpClor or
gpClL were not significantly different among
spontaneous and secretagogue-induced conditions (data not shown),
indicating an unaltered net driving force for Cl
. In the
combined presence of epinephrine and forskolin (3 patches), the rapid
kinetic mode was replaced by the flickering closures characteristic of
spontaneous activity (Fig. 7B).

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Fig. 6.
Epinephrine activation of gpClor.
Currents from gpClor were recorded while cell
attached. Pipette solution was high-K+. Dashed lines
indicate closed state. A: epinephrine (5 µM) was added to
the bath solution just before the start of the current trace. Within 1 min gpClor became active, after having been
quiescent during 17 min of recording in basal condition.
Vhold was +30 mV. B: current traces
are shown for gpClor activated by epinephrine
(A).
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Fig. 7.
Forskolin activation of gpClor.
Currents from gpClor were recorded while cell
attached. Pipette solution was high-K+. Dashed lines
indicate closed state. A: gpClor
became active ~9 min after forskolin (1 µM) addition;
Vhold was +40 mV. B: subsequent
addition of epinephrine (5 µM) together with forskolin activated a
second gpClor; cumulative PGE2
addition (10 µM) led to a single gpClor;
cumulative carbachol (CCh; 10 µM) addition reduced single-channel
current amplitude. Vhold was +60 mV.
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Linear-
Cl
channels also were activated by
K+ secretagogues. Epinephrine (5 µM) activated (in 57 quiescent patches) gpClL21 (2; 4%),
gpClL13 (1; 2%), and
gpClL8 (1; 2%). PGE2 (100 nM)
activated (in 18 quiescent patches) gpClL21 (1;
6%) and gpClL13 (1; 6%), but not
gpClL8. Forskolin (1 µM) activated (in 58 quiescent patches) gpClL21 (1; 2%),
gpClL13 (2; 3%), and
gpClL8 (2; 3%). CCh did not have a discernable
action on any gpClL. Addition of epinephrine or
PGE2 to patches with active gpClL13
increased channel Po by reducing the apparent
number of long closures (Fig. 8).

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Fig. 8.
Epinephrine stimulation of
gpClL13. Currents from
gpClL13 were recorded while cell attached for
the 13 min preceding epinephrine (5 µM) addition. The
epinephrine-stimulated trace was recorded 6 min after addition, and
similar activity was recorded for an additional 8 min during
epinephrine stimulation. Pipette solution was high-K+.
Vhold was 60 mV. Dashed lines indicate closed
state.
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The total incidence of Cl
channel activation (all channel
types) by epinephrine (5 µM) was 8 of 57 quiescent cell-attached patches (14%). For PGE2 (100 nM), the total incidence of
Cl
channel activation was 2 of 18 quiescent patches
(11%). The total incidence of Cl
channel activation with
forskolin (1 µM) was 8 of 58 quiescent cell-attached patches (14%).
These incidence rates likely underestimate secretagogue sensitivity
because many nonresponsive patches may not have contained
Cl
channels. These activation results do support that
secretagogues significantly increased g
but indicate that no particular Cl
channel type was
solely responsible.
Po of these Cl
channels was
secretagogue dependent. For spontaneously active
gpClor, Po was lower at
negative than at positive Vhold (Fig.
9A). In the physiological
range of Vcell (near
Vhold = 0 mV), Po
was ~0.4. Po in the forskolin-stimulated state
was not detectably different from spontaneous activity, but only values
at positive Vhold were measurable. Epinephrine
activation produced a lower Po than in the basal
or forskolin conditions (Fig. 9A), consistent with the
briefer open times (Figs. 1A and 6B); at
physiological Vcell, Po
of gpClor in the epinephrine condition was
~0.2. Spontaneously active gpClL21 and
gpClL13 also had voltage-dependent
Po, lower at negative
Vhold with the steepest slope near the
spontaneous Vcell (Fig. 9B). Neither 1 µM forskolin (n = 2) nor 100 nM PGE2
(n = 1) altered Po for gpClL21 (data not shown), but only values at
positive Vhold were measurable. Stimulation with
epinephrine (Fig. 8) or PGE2 (100 nM) removed the voltage
dependence for gpClL13, such that
Po was ~0.5 (Fig. 9B).
