Secretory modulation of basolateral membrane inwardly
rectified K+ channel in guinea pig distal colonic
crypts
Yingjun
Li and
Dan R.
Halm
Department of Physiology and Biophysics, Wright State University,
Dayton, Ohio 45435
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ABSTRACT |
Cell-attached recordings revealed
K+ channel activity in basolateral membranes of
guinea pig distal colonic crypts. Inwardly rectified currents were
apparent with a pipette solution containing 140 mM K+.
Single-channel conductance (
) was 9 pS at the resting membrane potential. Another inward rectifier with
of 19 pS was observed occasionally. At a holding potential of
80 mV,
was 21 and 41 pS,
respectively. Identity as K+ channels was confirmed after
patch excision by changing the bath ion composition. From reversal
potentials, relative permeability of Na+ over
K+ (PNa/PK)
was 0.02 ± 0.02, with
PRb/PK = 1.1 and
PCl/PK < 0.03. Spontaneous open probability (Po) of the 9-pS
inward rectifier (gpKir) was voltage
independent in cell-attached patches. Both a low
(Po = 0.09 ± 0.01) and a moderate
(Po = 0.41 ± 0.01) activity mode were
observed. Excision moved gpKir to the medium
activity mode; Po of
gpKir was independent of bath Ca2+
activity and bath acidification. Addition of Cl
and
K+ secretagogues altered Po of
gpKir. Forskolin or carbachol (10 µM)
activated the small-conductance gpKir in
quiescent patches and increased Po in
low-activity patches. K+ secretagogues, either epinephrine
(5 µM) or prostaglandin E2 (100 nM), decreased
Po of gpKir in active
patches. This gpKir may be involved in
electrogenic secretion of Cl
and K+ across
the colonic epithelium, which requires a large basolateral membrane
K+ conductance during maximal Cl
secretion
and, presumably, a lower K+ conductance during primary
electrogenic K+ secretion.
chloride secretion; potassium secretion; prostaglandin
E2; epinephrine
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INTRODUCTION |
ACTIVE SECRETION OF
IONS across colonic epithelia serves to produce a driving force
for fluid secretion and to modify the composition of secreted fluid
(7, 15). Excessive rates of secretion occur in
pathophysiological states such as secretory diarrhea and ulcerative
colitis. As in other fluid-secreting epithelia, electrogenic
Cl
secretion is a major mechanism for producing fluid
flow (14). Stimulating Cl
secretion requires
exit of K+ entering via the Na+-K+
pump and Na+-K+-2Cl
cotransport,
which can occur via basolateral membrane K+ channels
(14). In mammalian colon, K+ secretion is
stimulated together with Cl
secretion, contributing to
the relatively high luminal K+ concentration. Localization
studies support the presence of K+ and Cl
secretory capacity in columnar cells of colonic crypts (18, 19). The cellular mechanism for K+ secretion is
electrogenic and related to the mechanism for Cl
secretion. A key feature is that electrogenic K+ secretion
measured in vitro is entirely bumetanide sensitive, suggesting an
absolute requirement for
Na+-K+-2Cl
cotransport (15,
41). In addition, apical and basolateral membrane K+
channels allow exit of K+ from the cell. Because the rate
of K+ secretion can vary relative to that of
Cl
secretion, colonic secretory cells may control
K+ secretion, in part, by modulating basolateral
K+ channel activity to alter the amount of K+
exiting into the lumen.
Activity of K+ channels has been detected in colonic crypts
(55), a site for fluid and mucus secretion (7,
16). Channels have been observed both in the crypt base
(4, 5, 39) among the first progeny of the crypt stem cell
and in the tubular portion of the crypt (32, 44, 45) among
the rapidly dividing cells. The predominant cell types of the crypt are
columnar cells and goblet cells (16, 19). Goblet cells are
distinguished from columnar cells by dense apically located mucin
granules that are released during cholinergic stimulation.
Cl
secretion occurs with either cholinergic activation
that increases intracellular Ca2+ or secretagogues such as
vasoactive intestinal peptide and prostaglandin E2
(PGE2) that increase intracellular cAMP (7,
15). An increase in K+ conductance would serve to
maintain Cl
secretion by allowing K+ exit and
by developing a cell negative membrane electrical potential difference
to drive conductive Cl
exit through the apical membrane
into the lumen. Three major types of K+ channels have been
observed during secretory activation of isolated colonic crypts as well
as colonic tumor cells such as T84 and HT29 (55):
large-conductance, Ca2+-activated K+ channels
(slo or BK); intermediate-conductance, Ca2+-activated
K+ channels (IK1); small-conductance, cAMP-activated
K+ channels (KvLQT/minK). These K+ channels
belong to the greater group of K+ channel proteins that
have similar pore-forming domains but distinct regulatory domains and
components (10, 27).
Guinea pig distal colon can produce high rates of K+ and
Cl
secretion in response to secretagogues (17,
41). Both epinephrine and low concentrations of PGE2
stimulate electrogenic K+ secretion in the absence of
accompanying Cl
secretion, whereas high concentrations of
PGE2 or forskolin stimulate electrogenic secretion of both
K+ and Cl
. Thus guinea pig distal colon
provides a comparison between these two modes of secretion so that the
varied roles of basolateral K+ channels can be examined. In
particular, increased basolateral membrane K+ channel
activity would aid Cl
secretion by enhancing conductive
Cl
exit, whereas decreased activity would increase
K+ secretion by limiting exit of K+ into the
interstitial space. The present study has indicated that cells in
colonic crypts exhibit an inwardly rectified K+ channel,
gpKir, that has characteristics distinct from
other K+ channels previously observed in colonic epithelia.
Both cAMP- and Ca2+-dependent secretagogues activate
gpKir, whereas K+
secretagogues moderate channel activity to lower levels.
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METHODS |
Male guinea pigs (400-650 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. Portions of
mucosa were mounted, with the use of cyanoacrylate glue, on to Lucite
holders with apertures 1 cm wide and 4 cm long. Mucosal portions on
holders were incubated at 38°C in HEPES-buffered solution with
indomethacin (1 µM) to reduce spontaneous fluid and mucus secretion
(16, 17, 41). 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 crypt epithelium from the mucosa followed general
procedures developed previously (3, 5, 6, 44, 57).
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 if the EDTA solution was prepared the day of the
isolation, as noted previously (3). Mucosal portions were
consecutively incubated in 30 mM citrate Ringer with indomethacin (1 µM) for 15-30 min and then 30 mM EDTA Ringer for 15-20 min
at 38°C. Holders were then gently 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 the refrigerator until use and were suitable for patch-clamp
experiments up to ~36 h.
Isolated crypts were transferred onto a polylysine-coated plastic
coverslip in the electrical recording chamber mounted on the stage of
an inverted microscope (Diaphot; Nikon, Tokyo, Japan). 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) by using 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, Darmstadt, Germany)
via a 150 mM KCl agar salt bridge inside a holder containing a Ag-AgCl
electrode (12). 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 at 3-kHz filtering with a pulse
code-modulated videocassette recorder (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. Occasionally, cell
depolarization was produced with a high-K+ bath made by
substituting 135 mM K+ for Na+ in the standard
Ringer solution. Before patches were excised, the bath solution was
changed to one containing EGTA (Table 1) to maintain low free
Ca2+ (~10 µM) that would mimic intracellular
conditions. Lower levels of bath free Ca2+ (~100 nM and
<10 nM free Ca2+) were produced by adding only 0.1 mM or 0 mM Ca2+, respectively, to these bath solutions. Bath
solution pH was adjusted by titration with NaOH or HCl. Solution
osmolarity was 292 mosM (290-294 mosM), except for the 300 KCl
bath.
