From the August Krogh Institute, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark
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ABSTRACT |
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Chloride channels in the luminal membrane of exocrine gland acini from frog skin (Rana esculenta)
constituted a single homogeneous population. In cell-attached patches, channels activated upon exposure to isoproterenol, forskolin, or dibutyryl-cAMP and isobutyl-1-methyl-xanthine rectified in the outward direction with a
conductance of 10.0 ± 0.4 pS for outgoing currents. Channels in stimulated cells reversed at 0 mV applied potential, whereas channels in unstimulated cells reversed at depolarized potentials (28.1 ± 6.7 mV), indicating that
Cl was above electrochemical equilibrium in unstimulated, but not in stimulated, cells. In excised inside-out
patches with 25 mM Cl
on the inside, activity of small (8-pS) linear Cl
-selective channels was dependent upon
bath ATP (1.5 mM) and increased upon exposure to cAMP-dependent protein kinase. The channels displayed a
single substate, located just below 2/3 of the full channel amplitude. Halide selectivity was identified as PBr > PI > PCl from the Goldman equation; however, the conductance sequence when either halide was permeating the
channel was GCl > GBr >> GI. In inside-out patches, the channels were blocked reversibly by 5-nitro-2-(3-phenylpropylamino)benzoic acid, glibenclamide, and diphenylamine-2-carboxylic acid, whereas 4,4-diisothiocyanatostilbene-2,2-disulfonic acid blocked channel activity completely and irreversibly. Single-channel kinetics revealed one
open state (mean lifetime = 158 ± 72 ms) and two closed states (lifetimes: 12 ± 4 and 224 ± 31 ms, respectively). Power density spectra had a double-Lorentzian form with corner frequencies 0.85 ± 0.11 and 27.9 ± 2.9 Hz, respectively. These channels are considered homologous to the cystic fibrosis transmembrane conductance regulator Cl
channel, which has been localized to the submucosal skin glands in Xenopus by immunohistochemistry
(Engelhardt, J.F., S.S. Smith, E. Allen, J.R. Yankaskas, D.C. Dawson, and J.M. Wilson. 1994. Am. J. Physiol. 267:
C491-C500) and, when stimulated by cAMP-dependent phosphorylation, are suggested to function in chloride secretion.
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INTRODUCTION |
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With the advent of the patch-clamp technique, considerable effort has gone into the identification of the apical cAMP-dependent Cl channel involved in epithelial
secretion. Three types are suggested to participate in
cAMP-induced secretion: the outward rectifier Cl
channel (shark rectal gland, Greger et al., 1987
; colon
carcinoma cells, Hayslett et al., 1987
; airway cells, Frizzell et al., 1986
), the small conductance 5-11-pS linear
Cl
channel (shark rectal gland, Gögelein et al., 1987
;
colon carcinoma cells, Hayslett et al., 1987
; pancreatic
ducts, Gray et al., 1989
; airway cells, Frizzell et al., 1986
;
human tracheal gland, Becq et al., 1993b
), and a population of very small conductance channels (<2 pS) (airway cells, Kunzelmann et al., 1994
; and colon carcinoma cells, Fischer et al., 1992
; Kunzelmann et al.,
1992
). Whereas it is generally accepted that the 5-11-pS
channel is encoded by the cystic fibrosis transmembrane conductance regulator (CFTR)1 gene, controversy remains on the identity and involvement of the
other channels in secretion. The main interest in this
subject derives from the disease cystic fibrosis, which is
caused by mutation of the CFTR gene. Naturally, the
identity and distribution of secretory Cl
channels
seems crucial for an understanding of the pathology of
cystic fibrosis and for identification of redundant pathways that may aid in its therapy.
Due to the difficult anatomy of most secretory epithelia, in the majority of cases investigators must rely on
cultured cells, where the distribution of Cl channels
or the expression of as yet unidentified subunits may be
altered when compared with native tissue. An exception to this is the work done on freshly isolated shark
rectal glands. However, being an osmoregulatory organ
with specialized functions, the shark rectal gland may
have a physiology that is not comparable with most isotonic secreting glands affected in cystic fibrosis.
Here we present a patch-clamp study of the luminal
membrane of freshly isolated secretory gland acini
from frog skin, which has been shown to be a major site
for expression of CFTR in Xenopus (Engelhardt et al.,
1994). The frog skin glands consist of numerous acini
connected to the outside via very short ducts. They
therefore present the interesting possibility of investigating primary acinar secretions almost unmodified by
ductal activities. After adrenergic stimulation, the activity of the glands can be studied in isolation by blocking
Na+ absorption of the epithelium. Thus, the tissue has
been used for the study of secretion using transepithelial electrical measurement combined with tracer studies (Koefoed-Johnsen et al., 1952
; Thompson and Mills,
1983
) and measurement of the water flow (Bjerregaard and Nielsen, 1987
). Recently, a novel secretory model,
the Na+ recirculation model, was proposed for the frog
skin glands (Ussing et al., 1996
). The acini are easily
isolated and the apical membrane can be patched after
microdissection from the serosal side. We report that
the only apical Cl
channel found in this tissue is a 8-10
pS cAMP-regulated ion channel with properties similar,
but not identical, to human CFTR. Properties of Na+
channels found during the same investigation will be
published elsewhere (see also Sørensen et al., 1998
).