Spontaneously active gpClL8 had
voltage-independent Po with a value of either
0.54 ± 0.07 (n = 3) or 0.19 ± 0.04 (n = 3), suggesting the existence of two kinetic modes.

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Fig. 9.
Cell-attached open probability
(Po). Currents from Cl channels
were recorded while cell attached before and after secretagogue
addition. Po was calculated from amplitude
histograms of current records (see METHODS). Pipette
solution was high-K+. A: dependence of
gpClor Po on
Vhold is shown for basal condition
( ; n = 6) and in the presence of 1 µM
forskolin ( ; n = 3) or 5 µM
epinephrine ( ; n = 4).
Po of gpClL21 for basal
condition ( ; n = 3) is also shown. Fits
to Boltzmann distribution (dashed lines) allowed estimates
(28) of equivalent gating charge (zg) and
holding voltage at half-activation (V1/2) for
gpClor[basal] (zg = 1.6, V1/2 = +22 mV) and
gpClor[epi] (zg = 1.0, V1/2 = +63 mV). B: dependence of
Po on Vhold for
spontaneously active gpClL21 ( ;
n = 3) and gpClL13
( ; n = 3) is shown. Stimulation of
gpClL13 with either epinephrine (5 µM) or
low-concentration PGE2 (100 nM) altered
Po ( ; n = 3).
Fits to Boltzmann distribution (dashed lines) gave estimates of
zg and V1/2 for
gpClL21 (zg = 1.6, V1/2 = +6 mV) and
gpClL13[basal] (zg = 2.5, V1/2 = +9 mV).
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Excision into I/O configuration altered kinetic behavior for
gpClor, perhaps as cytosolic components were
lost into the bath solution. Rapid closing kinetics due to epinephrine
activation were slowed such that the channels spent more time open
(Fig. 10A). In addition,
Po became generally voltage independent after excision with a value of ~0.8 (Fig. 10B). Reducing bath
solution free Ca2+ did not alter Po
of excised gpClor (data not shown), similar to
previous reports for Clor (5, 37, 43).
Excision of quiescent patches rarely (2 of 231 patches; 1%) led to
activation of gpClor. Activity of all
gpClL types generally was lost after excision.

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Fig. 10.
Excision of gpClor . A: a cell-attached patch with an epinephrine-activated
gpClor was excised into NaCl bath solution.
Vhold was +50 mV. Pipette solution was
high-K+. Dashed lines indicate closed state. Openings of 2 gpClor were apparent after excision.
B: Po of
gpClor is shown for excised condition
( ; n = 10). Cell-attached conditions,
basal/forskolin ( ) and epinephrine ( )
from Fig. 9A, are also shown in relation to
Vm after correction for apparent
Vcell of 40 mV (39).
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Voltage dependence of
(Fig. 2C) and
Po (Fig. 9A) for
gpClor was used to calculate a time-averaged
current that would predict the voltage dependence for a steady-state
whole cell current due to gpClor (Fig.
11A). The combination of
voltage dependence from
and Po results in a
sharp outward rectification; simply multiplying by the number of active
channels would reproduce exact whole cell current amplitudes. Voltage
dependence of Po for
gpClL21 and gpClL13
(Fig. 9B) also resulted in time-averaged currents with
outward rectification (Fig. 11B). Although time-averaged
currents from gpClor and
gpClL13 are difficult to distinguish,
activation of K+ secretion with epinephrine or
low-concentration PGE2 would evoke a nearly linear
time-averaged current from gpClL13.

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Fig. 11.
Time-averaged conductive properties of basolateral membrane
Cl channels. A: I-V relation is
shown (means ± SE) for gpClor activity in
cell-attached condition ( ; from Fig. 2A).
Also shown are time-averaged currents,
iClPo, for
basal/forskolin state ( ) and for epinephrine state
( ). Cell-attached Po from Fig.
9A was used to calculate
iClPo.
Vm was calculated from
Vhold and apparent Vcell,
40 mV (39). B: time-averaged currents
(iClPo, calculated from
Figs. 2 and 9) are shown for gpClor[epi]
( ), gpClL21[basal]
( ), gpClL13[basal]
( ) producing outwardly rectified currents. Currents
were normalized to an inward conductance of 10; the dashed line
indicates the case for voltage-independent conductance.