Drugs were added in small volumes from concentrated stock solutions.
PGE2 was obtained from Cayman Chemical (Ann Arbor, MI), and
epinephrine was from Elkins-Sinn (Cherry Hill, NJ). All other chemicals
were obtained from Sigma Chemical (St. Louis, MO). PGE2 was
prepared in an ethanol stock solution that added 0.1% ethanol at 10 µM PGE2; additions of 1% ethanol alone did not alter
transepithelial measures of K+ or Cl
secretion (17).
Current data were transferred via DigiData-1200 interface to a computer
for analysis using pCLAMP6 software (Axon Instruments, Foster City,
CA). Currents were filtered at 700 Hz. Junction potentials at pipette
tip and bath reference bridge were calculated to correct holding
voltages (1, 38). Relative ion permeabilities were calculated by using the Goldman-Hodgkin-Katz potential equation together with the measured reversal potential and solution ion composition. Open probability (Po) was
calculated from all-points histograms of current amplitude. Area under
each current peak (A) 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 the
baseline and increasing to N, the number of active channels. A current-voltage relation was constructed from
the lowest current peaks to assure that the lowest peak at each voltage indicated the closed state. Records of sufficient length (5-10 min) were obtained for each stimulatory condition to allow a reliable measure of N from the number of observed peaks
(22). Currents recorded at holding potentials
(Vhold) more positive than about
10 mV were
generally too small to allow accurate measurement of
Po. Kinetic analysis was performed on records
containing only one channel 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
(Gaussian) to minimize noise but also to maximize bandwidth. Log
binning was used to improve fitting and display of exponential curves (24); maximal likelihood estimates were used to obtain
time constants from open and closed times. Results are reported as means ± SE. Statistical comparisons were made by using two-tailed Student's t-test for paired comparisons, with significant
difference accepted at P < 0.05.
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RESULTS |
Basolateral membranes of isolated crypts were readily accessible
to sealing with patch pipettes; all results were from seals on the
middle (tubular) section of isolated crypts. Differences between
columnar and goblet cells could not be discerned readily to identify
the cell type recorded. Spontaneous single-channel currents were
observed while cell-attached consistent with K+ channel
activity (Fig. 1), with both
high-Na+ and high-K+ pipette solutions (Table
1). The reversal potential in cell-attached recordings supported
identification of channel types producing currents. For crypt
epithelial cells (19, 31), currents from K+
and Cl
channels recorded with high-Na+
pipette solution would be expected to reverse at negative and positive
Vhold, respectively, as determined by the ion
concentration gradients. Thus, at resting membrane electrical potential
difference (Vhold = 0 mV), K+
currents would be outward and Cl
currents would be inward
(net outward Cl
flow). In addition, nonselective cation
channel currents would reverse at large positive
Vhold, corresponding to a cell membrane electrical potential difference of 0 mV. Use of high-K+
pipette solution had the advantage of increasing the size of inward
K+ currents, thus permitting better detection of
small-conductance channels. Because the equilibrium potential for
K+ would be near a membrane electrical potential difference
of 0 mV with roughly equal K+ concentrations inside and
out, cell-attached reversal potentials of K+ channel
currents with high-K+ pipette solution allowed a rough
estimate of cell membrane electrical potential difference.

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Fig. 1.
K+ channel currents in crypt cells. Current
traces are shown for K+ channels from cell-attached patches
of basolateral membrane in isolated colonic crypts. Closed states are
indicated by dashed lines. A: currents at several holding
potentials (Vhold) are shown for an ~9-pS
K+ channel with high-Na+ pipette solution,
containing 5 mM K+ (see Table 1). B: currents
for an inwardly rectified ~9-pS K+ channel with
high-K+ pipette solution, containing 140 mM K+
(see Table 1). C: currents for a larger conductance (~19
pS) inwardly rectified K+ channel with high-K+
pipette solution. D: currents for an ~85-pS K+
channel with high-K+ pipette solution.
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Currents consistent with Cl
channels, as distinguished by
reversal potentials, also were observed (30). Nonselective
cation channels (52) reversing at positive
Vhold were not observed in cell-attached
patches. Rarely (5 of 304 patches, 1.6%), nonselective cation channels
were observed reversing at Vhold of 0 mV,
suggesting a depolarization of membrane electrical potential difference
for these cells. Changing the bath solution to a high-K+
solution, which presumably depolarized cells, resulted occasionally (9 of 57 patches, 16%) in the appearance of nonselective cation channels
in cell-attached patches. For patches with nonselective cation
channels, two to six channels were present with a voltage-independent single-channel conductance of ~25 pS.
The most common K+ channel activity with
high-Na+ pipette solution (10 of 73 patches, 14%) had a
linear current-voltage relation with a single-channel conductance (
)
of 9 pS (Figs. 1A and 2). During recording with high-K+ pipette solution, inwardly
rectified current-voltage relations were observed (58 of 231 patches,
25%) with
of 9 and 19 pS at Vhold = 0 mV (Figs. 1, B and C, and 2). These two channel
behaviors with high-K+ pipette solution may be specific
conductance states of the same channel, because they were observed
together (3 of 6 patches with large-conductance
gpKir, 50%). Cell-attached currents consistent
with a K+ channel of ~85 pS also were seen in one patch
with the standard bath solution (Figs. 1D and 2A)
and in one other patch with high-K+ bath solution that
presumably depolarized cells. Apparent higher incidence of
K+ channels with high-K+ pipette solution may
only reflect the generally greater ease of identifying the resulting
larger inward K+ currents.

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Fig. 2.
K+ channel conductive properties.
A: current-voltage relations are shown for 4 types of
K+ channel activity observed in cell-attached patches of
crypt basolateral membrane (means ± SE). With
high-Na+ pipette solution, single-channel currents were
linearly dependent on voltage ( , n = 6). With high-K+ pipette solution, currents were inwardly
rectified, and those exhibiting both inward and outward currents were
averaged ( , n = 15). Larger inwardly
rectified currents forming a distinct group were averaged
( , n = 6). A larger conductance (~85
pS) K+ channel recorded with high-K+ pipette
solution also is shown ( , n = 1).
Symbols often obscure small error bars. I, current.
B: single-channel conductance (chord conductance, ) is
shown from current-voltage relations in A.
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Ion selectivity of gpKir.
Activity of gpKir generally persisted after
excision into an inside-out (I/O) configuration (17 of 18 patches,
94%), which allowed ion selectivity to be determined more precisely.
With a high-K+ pipette solution and a K-gluconate bath
solution (containing 50 mM Cl
, Table 1), the reversal
potential was near 0 mV (Fig.