Preliminary reports of this work have appeared (Sørensen and Larsen, 1998a
, 1998b
).
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MATERIALS AND METHODS |
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Preparation of Isolated Epithelial Sheets
Frogs (Rana esculenta) were killed by decapitation and double pithing. The skin was dissected off in one piece and the connective tissue (corium) was removed by a combination of enzymatic and hydrostatic treatment (Andersen et al., 1995). In brief, the skin was mounted in a chamber exposing the mucosal side to a bath of NaCl Ringer thermostatically controlled at 29-30°C. The serosal side was treated with 1-1.2 mg/ml crude collagenase in NaCl Ringer for 75 min (Collagenase A; Boehringer-Mannheim GmbH, Mannheim, Germany, or Collagenase type 2; Worthington Biochemical Corp., Lakewood, NJ). A tube attached to the chamber was then filled with NaCl Ringer (the enzyme was not removed) to give an over-pressure of 10-25 cm H2O on the serosal side. This treatment resulted in the detachment of the epithelium from the connective tissue in some areas. The skin was then transferred to a petri dish and the connective tissue was removed by gentle dissection from the serosal side, leaving a large area of epithelium intact. The epithelium was kept in aerated NaCl Ringer at room temperature (22-24°C) and used on the same day.
Preparation of Glands for Patch Clamp
A piece of the isolated epithelium was mounted in a chamber (area 0.79 cm2, volume ~0.5 ml) against a coverslip with the serosal side up and placed on the stage of an inverted microscope (Zeiss IM35; Brock & Michelsen, Birkerød, Denmark). The serosal, but not the mucosal, side was perfused continuously (2-5 ml/ min) with NaCl Ringer before and during seal formation, and perfusion persisted throughout the experiment in most cases. As viewed at low magnification (25×), the preparation consisted of an intact epithelial sheet with variable number of glands still attached, but stripped for connective tissue. The glands were viable as shown by trypan blue exclusion.
For patch clamp on the luminal side of the gland cells, the intact glands were microdissected from the serosal side using a discarded patch pipette (Fig. 1) under high magnification (400×). The pipette was shoved into the gland lumen and moved to either side to slit the gland acinus into two parts. In cases where this was difficult, the glands were softened by exposure to divalent cation-free Ringer (composition as NaCl Ringer but with 10 mM EGTA replacing CaCl2 and MgCl2) for ~5 min. The open gland acinus was pressed against and often adhered to the epithelium, exposing the luminal (apical) membrane. A new patch pipette could now be used for patch clamp of the luminal membrane. The firm attachment of the gland acinus to the duct and therefore to the epithelium made the use of a holding pipette superfluous and assisted in the correct identification of the polarity of the membrane.
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Patch-Clamp Methodology
Patch pipettes were fabricated from borosilicate glass tubes with
an inner filament (ModulOhm, Herlev, Denmark) on a vertical puller (Hans Ochotzki, Homburg, Germany) using a gravity-driven two-step pulling procedure. The pipettes were fire-polished (Micro Forge MF-90; Narishige, Tokyo, Japan) to a final resistance of 5-10 M when filled with NaCl Ringer. An EPC-9 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany) furnished
with a 50-G
headstage was used for voltage clamping and amplification of pipette currents before recording on a digital recorder (DTR-1204; Biologic, Claix, France).
Analysis
For analysis of the channel amplitude, currents were filtered at
100 Hz (in a few cases at 50 Hz) corner-frequency (3 dB, low
pass eight-pole Bessel filter; Frequency Devices Inc., Haverhill, MA) and digitized at 1 kHz sampling frequency through a
1401plus interface (Cambridge Electronic Design, Cambridge,
UK). Measurement of channel amplitude was performed using
Gaussian fits to all-points histograms and the current-voltage relationship constructed accordingly. The current-voltage relationships of Figs. 3, 4, and 6 were fitted using polynomial regression
of increasing order, including the highest order that was considered significant (P < 0.05) by analysis of variance (ANOVA; Sokal
and Rohlf, 1981
). The regression procedure was carried out by
including all data points from individual experiments and the error associated with the fitted parameters is the standard error of
the resulting fit. Since this procedure includes only one fitting
event, no error value is associated with derived variables such as
the reversal potential.