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Kinetic analysis of gpClor.
Patches with only one gpClor present allowed
detailed analysis of kinetic behaviors induced by secretagogue
activation. Open duration distributions (Fig. 12, A and
B) exhibited two
exponentials during spontaneous and forskolin activity, supporting the
presence of two open states, but during epinephrine activation
distributions had only a single exponential with a shorter time
constant (Table 2). Closed duration
distributions had three distinct exponentials (Fig. 12, C
and D, and Table 2), consistent with three closed states,
and a few long-duration closures (>1 s), suggesting an additional
longer-lived closed state of rare occurrence. Closed duration
distributions differed during epinephrine activation by a shift in
events from the short-duration C1 closed state to the
longer-duration C2 state (Fig.
13 and Table 2). Both the
shorter-duration open time and longer-duration closed time during
epinephrine activation would contribute to lower
Po (Fig. 9A). Thus the powerful
K+ secretagogue epinephrine induced a kinetic mode for
gpClor distinct from the basal/forskolin
kinetic mode.

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Fig. 12.
Opening and closing kinetics for gpClor . Histograms are shown of open durations (A and B)
and closed durations (C and D) with log binning
(see METHODS) for single gpClor
during secretagogue stimulation while cell attached.
Vhold was +60 mV. Each current record was ~20
s in length, with a large number of events in each condition: forskolin
(1,512) and epinephrine (4,041). Open time histograms for forskolin
condition had a peak fit well by a mixture of 2 exponentials; open time
histograms for epinephrine condition had a peak fit best by a single
exponential. Relative frequency was obtained by normalizing to the
number of open events, estimated from the fitted exponentials.
Individual fit components are shown as dotted lines. Fitted open time
constants and proportions of events (in parentheses) were forskolin,
1.6 ms (0.04) and 11.9 ms (0.96); epinephrine, 2.2 ms. Closed time
histograms exhibited multiple peaks requiring a mixture of exponentials
for an adequate fit; relative frequency was obtained by normalizing to
the number of open events for that condition. Fitted closed time
constants and proportions of events (in parentheses) were forskolin,
0.6 ms (0.82), 3.6 ms (0.17), 67.9 ms (0.01); epinephrine, 1.0 ms
(0.21), 2.8 ms (0.785), 171.6 ms (0.005).
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Fig. 13.
Closed states for gpClor. Time
constants and proportions of events for kinetic states obtained from
fits of duration histograms to mixtures of exponentials were averaged
from patches containing single gpClor (Table
2). Basal and forskolin-stimulated cell-attached activity were similar
and combined for averaging ( ; n = 4).
Activity during epinephrine ( ; n = 3)
and after excision ( ; n = 5) also are
shown. Asterisk indicates voltage-dependent time constant; value at +60
mV is shown.
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The time constant (
) of the most common open events was voltage
dependent during spontaneous activity, forskolin activation, and
epinephrine activation (Fig. 14).
Closed
were voltage independent, except during epinephrine
activation, when one closed time constant (C2) was voltage
dependent (Fig. 14). This extra voltage dependence contributed to the
epinephrine-induced shift in the dependence of
Po on voltage (Fig. 9A), such that
the voltage at half-activation (V1/2) was ~40
mV more positive than in the basal/forskolin kinetic mode. The
proportion of events making up each open and closed duration
exponential was voltage independent (data not shown), within the
variations of the measurement (~10%). After excision, the dominant
open
(for long-duration open state) became twofold longer and
voltage independent (Table 2 and Fig. 14). Closed
were similar to
basal spontaneous activity (Table 2 and Fig. 13) after excision,
supporting the presence of a diffusible mediator controlling the
epinephrine-induced closed state distribution.

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Fig. 14.
Voltage dependence of time constants for
gpClor. Open and closed kinetics were examined
for patches with single gpClor (as in Fig. 12).
Dependence on Vhold of open time constants
( O) are shown for basal/forskolin ( ;
n = 4), epinephrine ( ;
n = 3), and excised ( ;
n = 4) conditions. Also shown is voltage dependence of
C2 during epinephrine stimulation ( ;
n = 3).