3A), as expected for a cation
channel. Increasing bath solution KCl concentration shifted the
reversal potential to negative voltages (Fig. 3A) consistent
with cation selectivity. Relative anion permeability (PCl/PK) was <0.03
(n = 9). Lowering bath solution K+
concentration by substitution with Na+ shifted the reversal
potential to positive voltages, as expected for a
K+-selective channel (Fig. 3A). Relative
Na+ permeability
(PNa/PK) was 0.02 ± 0.02 (n = 6). Substituting bath K+
completely with Na+ produced steep inwardly rectified
currents without outward currents (Fig. 3C), also consistent
with high selectivity for K+ over Na+.
Similarly (Fig. 3A), relative Rb+ permeability
(PRb/PK) was 1.12 (n = 2, range 1.05-1.19). Currents from the 9-pS
channel with high-Na+ pipette and Na-gluconate bath
solution also were completely inwardly rectified (Fig. 3C),
supporting identification as a K+ channel. Ion selectivity
of the 19-pS gpKir also supported high
preference for K+ over Na+ or Cl
(data not shown). These current measurements indicate that the observed
channels were K+ selective with significant Rb+
permeability, suggesting a divergence from other inwardly rectified K+ channels (8, 49).

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Fig. 3.
Ion selectivity of inwardly rectified K+
channel. Current-voltage relations are shown with various bath ion
compositions (means ± SE). A: after excision into
inside-out (I/O) configuration, bath solution (see Table 1) was changed
among K-gluconate ( , n = 7), 70 mM
K+-85 mM Na+ (70K/85Na; ,
n = 6), 300 mM KCl ( , n = 2), and RbCl ( , n = 2). Pipette
solution was high-K+. Symbols often obscure small error
bars. B: single-channel conductance is shown from
current-voltage relations in A. C: only inward
currents were observed for I/O patches with Na-gluconate (Na-glcn) bath
solution with either high-K+ pipette solution
( , n = 2) or high-Na+
pipette solution ( , n = 5). Currents
from A with K-gluconate ( ) and 70K/85Na
( ) also are shown for comparison of variations in
K+ and Na+ concentration. D:
single-channel conductance is shown from current-voltage relations in
C. Chord-conductance for Na-gluconate conditions with
high-K+ pipette solution was calculated with a reversal
potential (Erev) of +90 mV, obtained from
Na+/K+ selectivity exhibited in A;
at membrane potentials (Vm) = 0 mV
ranged over ~1 pS when Erev changed from +80
mV to +100 mV. For high-Na+ pipette solution,
Erev of +70 mV was estimated by extrapolation.
E: single-channel conductance is shown for I/O condition
with K-gluconate bath ( ) together with those for
cell-attached conditions ( and ) from
Fig. 2B with inclusion of apparent cell membrane electrical
potential differences (Vcell) exhibited in Fig.
2A ( 40 mV): Vm = Vhold + Vcell.
Pipette solution was high-K+.
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Concentration dependence of gpKir.
Increasing K+ concentration at the cytoplasmic face of the
patch (0, 70, and 143 mM; with constant ionic strength) increased
of gpKir at positive membrane potentials
(Vm) (Fig. 3D), consistent with
saturation of outward flow at <70 mM K+. Further increase
of K+ concentration to 300 mM (with increased ionic
strength) led to ~45% larger
at positive
Vm (Fig. 3B). For inward
K+ flow from the pipette, decreasing K+
concentration in the bath (with constant ionic strength) did not alter
at large negative Vm (Fig. 3D),
but high bath K+ concentration (300 mM; with higher ionic
strength) increased
at negative Vm (Fig.
3B). One possible explanation for this apparent influence of
cytoplasmic K+ on K+ influx, and efflux, is
that the small-conductance state of gpKir was
converted to the large-conductance state by increased ionic strength,
since
at negative Vm with 300 mM bath
K+ (Fig. 3B) was similar to
of the
large-conductance gpKir (Fig. 2B).
During cell-attached recording with high-Na+ pipette
solutions (Fig. 2), outward rectification was not apparent for the 9-pS K+ channel even though pipette K+ concentration
was only 5 mM (Table 1). Lack of outward rectification with an
outwardly directed concentration gradient suggests that this
K+ channel actually was an inward rectifier, similar to
previous observations on colonic K+ channels
(39). Relative Rb+ conductance of
gpKir was similar to that with K+
(Fig. 3B), indicating that the mechanism producing
rectification (27) apparently was not altered appreciably
by Rb+ substitution for K+ at the cytoplasmic
face of the channel. Similarity of K+ efflux with
high-Na+ and high-K+ pipette solutions, as
indicated by
at positive Vhold (Fig. 2B), supports the suggestion that these two K+
channel activities (Fig. 1, A and B) were an
identical channel type, an inward rectifier.
Voltage dependence of
for gpKir.
Excision into an I/O configuration can lead to altered channel behavior
as cytoplasmic components are lost. Single-channel conductance of
gpKir was not changed dramatically by excision
into a high-K+ solution that mimicked intracellular ion
composition (Figs. 2B and 3B). Generally the
small-conductance state was present, although the large-conductance
state was observed (2 of 18 patches, 11%). However, a direct
comparison of conductance-voltage dependence for cell-attached and
excised conditions requires an estimate of resting cell membrane
electrical potential difference (Vcell). Assuming that cell-attached reversal potentials of K+
channels with high-K+ pipette solution represented a
Vcell of 0 mV, cell-attached
Vhold then can be adjusted to indicate actual
Vm (Fig. 3E). Excised
appeared to
conform best in size to the small-conductance state seen with
cell-attached recordings. The ~50 mV rightward shift in voltage
dependence for
indicates that cytoplasmic factors controlling
may have been lost upon excision. One possibility is that
Mg2+ in the bath solution (Table 1) during I/O conditions
blocks gpKir with different voltage dependence
than the native cytoplasmic components (27).
Kinetic modes of gpKir.
Po of gpKir during
cell-attached recording was voltage independent but occurred in two
distinct modes (Fig. 4A),
moderate activity (Po = 0.41 ± 0.01, n = 8) and low activity (Po = 0.09 ± 0.01, n = 6). Abrupt transitions between
low and moderate states were observed (4 of 58 patches with
gpKir, 7%) but were not reversed, suggesting
that a voltage-independent regulatory process controlled transitions
between these two kinetic modes of gpKir.
Excision into an I/O configuration increased Po
of low-activity gpKir, producing a kinetic mode
with brief open and closed events (Fig. 4B); transition to
moderate Po occasionally occurred only several
minutes after excision. In the excised I/O condition, Po was similar to the moderate-activity state
(Po = 0.42 ± 0.01, n = 5) regardless of whether cell-attached activity had been low or
moderate (Fig. 4C), indicating that excision may remove a
cytoplasmic component that acts to limit Po.

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Fig. 4.
Open probability (Po) of guinea
pig inward rectifier K+ channel
(gpKir). Currents from
gpKir were recorded before and after excision
into I/O configuration. Po was calculated from
current records (see METHODS). Pipette solution was
high-K+. A: dependence of
Po on Vhold is shown for
cell-attached condition (means ± SE). Spontaneous activity was
observed in either of 2 states, moderate ( ,
n = 8) or low ( , n = 6). B: a cell-attached patch was excised into K-gluconate
bath solution. Current traces are shown for
Vhold of 50 mV. Closed states are indicated by
dashed lines. Openings for 2 gpKir were
apparent after excision. C: Po is
shown for those patches (n = 5) in which
gpKir from basal conditions were recorded in
both cell-attached (open symbols) and excised I/O (filled symbols)
configurations. Cell-attached Po is shown with
inclusion of apparent Vcell, 40 mV (see Fig.