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Determination of the halide selectivity of the channel was
done in excised inside-out patches by measuring the reversal potential in symmetrical 120 mM Cl and after replacing 100 mM
bath Cl
with either Br
or I
. The change in reversal potential
upon replacement,
Vrev, was used for calculation of the permselectivity ratio for the other halide (X) and Cl
, using the formula
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(1) |
derived from the Goldman voltage equation. Since the substituted halides and Cl have almost identical mobilities, the resulting liquid junction potentials are insignificant (<0.3 mV, calculated following Barry and Lynch, 1991
) and have not been corrected for.
For analysis of the single-channel kinetics, currents were filtered at 200 Hz (3 dB corner frequency, eight-pole Bessel filter) and digitized at 5 kHz. According to double-Lorentzian fits
to power density spectra calculated from the same patches (see
below), the bandwidth of 200 Hz would include >98% of the
band-unlimited variance due to channel gating. The channel
open and closed times were determined using a single threshold
just below 50% of the full channel amplitude. A minimal time
resolution of 1 ms was superimposed on the distribution of open
and closed times, and events >4 ms were used for fitting (Colquhoun and Sigworth, 1995
).
For stationary noise analysis in multi- or single-channel
patches, currents were filtered at a corner frequency of 500 Hz (3 dB, low pass eight-pole Butterworth; Frequency Devices
Inc.) and digitized at 1 kHz. The current was divided into blocks
of 4,096 points each (fundamental frequency = 0.244 Hz) and
the power density spectrum was estimated using a Fast Fourier
Transform. After averaging of the spectra, Lorentzians were fitted using a Levenberg-Marquard routine. In practice, the formula fitted to the spectra was:
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(2) |
where So1, fc1 and So2, fc2 are the low frequency asymptote and corner frequency of the low and high frequent Lorentzian, respectively, and S is a plateau-level (formally attained as f
) indicating the background noise level in the patch. The (band-unlimited) variance in current ascribable to each Lorentzian was
calculated by integration of the Lorentzians at positive frequencies, yielding
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(3) |
Conventions and Software
In the figures, outward currents (corresponding to an inwardly
directed flux of Cl) are defined as positive and displayed as upward deflections. Potentials are given as bath referenced to the
pipette. Numbers are given as mean ± SEM. Currents used for
amplitude and single-channel kinetic analysis were digitized and
analyzed using the Patch and Voltage Clamp Software v. 6.24 (Cambridge Electronic Design). For stationary noise analysis,
currents were digitized and Fourier transformed using the SPAN-Spectral/Variance analysis program (J. Dempster, University of
Strathclyde, Glasgow, UK). Polynomial regression and fitting of
multiple Lorentzians (Eq. 2) was performed using Origin 5.0 (Microcal Software Inc., Northampton, MA), which was also used
for preparing graphical displays.
Solutions and Chemicals
The standard extracellular NaCl Ringer contained (mM): 113 Na+, 117.7 Cl, 3.7 K+, 3 acetate, 10 glucose, 5 HEPES, 1 Ca2+, 1 Mg2+, pH 7.4. The standard intracellular solution, denoted I-28,
contained (mM): 15 Na+, 27.9 Cl
, 105 aspartic acid, 115 Tris, 5 HEPES, 1 EGTA, 1.1 Mg2+, 0.371 Ca2+, pH 7.2, 10
7 M free-Ca2+,
10
3 M free Mg2+. For measurement of conductance under symmetrical [Cl
], the intracellular solution denoted "120.6 mM
Cl
" contained (mM) 117.7 Na+, 120.6 Cl
, 5 HEPES, 1 EGTA,
1.1 Mg2+, 0.371 Ca2+, pH 7.2, 10
7 M free Ca2+, 10
3 M free Mg2+.
For measurement of halide selectivity, 100 mM Cl
was replaced
by 100 mM Br
or I
.
The pipette used for patch clamp contained NaCl Ringer to
which in most cases was added 1 mM quinine to block larger K+
channels of the apical membrane, which otherwise compromised the resolution in cell-attached patches. Quinine inhibits the macroscopic K+ secretion when the glands are stimulated, whereas
Ba2+ is ineffective (M.S. Nielsen, personal communication). Cl
channels obtained with quinine in the pipette did not differ noticeably (e.g., in conductance) from Cl
channels obtained in
the absence of quinine.
Isoproterenol, forskolin, IBMX (isobutyl-1-methyl-xanthine), ATP, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid), and dibutyryl-cAMP(db-cAMP) were from Sigma Chemical Co. (St. Louis, MO). NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) and glibenclamide were from Research Biochemicals, Inc. (Natick, MA). DPC (diphenylamine-2-carboxylic acid) was from FLUKA Chemie (Buchs, Switzerland). cAMP-dependent protein kinase, catalytic subunit, was from Promega (Madison, WI).