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 |
DISCUSSION |
Electrogenic secretion of K+ across colonic epithelia
can occur without accompanying Cl
secretion or
Na+ absorption and produces a lumen-positive
transepithelial electrical potential difference
(Vt) via a cellular mechanism (Fig.
15), apparently employing
apical membrane K+ channels and basolateral membrane
Cl
channels (17, 19, 21, 50). Together these
conductive pathways contribute to a cell-negative electrical PD at
apical (Va) and basolateral
(Vb) membranes that aids in driving basolateral Cl
exit. Secretory exit of K+ into the lumen
is dependent on the relative K+ conductance of apical and
basolateral membranes as well as the electrochemical driving forces
(39, 42, 50). Basolateral exit of Cl
is
essential during this type of K+ secretion because
basolateral Na+/K+ pumps drive K+
uptake via continual extrusion of Na+ entering through
basolateral Na+-K+-2Cl
cotransporters, which results in Cl
entry
(19). Maintenance of cell volume during sustained
secretion requires a balance between these basolateral influx and
efflux pathways for Cl
(Fig. 15). Activation by
K+ secretagogues indicates that the Cl
channels observed in colonic crypts (Table
3) contributed to the exit pathway for
Cl
.

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Fig. 15.
Cellular transport model for electrogenic K+
secretion. The proposed mechanism for electrogenic K+
secretion across distal colonic epithelium includes 4 types of
transport proteins (17, 50). Prevailing electrochemical
gradients and phosphorylation potential determine the directions of net
flow (arrows). The model schematic shows apical membrane K+
channels acting in concert with a combination of basolateral membrane
transporters (Na+/K+-ATPases,
Na+-K+-2Cl cotransporters,
Cl channels, K+ channels) that permit net
K+ uptake from the serosal interstitium into the cell and
K+ exit from the cell into the lumen. Positive charge flows
across these epithelial cells from serosa to lumen carried by
K+ through apical membrane channels and by Cl
through basolateral membrane channels. [The standard Cl
secretory model (15, 18, 19) is similar, except that
Cl channels dominate the apical membrane conductance and
basolateral membrane Cl conductance would be assumed
minimal.]
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Conversion of transport function to active Cl
secretion
transforms cellular ion flow by redirecting Cl
exit to
apical membrane Cl
channels. Colonic epithelia may
contain separate cell types producing these two modes of ion secretion,
but crypts respond to secretagogues with changes in cell composition,
suggesting that both the electrogenic K+ secretory mode and
the electrogenic KCl secretory mode occur in columnar cells (24,
27). Cl
permeability of colonic crypts is distinct
between these modes, with a larger Cl
efflux capacity in
the K+ secretory mode (24). For epithelial
cells capable of producing both secretory modes, coordinated regulation
of Cl
channels in apical and basolateral membranes
permits Cl
exit to support either of these secretory
modes. Although opening apical Cl
channels is a key event
for initiating Cl
secretion, managing basolateral
membrane Cl
conductance (g
)
also would be necessary for the sustained ion secretion observed in
colonic epithelia. Presumably, closing basolateral Cl
channels would best serve promotion of Cl
secretion, but
cell volume control may necessitate specific amounts of
g
.
Basolateral membrane Cl
channels.
Two major Cl
channel types were observed while cell
attached on basolateral membrane of guinea pig distal colonic crypts
(Figs. 1 and 2 and Table 3): outwardly rectified
(gpClor) and linear
(gpClL) I-V relations, with multiple
conductance forms. Two conductance states likely exist within a single
gpClor type because patches with the larger-
form of gpClor had the smaller-
form after
excision (Fig. 4), even though outward currents were carried by 160 mM
Cl
from the pipette in both cases. Whether the three
sizes of gpClL (Fig. 2B) represent
distinct channel types or simply were conductance states of a
particular Cl
channel cannot be resolved conclusively
from the present data. However, voltage dependence of
Po also was different among the three
gpClL sizes (Fig. 9B and Table 3),
supporting functionally distinct channel activities. Single-channel
conductance (Fig. 2B) and voltage dependence of
Po (Fig. 9) can be used to compare these
Cl
channel activity patterns (Fig. 11B) with
those of other Cl
channel types.