2A).
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Secretagogue modulation of gpKir activity.
Numerous secretagogues stimulate electrogenic K+ and
Cl
secretion across distal colonic epithelia (7,
15). Electrogenic K+ secretion is stimulated by
epinephrine or PGE2, and at higher concentrations
PGE2 stimulates Cl
and K+
secretion (17, 41). In addition, the cholinergic
agonist carbachol (CCh) stimulates Cl
secretion.
Forskolin, which increases intracellular cAMP through activation of
adenylate cyclase, also stimulates electrogenic Cl
secretion. Spontaneous activity of gpKir was
observed with both high-Na+ (7 of 10 patches with
gpKir, 70%) and high-K+ pipettes
(42 of 58 patches with gpKir, 72%). Addition
of forskolin to the bath during cell-attached recording increased both
the number of open gpKir (N) and
apparent Po in quiescent patches (Figs.
5A and 7A) and in
patches with spontaneous activity (Figs.
6A and 7B). CCh added to the bath during cell-attached recording also activated gpKir in a quiescent patch (Fig.
5B).

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Fig. 5.
Secretagogue activation of gpKir.
Currents from gpKir were recorded during
cumulative addition of secretagogues to spontaneously quiescent
patches. Activity generally was recorded at 10-mV increments of
Vhold. Pipette solution was high-K+.
Closed states are indicated by dashed lines. A: a
cell-attached patch was sequentially treated with forskolin (1 µM)
and epinephrine (5 µM). Currents are shown 10.8 min after forskolin
addition for Vhold of 60 mV and 6.5 min after
epinephrine addition for Vhold of 50 mV;
because Po was voltage independent (see Fig.
4A), kinetic changes can be assessed from these current
traces. The number of open gpKir increased to 3 with forskolin. B: a cell-attached patch was treated with
carbachol (CCh; 10 µM) for Vhold of 50 mV.
Time after addition of CCh is noted. The number of open
gpKir increased to 7 with CCh.
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Fig. 6.
Secretagogue modulation of gpKir.
Currents from spontaneously active gpKir were
recorded during cumulative addition of secretagogues. Pipette solution
was high-K+. Closed states are indicated by dashed lines.
A: a cell-attached patch was sequentially treated with
forskolin (10 µM) and PGE2 (10 µM) for
Vhold of 50 mV. Currents are shown 13.0 min
after forskolin addition and 11.7 min after PGE2 addition.
The number of open gpKir increased from 1 in
basal to 4 with forskolin and PGE2. B: a
cell-attached patch was sequentially treated with epinephrine (5 µM),
PGE2 (10 µ), and forskolin (10 µM) for
Vhold of 60 mV. Currents are shown 12.8 min
after epinephrine addition, 9.3 min after PGE2 addition,
and 8.7 min after forskolin addition. The apparent number of open
gpKir in basal was 5.
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Fig. 7.
Secretagogues reduce activity of
gpKir. Currents from
gpKir were recorded during cumulative addition
of secretagogues. Pipette solution was high-K+. Closed
states are indicated by dashed lines. A: a cell-attached
patch in which CCh (10 µM) had failed to activate
gpKir after ~20 min was sequentially treated
with forskolin (10 µM) and PGE2 (10 µM) (upper
traces). Currents are shown ~19 min after forskolin addition for
Vhold of 80 mV and ~20 s after
PGE2 addition for Vhold of 40 mV.
Po was 0.01 ± 0.001 during forskolin
treatment. Open gpKir inactivated after ~1
min with PGE2 (lower trace) such that activity
at other Vhold was not obtained. B: a
cell-attached patch was treated sequentially with forskolin (1 µM),
epinephrine (5 µM), and PGE2 (100 nM) for
Vhold of 50 mV. Currents are shown 18.5 min
after forskolin addition, 17.4 min after epinephrine addition, and 17.6 min after PGE2 addition. Only 1 gpKir was open throughout treatment;
Po was 0.05 ± 0.02 (basal), 0.71 ± 0.02 (forskolin), 0.46 ± 0.02 (epinephrine), and 0.34 ± 0.01 (PGE2).
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Using epinephrine to produce a K+ secretory state decreased
apparent Po of forskolin-stimulated
gpKir (Figs. 5A and 7B)
and of spontaneously active gpKir (Fig.
6B). PGE2 reduced apparent
Po after forskolin stimulation (Fig.
6A) and in the presence of epinephrine (Fig. 6B
and 7B); forskolin addition in the presence of epinephrine
and PGE2 modestly increased apparent
Po, though not to basal level (Fig.
6B). In a patch with low apparent Po
stimulated by forskolin (Fig.
7A), PGE2 addition
led to complete inactivation of gpKir. A patch
with a single gpKir (Fig. 7B)
exhibited progressive modulation of Po during
cumulative addition of secretagogues, increase with forskolin, decrease
with epinephrine, and further decrease with PGE2.
Forskolin and CCh, which activate Cl
secretion, generally
increased N and Po of
gpKir, whereas epinephrine and
PGE2, which activate K+ secretion, generally
decreased N and Po (Figs. 5-7).
Forskolin activated gpKir (increased
N) in quiescent patches (10 of 80 patches, 12.5%); CCh also
increased N of gpKir (4 of 43 quiescent patches, 9%). PGE2 did not activate
gpKir (0 of 30 quiescent patches); epinephrine
rarely activated gpKir (2 of 57 quiescent
patches, 3%). These observed proportions of
gpKir activation include secretagogue additions
to patches that may not have contained gpKir so
that actual efficacy may have been higher. Comparisons among these
secretagogue results, however, do provide a relative assessment of
action on gpKir, because all patches were
sampled from the same group of crypts.
Secretagogue actions also were compared for patches containing
spontaneously active gpKir, which eliminated
the confounding effect of blank patches. Forskolin activated additional
gpKir in patches with spontaneously active
gpKir (3 of 12 active patches, 25%); CCh did
not activate gpKir in patches with
spontaneously active gpKir (0 of 6 active
patches). Both epinephrine (2 of 12 active patches, 17%) and
PGE2 (4 of 17 active patches, 23%) inactivated gpKir in patches with spontaneously active
gpKir; these K+ secretagogues did
not activate further gpKir in active patches.
Large-conductance gpKir (Figs. 1C
and 2) also were activated by forskolin (4 of 6 patches with
large-conductance gpKir recorded, 67%); other
secretagogues did not activate this large-conductance
gpKir. These proportions for secretagogue
action may include some patches with every
gpKir present already activated such that
further increases in N were not possible. In addition, these
spontaneously active patches may have been in a state that altered
sensitivity to secretagogues.
Secretagogue-induced changes in Po and
N for gpKir, similar to the patches
shown in Figs. 5-7, are summarized in Fig.
8. Po remained independent of Vhold after addition of
forskolin, CCh, epinephrine, or PGE2 (Fig. 8C).