DPC was dissolved in 0.1 M NaOH and titrated to pH 7.8 with
HCl and H2SO4 to a total [Cl] of 24 mM and [DPC] of 20 mM.
NPPB and glibenclamide were dissolved at 0.1 M in DMSO. DIDS
was dissolved in water at a nominal concentration of 10 mM and
the dissolution was verified by measurement of the optical density
at 342 nm, yielding a concentration of dissolved DIDS of 9.44 mM.
Reference Electrode and Measurements of Liquid Junction Potentials
The reference electrode was connected to bath via an agar bridge made up of NaCl Ringer. When exchanging the solution in the bath for I-28, a well-defined liquid-junction potential appears at the agar bridge, making the potential across the patched membrane, VM:
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(4) |
where V2,1 is the potential of I-28 with respect to NaCl Ringer
(Neher, 1992). This potential was measured against a free-flowing 2 M KCl electrode according to Neher (1992)
, yielding V2,1 =
3.71 ± 0.05 mV (n = 6). All accepted measurements were reversible within 0.11 mV.
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RESULTS |
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Cell-attached Patches
Activation of channels.
In cell-attached patches obtained
on unstimulated (resting) gland cells, the application
of 2 µM isoproterenol induced activity by small Cl-selective channels in ~60% of the cases (20 of 34 patches)
(Fig. 2). Similar channels were induced by application
of 12.5 µM forskolin (14 of 31 patches) or by 0.5 mM
db-cAMP and 0.1 mM IBMX (12 of 22 patches). Fig. 2
shows the characteristic long bursts of activity upon
stimulation. The time-delay before the first burst of activity lasting at least 1 s was significantly shorter with isoproterenol (56 ± 7 s, n = 20, the "dead time" in the
perfusion system was 20-30 s and is included in the
time-delay) than with either forskolin (105 ± 12 s, n = 14; P < 0.001, Mann-Whitney U test) or db-cAMP and
IBMX (110 ± 13 s, n = 12, P < 0.005, Mann-Whitney U
test).
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Current-voltage relationship.
Fig. 3 shows the average
current-voltage relationship (A) of cells stimulated
with cAMP-inducing agents and an example of channel
gating (B) at different holding potentials. Note the
long bursts of activity and the increased number of
brief closings ("gaps") at hyperpolarized potentials,
which were seen consistently in cell-attached patches.
The current-voltage relationship saturated at negative
potentials (Fig. 3 A), and a cubic polynomium was required for an accurate description (ANOVA: linear and
quadratic regression, P < 0.001; cubic regression, P < 0.005; quintic regression, P > 0.10; 110 data points from
nine experiments). When considering only positive potentials, the current-voltage relationships showed no
significant deviations from linearity (ANOVA: linear regression, P < 0.001; quadratic regression, P > 0.50; 51 data points from nine experiments) and had a conductance of 10.0 ± 0.4 pS. The estimated reversal potential
was 1.2 mV (i.e., close to 0 mV), indicating that Vc ECl
(since Vrev = ECl
Vc). Thus, Cl
is apparently in equilibrium after activation by cAMP (see DISCUSSION).
Inside-Out Patches
Regulation of channels.
In most cases, channel activity
was lost upon patch excision into intracellular solution
(I-28) devoid of ATP. In 31 of 68 patches (patches with
and without activity in cell-attached configuration are
included), channel activity was noted during exposure
of the cytosolic side of the membrane to 1.5 mM ATP.
Channel activity was quantified in terms of NPo (N = number of channels, Po = open probability) by measuring mean macroscopic current I (leakage current subtracted), and dividing by the mean current through one channel, i (see Fig. 6, below) measured at the
same potential. The resulting activity measurements
show that channel activity was dependent on ATP after
excision to the inside-out configuration (Fig. 5 B, left).
Channel activity increased further in 11 of 16 cases
(only counting patches where unambiguous channel
activity was observed after excision) when the patch was
exposed to the catalytic subunit of cAMP-dependent
protein kinase (16-66 nM, corresponding to 50-200
casein U/ml) (Fig. 5, A and B). After phosphorylation,
the channels were active even in the absence of protein
kinase, as long as ATP was present at the inside of the
membrane. Removal of ATP, however, caused a reversible loss of activity (Fig. 5, A and B). This mode of regulation is identical to the dual phosphorylation/ATP dependence of the CFTR Cl channel (Tabcharani et al.,
1991
; Anderson et al., 1991
). No rundown of channel
activity was noted in the presence of ATP even after removing cAMP-dependent protein kinase. Thus endogenous protein phosphatase activity was apparently not
present in excised patches.
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Current-voltage relationship.