Clor has been observed in basolateral membranes of rat and
mouse colonic crypts (3, 43) as well as mouse jejunal
crypts (5). Those studies focused primarily on the cells
at the base of the crypt near the stem cell, whereas the results
reported here were from cells making up the cylindrical portion of the crypt where rapid cell division and secretion occur. The
of mClor in basolateral membranes of mouse colonic
crypts (43) is similar to gpClor
at negative Vhold and at positive
Vhold is intermediate to the two forms of
gpClor (Fig. 2B). Excision permits
comparison of Clor under well-defined conditions of
Cl
concentration and Vm; at
Vm = 0 mV and 160 mM Cl
,
is similar for gpClor,
mClor, and T84Clor: 47 (Fig. 3B), 36 (5, 43), and 41 (20, 26) pS, respectively. On the basis of outward rectification and
ion selectivity, CLC-3 appears at present the most likely candidate for
producing Clor (30, 46, 69) and therefore also
gpClor.
Po for Clor generally is larger at
more positive Vhold in cell-attached recording
(26, 37, 43), similar to gpClor
(Fig. 9A). Consequently, the time-averaged I-V
relations for gpClor were steeply rectified
(Fig. 11). After excision this voltage dependence of
Po often is retained (26, 37, 43)
but sometimes is lost (5, 48), as with
gpClor (Fig. 10B). Clearly,
time-averaged currents would be less rectified with voltage-independent
Po. Po for CLC-1 is
largest at more positive Vm, with an equivalent
gating charge of ~1 (33) similar to
gpClor (Fig. 9A); however,
for
CLC-1 is only ~2 pS (33). Po for
CLC-2 is largest at more negative Vm and for
CLC-3 is relatively voltage independent (9, 33). This
range of voltage sensitivities for CLC members suggests that this
general channel structure could produce the characteristics of
gpClor, but the exact features of
gpClor cannot be assigned conclusively to any
of the CLC members.
Both gpClL21 and
gpClL13 had voltage-independent
with
Po that was larger at more positive
Vhold (Figs. 2C and 9B).
These conduction characteristics produce outwardly rectified
time-averaged currents (Fig. 11B) that are similar to many
Cl
currents recorded in whole cell mode (16, 33,
47, 57, 62). The ~15-pS
of CLCA is comparable to that of
gpClL13 (16). A basolateral
membrane Cl
channel, with linear
of 28 pS, activated
by cell swelling in rat colonic crypts (12, 13), may
correspond to gpClL21. Basolateral membrane
Cl
channels in kidney tubular cells have linear
of
20-28 pS, and those from the thick ascending limb of Henle's loop
have Po that is larger at more positive
Vm (53, 56). Although expression of
CLC-K has only been demonstrated in the kidney and inner ear (33,
52, 55, 66), modest outward rectification of CLC-Kb/barttin whole cell current (66) is consistent with the behavior of
gpClL21 (Fig. 11B).
The Cl
channel formed by CFTR has voltage-independent
of 6-10 pS (33, 60) as well as voltage-independent
Po (59). Both of these
characteristics are consistent with gpClL8
behavior (Fig. 2C). However, CFTR has modestly outwardly rectified currents when recorded while cell attached compared with the
linear I-V relation for gpClL8. As
with the other gpClL,
was voltage
independent either because the low intracellular Cl
concentration was saturating for conduction (consistent with relatively
high-affinity Cl
interaction in the pore) or currents
would be modestly inwardly rectified with equal Cl
concentrations on both sides. For CFTR the outward rectification appears to include influences from phosphorylation and permeation by
other intracellular anions (60). If
gpClL8 were produced by CFTR, then other
regulatory influences would be necessary to account for these
differences in ion conduction. The voltage-independent steady-state
conductance of CLC-Ka/barttin (66) also is consistent with
the behavior of gpClL8.
Uncertainty concerning the molecular identity of
gpClor and gpClL
exists, in part, because the functional properties differ somewhat from
those of presently defined Cl
channel families. Channels
observed in native tissue may occur as heteromultimeric assemblies of
channel subunits together with regulatory components that produce
distinct behavior. Combinations of CLC-1 and CLC-2 result in
biophysical properties that are not a simple sum of the homomultimeric
channels; in particular, voltage dependence is largely lost
(41). The presence of the
-subunit barttin also alters
the properties of CLC-K (66). Isolation of crypts may
initiate a progressive alteration in channel subunits present such that
biophysical and regulatory properties might change. However, colonic
mucosa retains secretagogue sensitivity for K secretion for >8 h after
isolation (51), and channel responses were similar over
the ~36-h patch-clamp study period. Species-dependent differences in
channel function also may contribute to difficulty in precisely
identifying these guinea pig Cl
channels. Although the
identities of the Cl
channels observed in this study
remain undetermined, a basolateral location does allow contributions to
cell functions ranging from cell volume control to transepithelial ion flow.