Forskolin and CCh, on average, increased Po to a
level (0.38 ± 0.10, n = 8, and 0.28 ± 0.05, n = 3, respectively) consistent with the
moderate-activity mode of spontaneously active patches (Fig.
4A). PGE2 and epinephrine, on average, decreased Po to a level (0.22 ± 0.04, n = 10, and 0.21 ± 0.07, n = 6, respectively) between the two activity modes of spontaneously active
patches (Fig. 4A). Some of the variation in secretagogue
responses may depend on the order of secretagogue addition, the cell
type sealed, and the prior, unknown state of each crypt. Epinephrine
once activated gpKir (N = 3, Fig. 8B) only after forskolin had failed to stimulate a
quiescent patch, and Po increased only to ~0.1
(Fig. 8A), similar to the low-activity mode of spontaneously
active patches (Fig. 4A). Forskolin produced the smallest
Po increases when in the presence of other
secretagogues or with Po near 1.0. Increasing PGE2 concentration from 100 nM to 10 µM did not lead to
further change in Po (from 0.27 ± 0.04 to
0.24 ± 0.07, paired difference
0.03 ± 0.04, n = 3). Po and N
changed in a manner consistent with the expected demands of
transepithelial ion secretion, increasing during Cl
secretion and decreasing during K+ secretion.

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Fig. 8.
Secretagogue dependence of Po for
gpKir. Po and number of
active channels (N) for gpKir (12 patches) were determined from current records (see
METHODS). Values for Po and
N before and during each secretagogue treatment are
connected and plotted in pairs for comparison: solid lines connect
changes from patches that had spontaneous activity, and dashed lines
connect changes from patches that were quiescent spontaneously.
A: secretagogue-induced changes of Po
are shown for previously untreated patches: forskolin
( ; f), CCh ( ; C), PGE2
( ; P), and epinephrine ( ; e).
Subsequent secretagogue responses of these treated patches also are
shown (open symbols), with a label at the "before" value indicating
the order of prior cumulative secretagogue additions. Average
Po for each patch was calculated from
determinations at several Vhold (means ± SE), because Po was voltage independent
(C). Symbols often obscure small error bars. *Significant
changes (P < 0.05). B: secretagogue-induced
changes of N are shown for experiments in A.
Counting current levels provides an accurate measure of N
for values of 4 or smaller (20). N was >4 in
only two patches, but both had Po of ~0.4 so
that N probably was not seriously underestimated.
C: dependence of Po on
Vhold is shown for the cell-attached condition.
Activity was averaged for periods after addition of each secretagogue
(means ± SE): forskolin ( , n = 6), CCh ( , n = 3), PGE2
( , n = 9), and epinephrine
( , n = 5); averages include those
experiments that had Po measured for 3 or more
Vhold.
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Kinetic mechanism of gpKir.
Open and closed durations of gpKir activity
were examined to obtain a preliminary mechanism controlling
Po changes by secretagogues. Histograms of open
durations from a patch containing a single gpKir (Fig. 7B) exhibited one peak.
In the basal and forskolin condition, the peak was fit well by one
exponential, and after K+ secretagogue additions, two
exponentials were required for an adequate fit (Fig.
9,
A-D) consistent with at least two open states for
gpKir. Histograms of closed durations exhibited
several peaks, indicating multiple closed states for
gpKir (Fig. 9, E-H). Closed
durations for the basal condition were fit well by four exponentials
(Fig. 9E) with widely separated time constants, indicating
at least four closed states for spontaneously active
gpKir. Closed durations during forskolin,
epinephrine, and PGE2 addition also were fit best by four
exponentials (Fig. 9, F-H), indicating at least four
closed states for secretagogue-stimulated conditions. Forskolin
stimulation of Po (Fig. 8A) occurred
through elimination of closed events longer than ~400 ms with a small relative increase in closed events of intermediate duration (Fig. 9F). Reduction of Po by epinephrine
and PGE2 occurred through a further increase in the number
of closed events of intermediate duration (Fig. 9, G and
H) and a decrease of time constants for open events (Fig. 9,
C and D). Activation of
gpKir appears to have occurred largely by
altering residence in various closed states, which can be seen
qualitatively in the current records (Figs. 5-7). Modulation of
Po at intermediate levels also occurred through
changes of relative residence in closed states, together with
shortening of the longer open time constant.


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Fig. 9.
Secretagogue dependence of
kinetics for gpKir. Histograms of open
(A-D) and closed durations (E-H) are
shown in which log binning was used (see METHODS) for a
single gpKir during cumulative addition of
several secretagogues while cell-attached (see Fig. 7B).
Vhold was 70 mV. Each current record was ~50
s in length with a various number of events in each condition: basal
(740), forskolin (5,479), epinephrine
(8,252), and PGE2 (8,380).
Open-duration histograms for basal and forskolin conditions had a peak
fit well by a single exponential; open-duration histograms for
epinephrine and PGE2 conditions had a peak fit best by a
mixture of 2 exponentials. 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 basal, 9.4 ms; forskolin, 7.7 ms; epinephrine, 1.1 ms (0.38) and
2.4 ms (0.62); and PGE2, 0.7 ms (0.40) and 1.5 ms (0.60).
Closed-duration 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
basal, 0.3 ms (0.94), 8.6 ms (0.03), 187 ms (0.02), and 14.5 s
(0.01); forskolin, 0.3 ms (0.87), 3.7 ms (0.06), 10.8 ms (0.06), and
101 ms (0.01); epinephrine, 0.3 ms (0.45), 2.3 ms (0.30), 6.6 ms
(0.24), and 59.3 ms (0.01); and PGE2, 0.3 ms (0.54),
3.4 ms (0.30), 8.3 ms (0.14), and 50.4 ms (0.01). Kinetic parameters
from currents at Vhold of 80 mV were
similar.
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The kinetic mechanism for gpKir was
analyzed further with other patches containing only one active channel;
10 gpKir were examined from 16 fits for
conditions including spontaneous activity, secretagogue stimulation,
and excision. One open state was apparent in basal conditions with low
Po, as judged by open-duration histograms fit by
a single exponential (8.0 ± 0.6 ms, n = 3). Spontaneously active gpKir in the
medium-Po mode (n = 3) had
open-duration histograms fit by two exponentials with time constants
0.8 ± 0.2 ms (32 ± 11%) and 2.6 ± 0.1 ms (68 ± 11%). These two time constants further support the presence of at
least two open states for gpKir (Fig.
10A), a short-duration state
(OS) and a longer duration state (OL). Fits of
closed-duration histograms from various conditions suggest the presence
of at least five closed states, even though many were fit well
individually by four exponentials.

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Fig. 10.
Open and closed states of gpKir.
Time constants and proportions of events for kinetic states obtained
from fits of duration histograms to mixtures of exponentials were
averaged from patches containing only 1 gpKir
(means ± SE, range is shown for n = 2).
Spontaneous cell-attached activity with low Po
(n = 3) and medium Po
(n = 3) together with excised I/O activity
(n = 3) are shown. Activity during PGE2
(n = 2) and epinephrine (n = 1)
treatments also is shown. A: open states of shorter
(OS) and longer (OL) time constants are
connected for each condition. Burst analysis (1-ms delimiter) yielded
burst-duration histograms fit by a mixture of 2 exponentials.