When bathed with intracellular solution and ATP, the channel reversed close
to the calculated reversal potential for Cl (Fig. 6). In
spite of the skewed Cl
concentrations in this case, the
current-voltage relationship showed no significant deviations from linearity (ANOVA: linear regression, P < 0.001; quadratic regression, P > 0.75; 158 data points
from 14 experiments). Linear regression yielded a conductance of 8.0 ± 0.1 pS and a reversal potential at
38.9 mV when corrected for liquid junction potential
(Eq. 4). Since the equilibrium potential for Cl
was
36.4 mV, no aspartate permeability was detected.
Substates. The channels displayed a characteristic substate (Fig. 7 A) in most patches. The amplitude of this substate was measured using Gaussian fits to all-points histograms (Fig. 7 B) and expressed as a fraction of the full amplitude. This measurement was feasible at positive holding potentials only, where amplitudes were high and activity levels stationary over a long period of time. The distribution of relative substate amplitudes from 25 patches showed a variable substate level, from 0.37 to 0.67, with predominance around or just below 2/3 of the full amplitude (Fig. 7 C). Thus, the substates did not reveal any obvious oligomeric organization of the channel, in which case the substates would be expected to assemble around levels that would constitute multiples of an integer fraction of the full amplitude (e.g., if three parallel pores were to form the functional channel, two peaks in the substate histogram would a priori be expected, at 1/3 and 2/3 of the full amplitude).
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Halide selectivity.
In inside-out patches bathed in 120.6 mM Cl (MATERIALS AND METHODS), the channels
(shown to be Cl
selective by a control period in I-28)
displayed a linear current-voltage relationship with a conductance of 10.3 ± 0.3 pS (five experiments). When
100 mM Br
substituted for Cl
in the bath, the current-voltage relationship was still linear, when considering either positive or negative potentials, however, the slopes were different (Fig. 8). To be able to estimate conductance at positive and negative potentials
(denoted
+ and
, respectively) together with the reversal potential, Vrev, the following equations were fitted to the data:
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(5) |
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Pharmacology.
Inside-out patches with stationary channel activity when exposed to I-28 and 1.5 mM ATP on
the inside were used for investigating the pharmacology of the channel. Putative blockers were added to I-28
in the presence of ATP and applied via the perfusion
system. Treatment of the channels with 100 µM NPPB
caused a decrease in current in single- or multichannel
patches (seven of seven patches investigated), apparently caused by brief interruptions of the open state
(Fig. 9). The block was relieved when the patch was perfused with control solution (I-28 and 1.5 mM ATP).
Treatment with glibenclamide (100-200 µM, effective
in nine of nine patches) or DPC (1 mM, effective in six
of six patches) likewise induced a partial and fully reversible block characterized by increased flickering
(Fig. 9). Especially in the case of DPC, it appeared that
the amplitude of the channel was also depressed, but
this could be caused by high frequency flickering beyond the bandwidth investigated (100 Hz in Fig. 9). Indeed, McCarty et al. (1993) reported that DPC block of
CFTR results in interruptions of the open state with a
time constant <1 ms, which would not have been resolved by our experiments.
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Single-channel kinetics. The kinetics of the channel was likewise investigated in inside-out patches exposed to I-28 and ATP and held at positive voltages. This yielded the most stationary channel activity, and the best signal to noise ratio. Two patches where only a single channel was observed were found to be of sufficient quality to allow analysis. The open time distribution was in both cases characterized by a single peak in a logarithmically binned histogram (Fig. 10 B), which could be satisfactorily fitted with a single exponential component, indicating a mean open time of 85.6 ms (Fig. 10 B) or 230 ms (data from the other patch, not shown). The closed time distribution exhibited two peaks and was fitted using a double exponential model. The fast time constant was 16.4 ms (7.6 ms in the other patch) and the slow one was 255 ms (193 ms in the other patch). Thus, a minimum kinetic model of the channel would comprise one open and two closed states.
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Analysis of stationary current fluctuations.
In single- or
multichannel patches, the kinetic analysis was extended
by calculating the one-sided power density spectrum (PDS) using Fast Fourier Transformation of the current traces. In each case, the PDS was initially fitted
with a single Lorentzian and a plateau-level and the fit
compared with the PDS data points. In 12 of 14 patches,
a second Lorentzian was required to obtain a reasonable fit. When fitting the second Lorentzian (Eq. 2), the parameters of the first (low frequency) Lorentzian
were fixed and only the second Lorentzian and the plateau-level, S, were allowed to vary. The fit of two
Lorentzians described the PDS well in the 12 remaining patches (Fig. 11). The fitted corner frequencies in
double-Lorentzian patches were 0.85 ± 0.11 Hz (n = 12) and 27.9 ± 2.9 Hz (n = 12) for the low and
high(er) frequency Lorentzian, respectively. Calculation of the band-unlimited current variance contributed by each Lorentzian by Eq. 3 revealed a ratio of
slow to fast component variance (
s/
f) of 16.1 ± 4.7 (n = 12), thus the low frequency Lorentzian contributed most of the noise.