Regulation of K+ secretion.
Control of g
contributes to determining
the rate of electrogenic K+ secretion by providing an exit
path for Cl
entering via
Na+-K+-2Cl
cotransporters (Fig.
15) and by influencing the electrical driving force for apical
K+ exit. The Cl
channels
gpClor and gpClL (Table
3) are likely involved in augmenting this
g
needed for K+ secretion.
Stimulation of electrogenic K+ secretion occurs through
-adrenergic receptors (17) and prostaglandin EP2 receptors (23), both of which act through
increasing intracellular cAMP. Activation of prostaglandin DP
receptors, which also increase cAMP production, stimulates electrogenic
KCl secretion (23), suggesting a functional
compartmentalization of cAMP allowing selective induction of
Cl
secretion. The concentration dependence of the
forskolin response supports the existence of two interactions with
distinct affinity (Fig. 5B) similar to adenylyl cyclase
activation in human colonic crypts (4). This divergence of
sensitivities to forskolin may represent distinct forms of adenylyl
cyclase, because activation with the G protein GS
increases forskolin affinity of adenylyl cyclase from an
EC50 of >10 µM to ~100 nM (64). Specific
activation of Cl
channels to support each ion secretory
mode may occur via adenylyl cyclases that are functionally
compartmentalized through the action of other intracellular signaling cascades.
Secretagogues for electrogenic K+ secretion, epinephrine
and either PGE2 or forskolin at low concentration,
increased the number of active gpClor and
gpClL (Table 3), consistent with higher
g
. The epinephrine secretory state of
gpClor had lower Po due
to a shift in the Po voltage dependence to more
positive Vhold (Fig. 9A).
gpClL13 also supported secretagogue activation
of g
with increased
Po at the spontaneous
Vcell (Fig. 9B). In addition, high-concentration PGE2 that stimulates Cl
secretion (23, 50) appeared to decrease N for
gpClor (Fig. 7B) such that
g
would be reduced. Cholinergic
stimulation of a Cl
secretory state increased flickering
of gpClor, consistent with reduced
Po (Fig. 7B). GTP induced a similar flickery state with reduced current amplitude in
mClor excised from colonic crypts
(43). Overall, K+ secretagogues increased
N for gpClor and
gpClL, but epinephrine produced a distinct
secretory mode for gpClor with lower
Po than with forskolin (Fig. 9A).
Therefore, other intracellular signals in addition to cAMP may be
needed to regulate the basolateral membrane Cl
channels
involved in electrogenic K+ secretion.
Kinetic analysis of gpClor activity (Figs. 12,
13, and 14) provided further insight into regulation of K+
secretion. Epinephrine activation of gpClor
occurred with flickery kinetics distinct from basally active or
forskolin-activated channels (Figs. 6 and 7). Open durations were
shorter (Fig. 12 and Table 2), and closed durations were dominated by
events with a medium-length
(Figs. 12 and 13 and Table 2), leading
to a shift in the voltage dependence of Po (Fig.
9B). Absence of the flickery epinephrine mode in the
presence of forskolin (Fig. 7B) suggested that a
basal/forskolin-induced factor can suppress entry into this flickery
kinetic mode. Because excision of patches with
gpClor in the flickery mode led to slower
kinetics similar to the basal/forskolin mode (Fig. 10 and Table 2), a
component of this kinetic control factor apparently was readily
exchangeable. Excision of patches also led to a loss of voltage
dependence for Po (Fig. 10) and reduction in
at positive Vm (Fig. 4), consistent with readily
exchangeable regulators.