B: closed time constants clustered into groups. For those
gpKir that did not exhibit a fit in a
particular closed-duration group, a proportion of zero was included in
the group average; n is indicated on plots for those cases.
*Significantly different from medium-Po or
excised conditions (P < 0.05). Vertical dotted lines
demarcate apparent ranges of observed time constants; average values
(from spontaneous activity) for each group were C1,
0.3 ± 0.01 ms, n = 10; C2, 3.2 ± 0.1 ms, n = 9; C3, 12.7 ± 1.5 ms,
n = 8; C4, 148.4 ± 35.2 ms,
n = 8; C5, 1.31 ± 54 s,
n = 4; and C6, 25.2 ± 10.7 s,
n = 2.
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Closed time constants clustered into six distinct groups (Fig.
10B). All of the gpKir examined had
a component with a time constant in the first group, C1.
Many gpKir (7 of 10) also had contributions in
the next three groups, C2 through C4. Most
gpKir (8 of 10) had fitted components in both
C2 and C3. Together, the simultaneous presence
of the four shortest components suggests that these states were
distinct and not variations within fewer states. In particular, the
closely spaced groups C2 and C3 occurred in six
successive 10-s intervals of the record in Fig. 9G (data not
shown), indicating that the time constant for a single state was not
simply drifting within this range unless the change oscillated continuously on a time scale much shorter than 10 s. Group
C5 appeared distinct because two
gpKir were fit well with five components,
C1 through C5, and one other had both
components C4 and C5. A group C6
component was observed for two gpKir with low
Po, one in combination with C4 and
C5. Thus, although secretagogue-induced changing of rates
for entering and exiting a state could blur groupings of time
constants, the minimum number of closed states for
gpKir was five, but possibly six.
Burst analysis supported the presence of two open states. Bursts were
delimited by closed durations longer than 1 ms. This duration was
chosen to ignore most of the closures due to the shortest apparent
closed state, C1. All of the gpKir
with spontaneous activity had burst-duration histograms fit by two
exponentials (Fig. 10A). Prolongation of the longer bursts in the low-Po mode compared with the
medium-Po mode was consistent with the relative
paucity of closures longer than 1 ms (Fig. 10B) such that
burst termination is delayed substantially. Distributions of open
durations during bursts were similar to all openings (data not shown).
Presence of the brief burst mode (0.9-1.2 ms) during spontaneous
low Po activity (Fig. 10A) suggests
that a low proportion of events (<5%) occurred in the short open
state, OS (Fig. 9A).
Excision led to increased Po of
gpKir similar to the
medium-Po spontaneous activity mode (Fig. 4).
Open time constants for the excised condition (1.1 ± 0.2 ms,
24 ± 5%, and 3.5 ± 0.8 ms, 76 ± 5%,
n = 3) were indistinguishable from those in the
medium-Po mode but significantly shorter
(P < 0.05) than that for the
low-Po mode (Fig. 10A). The
distribution of closures for the excised condition and
medium-Po mode differed from the
low-Po mode by a decreased proportion of events
in C1, an increase in C2, and a lack of events in C5 and C6 (Fig. 10B). Excision
appeared to have produced an increase in Po by
recreating the kinetic conditions of the spontaneous medium-Po mode.
Reductions in Po by epinephrine and
PGE2 after forskolin stimulation (Fig. 9, G and
H) occurred with a roughly equal and relatively large
proportion of closures in C2 and C3. For
another gpKir, PGE2 reduced
Po from a spontaneous
medium-Po mode by decreasing the proportion of
closures in C2 and increasing the proportion in
C3 (data not shown) such that the final distribution was
similar to that during forskolin/PGE2 stimulation (Figs.
9H and 10B). Epinephrine and PGE2
appeared to decrease Po largely through a shift
of closed events into C2 and C3 as well as a
relative shift of open events into OS.
Cytoplasmic regulators.
Distinctions between known inwardly rectified K+ channels
can be made, in part, from sensitivities to solution composition at the
cytoplasmic face of the channel. Notably, dependence on Ca2+ and pH can be used to aid in distinguishing among
intermediate-conductance Ca2+-activated K+
channels (IK1; KCNN4), inward rectifier K+
channels (Kir; KCNJ), and so-called background
K+ channels (TWIK; KCNK1) (10).
Activity of gpKir was similar with bath pH of
7.2 and 6.6 (Fig. 11A),
whereas increasing bath pH to 8.1 reduced gpKir
activity modestly (n = 3). Because renal epithelial
K+ channel (ROMK, Kir1.1; KCNJ1) activity
decreases with acidification (9, 36), another channel type
apparently was responsible for this gpKir
observed in crypts. Reduction in bath solution free Ca2+
(Fig. 11B) did not lead to lower activity for
gpKir (n = 4), indicating that
IK1 (13, 23) alone could not be responsible for producing
these currents.

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Fig. 11.
Control of gpKir. Currents from
gpKir were recorded in excised I/O
configuration during changes in bath solution. Pipette solution was
high-K+. Closed states are indicated by dashed lines.
A: currents are shown for bath solution pH (see methods) of
7.2, 8.1, or 6.6 for Vhold of 50 mV.
Po was voltage independent: 0.39 ± 0.02 (pH 7.2), 0.39 ± 0.03 (pH 6.6), and 0.28 ± 0.03 (pH 8.1);
the number of active gpKir was 4. B:
currents are shown for bath solution free Ca2+
concentration (see METHODS) of ~10 µM (basal), ~100
nM (low Ca2+), or <10 nM (0 Ca2+) for
Vhold of 50 mV. Po was
voltage independent: 0.46 ± 0.01 (basal), 0.45 ± 0.01 (low
Ca2+), and 0.42 ± 0.01 (0 Ca2+); the
number of active gpKir was 5.
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 |
DISCUSSION |
Active ion secretion across epithelia produces an osmotic gradient
that drives fluid secretion (14, 15). The lumen negative transepithelial electrical potential difference produced by
Cl
secretion results from a cellular mechanism employing
apical membrane Cl
channels and basolateral membrane
K+ channels. Together with the K+ concentration
gradient developed by operation of the Na+-K+
pump, basolateral membrane K+ conductance serves to
generate a cell negative basolateral membrane electrical potential
difference (Vb). Apical membrane
Cl
channels allow Cl
exit into the lumen,
driven by the electrochemical gradient. This gradient is directed
outward because of the influence of basolateral membrane K+
channels on apical membrane electrical potential difference
(Va). Continued Cl
secretion, and
thus fluid secretion, depends on a basolateral membrane K+
conductance large enough to maintain Cl
exit across the
apical membrane by ensuring that Va exceeds the size of the inwardly directed concentration gradient.