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DISCUSSION |
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Function of Luminal Cl Channels in Secretion
According to the current model (e.g., Petersen, 1992;
Greger, 1996
), Cl
secretion is accomplished by the serial arrangement of a luminal conductance together
with a driving force for Cl
and a basal conductance
and driving force for K+. Activation of the luminal Cl
conductance drives Cl
out in the lumen. Due to the
electrical shielding of the lumen by the resistance of
the paracellular pathway to anion flow, this will cause a
lumen-negative potential, which will cause Na+ to follow paracellularly. In the frog skin glands, the mucosal potential drops close to zero after 30-60 min of stimulation, and it was recently suggested that in this state of
the gland, Na+ secretion is active, not passive (Ussing et
al., 1996
). However, in both models, the serial arrangement of batteries favoring Cl
and K+ efflux is assumed.
In the present study, we have succeeded in identifying an apically located conductance for Cl, consisting
of small 8-pS channels in the luminal membrane.
These channels were demonstrated to be activated by
the cAMP cascade on the following levels: activation of
-adrenergic receptors using isoproterenol, activation
of the adenylate cyclase using forskolin, direct elevation of cellular [cAMP] using a membrane-permeable cAMP analogue together with the phosphodiesterase
inhibitor IBMX, and finally activation of channels in
excised inside-out patches by exposure to the catalytic
subunit of cAMP-dependent protein kinase. We conclude that in the frog skin gland a CFTR-like channel-type mediates cAMP-dependent Cl
secretion.
It should be noted that we did not encounter any
other type of Cl channel in the luminal membrane on
a regular basis. Only in two to three patches obtained
in the beginning of the study was a very large Cl
channel (conductance 150-200 pS) seen. The identity, regulation, and function of this channel are unknown.
Thus, it would appear from the current data that a single population of Cl
channels is present in the apical
membrane to serve the function of Cl
secretion. This
is an important finding in view of the different types of
cells found in the mucous/seromucous glands of frog
skin: a (mucous/seromucous) cell type with secretory
granules, a cell type without granules but with pronounced amplification of the basolateral membrane,
and a mitochondria-rich cell type (Mills and Prum,
1984
). Either these cells have identical Cl
channels in
the apical membrane or, for whatever reason, we have only been patching one type of cells. Furthermore,
~50% of cell-attached patches did not activate when
stimulated with cAMP-inducing agents, thus it is possible that at least one cell-type could be devoid of cAMP-stimulated Cl
channels in the luminal membrane. Engelhardt et al. (1994)
reported that CFTR message and
protein was predominantly expressed in the mucous
cell type in Xenopus glands.
To achieve sustained secretion, active K+ channels
are assumed in the secretory model, the function of
which are to recycle K+ that enters the cell through the
basolateral Na/K/2Cl cotransporter and the Na/K-ATPase and further to provide a driving force for Cl by upholding the inequality Vc < ECl. In cell-attached
patches, this inequality would correspond to a reversal
potential for Cl
channels at positive potentials. Data
from unstimulated cells showed that the inequality was
satisfied in the resting state of the gland. In stimulated
cells, however, Cl
channels reversed at 0 mV applied
potential, meaning that Vc
ECl after activation by
cAMP-inducing substances. The same result was obtained in whole-cell patches (Sørensen and Larsen,
1997
). Thus, there is no apparent driving force for Cl
exit after stimulation, meaning that K+ channels were
not substantially activated. An alternative is that the
driving force is so low, probably <4 mV, that we cannot find it in our studies. In a study of basolateral K+ channels in the same kind of preparation, Andersen et al.
(1995)
found a "maxi" K+ channel of unitary conductance ~200 pS. However, this K+ channel had very low
basal open probability (0.018 ± 0.005), and a rise in cytosolic Ca2+ by muscarinic stimulation was required to
achieve activation. Therefore, in this gland type it
would appear that after stimulation by cAMP-inducing
substances the magnitude of the K+ conductance is limiting net secretion. This contrasts to the situation in
shark rectal gland, where cAMP-dependent stimulation causes depolarization to
63 mV (apical membrane
potential) where ECl =
48 mV (calculated from
Greger et al., 1984
) and where Cl
channel currents in
activated cells reversed at +25 mV applied potential
(Greger et al., 1987
). Likewise, in colon crypts, the stimulation by cAMP causes a primary depolarization
and a secondary hyperpolarization caused by activation
of cAMP-dependent K+ channels (Lohrmann and
Greger, 1993
). However, Becq et al. (1993b)
, using primary cultures of human tracheal gland cells, obtained results similar to ours, that small cAMP-activated Cl
channels reversed at 0-mV applied potential.