Activation of Clor after excision is a general observation
in many cell types (3, 5, 26, 30, 33, 43, 61), supporting
the existence of cytosolic inhibitory regulators (35, 36,
38). The exact nature of these regulators is presently unknown,
but several classes of compounds have been implicated. Extracts of
cytosol appear to contain both a high-molecular-mass (10-300 kDa)
substance, likely a protein, and a lower-molecular-mass (<1 kDa)
substance similar to steroids (36). Addition of cytosol to
excised HT29Clor reduces
O from
32 to 7 ms (49), similar to the difference between excised
and basal/forskolin modes for gpClor (Table 2).
Closed
for HT29Clor are essentially
unchanged by cytosol (49) similar to excised and
basal/forskolin modes for gpClor (Figs. 13 and
16). Fatty acids such as arachidonic
acid can reduce Po of Clor, but the
mode of action and molecular size suggest that the inhibitor present in
cytosol is distinct (31, 36, 38).

View larger version (23K):
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|
Fig. 16.
Regulation of closed states for Clor. Time
constants and proportions of events for kinetic states are shown for
gpClor (Fig. 13) and Clor recorded
from the human colonic tumor cell line HT29 (49). All
kinetic constants for HT29Clor are from excised
I/O patches; aldosterone (100 µM) and cytosol were added to the bath
solution.
|
|
The flickery nature of the epinephrine-induced mode for
gpClor can be mimicked in excised
HT29Clor by aldosterone or glibenclamide
(48, 49);
O is reduced to ~1 ms, and
events with
C of ~2 ms dominate closures (Fig. 16).
The high EC50 for aldosterone action (~20 µM) led Rabe
and Frömter (49) to suggest that another lipid
component may be the actual endogenous control component. Both
aldosterone and glibenclamide are proposed to act by moving
HT29Clor into a specific kinetic mode rather
than by blocking (48, 49), which was supported by the
action of the secretagogue epinephrine to induce a nearly identical
kinetic mode in gpClor (Fig. 16 and Table 2).
Furthermore, such a steroidlike lipid may be the specific mediator of
epinephrine activation.
Secretory control of gpClor and
gpClL included increased N and
Po such that a range of g
was
possible. Each of these Cl
channel activities may
contribute a unique set of regulatory responses to adjust for demands
on secretory rate and cell volume control. Regulatory subunits
associating with the observed Cl
channels may be
necessary to produce the specific types of secretagogue sensitivities.
The CLC-K
-subunit barttin promotes channel activity (66), and subunits alter responsiveness of K+
channels to cytoplasmic signals. In particular, CFTR belongs to a
protein family that includes the sulfonylurea receptor that confers ATP
sensitivity to K+ channels, and CFTR has been shown to
associate with K+ channels (54). The
coincident presence of gpKir with
gpClor (Fig. 1A) and
gpClL suggests that a tight regulatory
connection potentially could exist between
g
and g
in
colonic crypts. Similarly, association of gpClL
with gpClor might confer control like that
between CFTR and Clor (30).
A basolateral membrane location of Cl
channels in
epithelia presumably serves both volume-regulatory needs and
transepithelial flow requirements. Whether volume-activated channels
open during K+ secretion was not determined in the present
study, but conductive Cl
exit would aid maintenance of
cell volume during secretory stimulation of Cl
influx
through Na+-K+-2Cl
cotransporters. As noted numerous times, transepithelial solute flow
combines aspects of regulatory volume increase (RVI) and regulatory
volume decrease (RVD). The distinct situation in epithelia, as during
electrogenic K+ secretion (Fig. 15), is that cell volume is
maintained near control values while transport processes resembling RVI
and RVD remain active. Simultaneous and continual operation of influx
and efflux pathways is what produces sustained transepithelial flow.
Thus a complete control scheme for basolateral Cl
channels will include not only the initiating events but also the
modulating signals that ensure stable cellular conditions during
ongoing secretion.
 |
ACKNOWLEDGEMENTS |
This study was supported by grants from the National Institute of
Diabetes and Digestive and Kidney Diseases (DK-39007) and the Wright
State University Research Challenge Program.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
D. R. Halm, Dept. of Physiology and Biophysics, Wright State
Univ., 3640 Colonel Glenn Hwy., Dayton OH 45435 (E-mail:
dan.halm{at}wright.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 27, 2002;10.1152/ajpcell.00464.2002
Received 3 October 2002; accepted in final form 5 December 2002.
 |
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