Colonic Cl
secretion in mammals is accompanied by
electrogenic K+ secretion, apparently by inclusion of
apical membrane K+ channels in Cl
secretory
cells (15, 41). For colonic secretory cells, therefore, K+ channels in both apical and basolateral membranes
contribute to maintaining a cell negative Va
that promotes Cl
secretion. In addition, K+
conductance of apical membrane relative to basolateral membrane contributes to determining the proportion of intracellular
K+ exiting into the lumen. Elevated luminal K+
concentration (19) would lower the K+
concentration gradient across the apical membrane and thereby reduce
the ability of apical K+ channels to ensure conductive
Cl
exit. Relative rates of Cl
and
K+ secretion vary among mammalian species
(15), but in guinea pig distal colon these electrogenic
flows are roughly equal when stimulated by high concentrations (>100
nM) of PGE2 (17, 41). Activation by
epinephrine (15, 41) or low concentrations (<100 nM) of
PGE2 (17, 41) produces electrogenic
K+ secretion without accompanying Cl
secretion. Cl
entering via basolateral membrane
Na+-K+-2Cl
cotransporters during
this type of sustained electrogenic K+ secretion apparently
exits across the basolateral membrane through Cl
channels
(15, 30, 41). Control of basolateral K+
channels during primary electrogenic K+ secretion, thus,
also would contribute to maintaining a driving force for
Cl
exit from the cell as well as to determining the
proportion of K+ exiting into the lumen.
K+ channel types.
Several classes of K+ channels have been identified by
amino acid sequence homology (10, 27). Although all seem
to conserve a general pore-forming component, wide variation occurs in
other portions of the sequence that presumably control channel
activation and kinetic behavior. Three of these K+ channel
types exhibit distinct inwardly rectified
: Kir (KCNJ), IK1 (KCNN4), and TWIK (KCNK1) (10).
With symmetrical 150 mM K+ concentration,
at
Vm = 0 mV is 30, 16, and 28 pS for Kir1.1 (9), IK1 (23, 25), and TWIK
(29), respectively. Specific sensitivities to pH,
Ca2+, and ATP also contribute to a functional
identification of these channel types (10). In particular,
the ROMK channel (Kir1.1; KCNJ1) is inhibited by
acidification at the cytoplasmic side of the channel (9,
36). Increasing cytoplasmic ATP also inactivates ROMK (Kir1.1;
KCNJ1) (9, 36); association with a regulatory subunit, the sulfonylurea receptor, confers even greater ATP
sensitivity to Kir6 (10). Activation by cytoplasmic
Ca2+ is a key feature of IK1 shared with BK (slo;
KCNMA) (10). The TWIK channel has been termed a
background channel because it does not have any direct dependence on
pH, Ca2+, or Vm (10,
29). Behavior of K+ channels in native circumstances
may vary from characteristics of overexpressed versions, however,
because K+ channels may exist as heteromultimeric
assemblies of channel and regulatory subunits that result in divergent
properties (10, 27, 43, 51).
Colonic K+ channels.
Three major types of K+ channels have been observed in
cell-attached patches on basolateral membrane of colonic and small
intestinal crypts (55): large-conductance K+
channels (5, 6, 26, 32, 33, 35, 42),
intermediate-conductance Ca2+-activated K+
channels (4, 5, 20, 35, 42, 44), and very small conductance K+ channels (57). Nonselective
cation channels, with
of 20-40 pS, also have been observed in
basolateral membranes of colonic epithelial cells (4, 6, 42,
47), similar in size to those observed in guinea pig crypts.
Large-conductance K+ channels in crypts have
ranging
from ~100 pS to >200 pS, and some are activated by increases in
Ca2+ activity at the cytoplasmic face of the channel
(5, 26, 32, 33, 35). Kinetic behavior and large
conductance suggest that these K+ channels, and those
observed in guinea pig crypts (Figs. 1D and 2), are
intestinal assemblies of the BK (slo; KCNMA) channel
(10). Colonic intermediate-conductance
Ca2+-activated K+ channels often are inwardly
rectified (5, 20, 39, 42) with
of 30-35 pS in
symmetrical 150 mM K+ concentrations at
Vm = 0 mV. Inwardly rectified
Ca2+-activated K+ channels with
of ~20 pS
have been observed as well in the colonic tumor cell line T84
(11, 49). Identification of these K+ channels
as IK1 (KCNN4) (10) is supported further by
detection of mRNA for IK1 in T84 cells and colonic epithelial cells
(56). Another K+ channel with very small
conductance (<4 pS) also has been detected in colonic crypts, with the
use of noise analysis (57), similar in size to KvLQT1
(KCNQ1) assembled with the regulatory subunit minK+ (KCNE) (58). Identity of this
crypt K+ channel as KvLQT1/minK+
(KCNQ1/KCNE) is supported by in situ hybridization
localizing mRNA for both subunits in colonic crypt cells
(46).
The predominant K+ channel type found in this study of
guinea pig distal colonic crypts was an inwardly rectified
K+ channel, gpKir (Fig.
1B). A larger conductance K+ channel was
observed rarely (Fig. 1D) that may correspond to the BK
channel (slo; KCNMA) (10). Identity of the
gpKir observed in basolateral membranes is
uncertain. With physiological external K+ and
Na+ concentrations (Figs. 1A and 2),
gpKir apparently had a linear current-voltage
relation. A less commonly observed inwardly rectified K+
channel (Fig. 1C), which may be another conductance state of gpKir, had steeper rectification than
gpKir at negative Vm
(Fig. 3E). Insensitivity to changes in Ca2+
activity on the cytoplasmic side of gpKir (Fig.
10B) suggests that IK1 (KCNN4) (10)
is not a candidate protein unless other associating components could
modify Ca2+ dependence. Insensitivity to acid pH (Fig.
10A) suggests that ROMK (Kir1.1; KCNJ1) (9,
36) also is not the protein forming gpKir. The lack of these specific controlling
factors for gpKir suggests TWIK
(KCNK1) (10) as a possibility, but
for TWIK with symmetrical 150 mM K+ concentrations (at
Vm = 0 mV) is approximately twofold larger (29) than for gpKir (Figs.
3B). Thus gpKir does not conform
easily to any of these known inwardly rectified K+ channel types.
Conduction (Fig. 3E) and activation (Fig. 11) properties for
gpKir suggest that guinea pig colonic crypts
exhibit a K+ channel distinct from those already
characterized in basolateral membranes for this type of epithelial cell
(55). However, several commonalties can be found between
gpKir and previously observed
intermediate-conductance K+ channels. ROMK has a
subconductance state of ~13 pS corresponding to a particular
phosphorylated state (34). Coexpression of the Cl
channel cystic fibrosis transmembrane conductance
regulator with ROMK leads to inwardly rectified K+ channels
with
of about half (~15 pS) that for ROMK alone
(43). In addition, heteromeric assembly of Kir4.1 with
Kir5.1 increases
by approximately twofold (51).
Ca2+-dependent K+ channels in T84 cells have
low Po independent of Vm
(50), similar to that for gpKir
(Fig. 4A). Other Ca2+-dependent K+
channels of intermediate conductance exhibit Po
that increases (5) or decreases (56) at more
positive Vm. ROMK has high Po (~0.9) that is relatively independent of
Vm (40). High relative permeability
to Rb+ (Fig. 3) appears to distinguish
gpKir from both ROMK (8) and IK1
(49). Although none of the currently established
K+ channels (10) has identical characteristics
to gpKir , a distinct assembly of channel and
regulatory subunits (27) presumably confers the properties
observed for gpKir.
Regulation of Cl
and K+
secretion.