In the whole frog skin preparation, the glands can
be stimulated to stationary secretion by isoproterenol
(Thompson and Mills, 1983) or prostaglandin E2 (Bjerregaard and Nielsen, 1987
), which apparently acts exclusively on the cAMP pathway. This would seem to require activation of K+ channels. Thus, we cannot rule
out the possibility that the collagenase treatment and/
or isolation of the glands from the connective tissue
have impaired a K+ conductance in the basolateral
membrane.
Identity of the Small Luminal Chloride Channel
The small unitary conductance (8.3 pS), together with
the dependence of the channel on cAMP in cell-attached
patches and of protein kinase A-dependent phosphorylation in inside-out patches and the obligatory requirement for ATP in the phosphorylated state, constitute
the functional distinguishing properties for the CFTR-encoded Cl channel (Tabcharani et al., 1991
; Anderson et al., 1991
). As previously noted, the CFTR message and protein have been localized to the submucosal glands in Xenopus (Engelhardt et al., 1994
), and
the conclusion that the apical Cl
channel in the frog
skin glands is CFTR is, therefore, in our view unescapable.
Rectification and Conductance
Single-channel CFTR currents obtained in cell-attached
patches have been reported to rectify slightly in the
outward direction (Haws et al., 1992; Gray et al., 1989
;
Tabcharani et al., 1991
), which was originally ascribed
to the different Cl
concentrations present on the two
sides of the membrane. In the frog skin gland, Cl
currents saturated at negative potentials, giving the current-voltage relationship an outwardly rectifying appearance. When we fitted the Goldman-Hodgkin-Katz
(GHK) current equation to the data by allowing the intracellular Cl
concentration and membrane potential
to vary, the values obtained (fitted parameters were:
[Cl
]c = 46.4 ± 6.3 mM, Vc =
26.8 ± 4.2 mV, PCl = 3.3[±0.3] [10
14 cm3 s
1]) agree well with values obtained by other methods ([Cl
]c = 38.5 mM (mucous
cells), 43.6 mM (gland cells, electron microprobe technique, Mills et al., 1985
) and Vc =
28.4 mV (whole-cell patches, Sørensen and Larsen, 1997
). Nevertheless,
we are able to conclude that the rectification seen in
cell-attached patches cannot result from Goldman rectification based on the following observations. (a) The
current-voltage relationship when considering only
positive currents did not rectify but could be fitted with
linear regression. This is not predicted by the GHK
equation. (b) When fitting the current-voltage relationship in the inside-out configuration under conditions
of symmetrical Cl
concentrations (Fig. 8) with the
Goldman-Hodgkin-Katz current equation, the resulting estimated single-channel permeabilities was 2.2 × 10
14 cm3 s
1. Thus, the channel has much lower permeability in the inside-out configuration, compared
with that predicted from cell-attached measurements
(3.3[±0.3] × 10
14 cm3 s
1). (c) In inside-out patches
exposed to a larger Cl
concentration gradient (inside:
25 mM, outside: 118 mM) than experienced in cell-attached patches (inside: ~40 mM, outside: 118 mM),
the current-voltage relationship was linear.
By the use of dialyzed whole-cell patches (Overholt et
al., 1993, 1995
) or excised inside-out patches (Linsdell
and Hanrahan, 1996a
; Linsdell et al., 1997
) it has been
demonstrated that CFTR currents under a range of
conditions do not obey the GHK equation, but that excess outward rectification results from a voltage-dependent block by the anions (gluconate, glutamate) usually substituted for Cl
in making up intracellular-like
solutions. It was suggested that during cell-attached recordings outward rectification could result from both
the Cl
concentration gradient and from block by large
intracellular anions (Overholt et al., 1993
; Linsdell and
Hanrahan, 1996a
). In our case, we fail to find any evidence of GHK behavior. Instead, we suggest that rectification in cell-attached patches is due solely to a voltage-dependent block. This block can apparently not be
mimicked by aspartate, which was used at the inside of inside-out patches in the present study.
The conductance of the frog CFTR channels was
found to be 10.0 pS for outgoing currents obtained in
cell-attached patches, 8.0 pS when bathed with 28 mM
Cl on the inside, and 10.4 pS in symmetrical 125 mM
Cl
. These are higher than the typical values reported
for mammalian CFTR (4-8 pS, Gray et al., 1990
; Larsen
et al., 1996
; Price et al., 1996
; Linsdell and Hanrahan,
1996a
; Tabcharani et al., 1997
), but agrees with measurements on Xenopus CFTR under conditions of high
cytosolic Cl
(9.6 pS, Price et al., 1996
).