Control of gpKir in the basolateral membrane
participates in production of Cl
and K+
secretion by contributing to maintenance of Va
that drives Cl
exit into the lumen while also adjusting
the proportion of K+ exit into the lumen. Activation of
gpKir occurred with two Cl
secretagogues, forskolin and CCh (Figs. 5-7), consistent with this channel being used to augment basolateral membrane K+
conductance. Both the number of active gpKir
(NK) and Po increased
(Fig. 8) such that basolateral membrane K+ conductance
(gK) would increase:
gK = NKPo
K. The
K+ secretagogues PGE2 and epinephrine led to
decreased gK primarily by reducing
Po (Fig. 8) and occasionally by deactivating gpKir (NK). This type of
control would suit the requirements of balancing K+ and
Cl
secretory rates by moderating basolateral membrane
K+ conductance within a precise range.
Regulatory modes for gpKir can be distinguished
on the basis of the kinetics producing Po.
Spontaneous activity exhibited two modes, with low and moderate
Po (Figs. 4A and 10). The
cAMP-dependent agent forskolin produced moderate
Po with kinetics different from the spontaneous
mode (Figs. 8A, 9, and 10). The K+ secretagogues
PGE2 and epinephrine reduced Po to
an intermediate level with kinetics distinguishable from the other
three modes (Figs. 8A, 9, and 10). These four kinetic modes
of gpKir presumably are produced by distinct
regulatory mechanisms characteristic of the components making up this
channel complex.
A kinetic scheme for gpKir must include two
open states and at least five, possibly six, closed states (Fig. 10).
Although the connections for these states were not determined uniquely
by the data available, several features controlling each mode were
apparent. Long-duration closures in the C5 and
C6 groups were associated only with the
low-Po mode. The
medium-Po mode also differed from the
low-Po mode by having similar contributions from
C1 and C2. Intermediate
Po of the K+ secretory mode occurred
with a relatively high contribution from C3. Both the
low-Po and forskolin modes had open durations
dominated by a single state with a time constant approximately four
times longer than OL of the
medium-Po and K+ secretory modes.
A common feature of many K+ channels, including
gpKir, appears to be activation through reduced
numbers of long-duration closures, although the mechanism producing
this kinetic change is distinct for each channel type. Similar to the
loss with forskolin of gpKir closed durations
ranging from 0.4 to 10 s (Fig. 9, E and F), cAMP-dependent activation eliminated longer closed durations of an
ATP-dependent, inwardly rectified K+ channel in the
basolateral membrane of proximal tubules (37). Protein
kinase A (PKA) together with ATP stimulates an 85-pS K+
channel from the lateral membrane of cortical collecting duct largely
through a decreased number of the long-duration closures as well as an
increased number of the long-duration openings (53). Modulation of ROMK (Kir1.1; KCNJ1) via alkalinization
increases Po by decreasing the number of
long-duration closures without changing open durations (9,
36). Heteromeric Kir4.1-Kir5.1 (KCNJ10-KCNJ16)
also increases Po upon alkalinization through a
loss of long-duration closures (59). Stimulation
of IK1 (KCNN4) by the K+ channel opener 1-EBIO
occurs through a decrease in long closed durations without any change
in open durations (48). A small-conductance Ca2+-activated K+ channel (SK2;
KCNN2) activates with increased cytoplasmic
Ca2+, in part, by losing long duration closures
(21).
Activation of K+ channels can result from phosphorylation
via cellular kinases (54). Both the cAMP-dependent PKA and
the Ca2+-dependent protein kinase C (PKC) have been
implicated in K+ channel regulation (10, 27,
54). Increasing cAMP either directly or with forskolin
stimulates activity of BK channels in rabbit colonic crypts
(33), intermediate-conductance K+ channels
(IK1) in human colonic crypts (44), and very small conductance K+ channels (KvLQT1/minK) in rat colonic crypts
(57). Loss of channel activity after excision into an I/O
configuration often can be returned by adding ATP to the bathing
solution or by inhibiting phosphatase activity (2),
suggesting that channel Po can be controlled
through a balance between kinase and phosphatase activity (13,
28, 39, 50, 54, 57).
The tendency for excision to increase Po of
gpKir (Fig. 4) suggests that a cytoplasmic
inhibitory component was lost. One possibility could be that a
membrane-resident phosphatase (2) dephosphorylated gpKir at an inhibitory site, similar to the
negative influence of PKC on ROMK (54). Excision
activation (Fig. 4B) and forskolin activation (Figs.
6A and 7B) both increase
Po of gpKir into the
range of the spontaneous medium-Po mode (Fig.
4A). In part, Po increased because
gpKir did not enter long-lived closed states.
However, during forskolin treatment gpKir
openings were predominately to the longer open state (Fig. 9B), whereas after excision openings occurring to both
states and OL had a shorter time constant (Fig.
10A). These two modes of activation could be reconciled with
a phosphorylation/dephosphorylation mechanism if at least two
phosphorylation sites were to exist with opposing action on
Po such that relieving the negative control is
sufficient for increased activity.
Cholinergic activation of gpKir apparently does
not proceed through direct influence of cytoplasmic Ca2+ on
the channel (Fig. 10B), as occurs for other
intermediate-conductance K+ channels (4, 11, 13, 23,
44, 49). Instead, Ca2+ dependence may be indirect,
conferred perhaps via a Ca2+-activated kinase such as PKC
or calmodulin-dependent kinase. Reduction of
gpKir Po by the
K+ secretagogues PGE2 and epinephrine (Fig. 8)
generally did not drop to the low spontaneous levels (Fig.
4A). Rather than returning to a condition with long-duration
closures, closures to states with intermediate time constants increased
in number (Figs. 9 and 10). Somatostatin also reduces
Po to a non-zero value for a cAMP-activated
K+ channel in human colonic crypts in concert with only a
~14% decrease of intracellular cAMP levels (45).
Although PGE2 and epinephrine can act through receptors
that increase cytoplasmic cAMP (15), this inhibitory
control of gpKir may occur through another
second messenger pathway such that several separable mechanisms may be
involved in controlling gpKir kinetics.
Colonic crypts possess several types of K+ channels
(55), and each may serve specific facets of the cellular
requirements for K+ conductance. These needs may not be
identical for the two predominant cell types in the crypt, columnar and
goblet. Because mucus release from columnar cells and goblet cells is
controlled by distinct secretagogues (16), responsiveness
to cholinergic stimulation by CCh may indicate a goblet cell regulatory
mode. However, attempts to examine cell type-specific responses may
have been complicated by differences in patching efficacy. Also in this
study, incidences of channel appearance and activation were low enough
to obscure further any cell type distinctions that might exist.
However, the contribution of gpKir to
basolateral membrane K+ conductance of crypt epithelium
would be a 9-pS channel (Fig. 2) with a Po that
can be modulated in the range from 0.1 to 0.5 (Fig. 8). For the
situations of Cl
and K+ secretion, this
feature of subtle modulations in Po may allow secretory cells to adjust basolateral K+ conductance for a
precise balance in rates of K+ exit and maintenance of
membrane electrical potential differences (Va
and Vb).
 |
ACKNOWLEDGEMENTS |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-39007.
 |
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.
10.1152/ajpcell.00065.2001
Received 7 February 2001; accepted in final form 6 November 2001.
 |
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