Halide Selectivity
Experiments on halide selectivity showed that even
though PBr/PCl > 1, the conductance when Br is permeating the channel is only half of that when the permeating anion is Cl
. Tabcharani et al. (1997)
using
human CFTR expressed in CHO cells obtained the
same result. Qualitatively similar results were obtained by I
substitution, but in this case the conductance for
I
was not measurable. These data are in agreement
with previously reported results obtained by cytosolic I
substitution in human CFTR (Tabcharani et al., 1992
).
However, more recently it was reported that two states
exist in the human CFTR Cl
channel: one (Iunbl) that
has simultaneously PI/PCl > 1 and GI/GCl > 1, and another (Ibl) that has PI/PCl << 1 and GI/GCl < 1 (Tabcharani et al., 1997
). This is not in agreement with our
data, which shows PI/PCl > 1 and GI/GCl << 1. However, for Xenopus CFTR, Price et al. (1996)
showed that
in the whole-cell situation the permeability for I
is
higher than for Cl
, whereas the conductance is less;
this was ascribed to a difference in amino acid sequence in the first and second membrane-spanning domain between human and Xenopus CFTR.
The results on conductance, rectification, and halide
selectivity when taken together are consistent with the
idea that two or more anion binding sites are present at
the cytosolic side of the channel (Linsdell et al., 1997).
Binding of anions other than Cl
to these sites may
cause rectification as seen in cell-attached patches and
when substituting Br
for Cl
. The intracellular Cl
concentration may in turn affect the conductance even
for outgoing currents; i.e., for an inwardly directed Cl
flux. Thus, a lower conductance for Cl
was observed in
inside-out patches exposed to 25 mM Cl
on the inside
(Fig. 6) than in cell-attached patches (measured intracellular Cl
concentration: 38-44 mM, Mills et al.,
1985
) or when exposed to 120 mM Cl
on the inside
(Fig. 8). Finally, a higher affinity at the first binding site
for a weakly permeant ion (Br
, I
) may cause the reversal potential to shift in favor of that ion, even when
the ion is not or barely able to carry any current
through the pore itself.
Pharmacology
The pharmacology of CFTR is usually described as
block by one or more of the compounds NPPB, DPC,
or glibenclamide, and insensitivity to DIDS. In the
present investigation, we found that NPPB, DPC, and
glibenclamide added to the cytoplasmic face of inside-out patches caused a partial block characterized by increased flickering, suggesting an interaction with the
open channel. This is consistent with investigations on
the exact mode of interaction of these blockers with
CFTR (DPC: McCarty et al., 1993; glibenclamide: Schultz
et al., 1996
; Sheppard and Robinson, 1997
). However,
we also found that DIDS caused an irreversible block of
the channels when added to the cytosolic side of inside-out patches. This was an unexpected finding, since
DIDS sensitivity is usually ascribed to the outwardly rectifying chloride channel (Bridges et al., 1989
) and, indeed, DIDS insensitivity is widely used as a distinquishing property of CFTR in whole epithelia (Shen et al.,
1995
), in whole-cell patches (Schwiebert et al., 1994
),
and when making single-channel measurements (Jovov
et al., 1995
). In most cases, DIDS has been added to the
extracellular side of the membrane (e.g., Gray et al.,
1990
; Schwiebert et al., 1994
; Shen et al., 1995
; Larsen et al., 1996
). Some investigators, however, have used a
protocol similar to ours (i.e., adding DIDS to the cytoplasmic side of inside-out patches or to both sides of reconstituted CFTR) and they failed to see any effect
(Egan et al., 1992
; Becq et al., 1993a
; Jovov et al., 1995
).
We have obtained evidence from the frog skin gland
that DIDS is ineffective at concentrations up to 500 µM
when added to the outside of outside-out patches (data not shown). Linsdell and Hanrahan (1996b)
reported
that, when added to the inside, DIDS was able to block
macroscopic CFTR currents recorded from excised
patches of stably transfected Chinese hamster ovary
cells. Even though they did not comment on reversibility, the block described by Linsdell and Hanrahan
(1996b)
was voltage dependent and would therefore
probably involve a reversible interaction with the channel pore. Further, they state that DIDS caused brief interruptions of the channel's open state, again indicating reversibility. In the present study, the block was irreversible and, within seconds, the currents were blocked
completely, even in multi-channel patches with many
active channels. Thus, apparently, this is a phenomenon distinctive from that described by Linsdell and Hanrahan (1996b)
and more resembling the block of the
outwardly rectifying Cl
channel (Bridges et al., 1989
).
![]() |
FOOTNOTES |
---|
Address correspondence to Jakob Balslev Sørensen, August Krogh Institute, Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark. Fax: +45 3532 1567; E-mail: JBSorensen{at}aki.ku.dk
Received for publication 29 December 1997 and accepted in revised form 26 March 1998.
2 The nonresponsive patch also did not respond to removal of ATP with a decreased channel activity; thus, apparently, the channels or the patch configuration were unusual.This study was supported by the Danish Natural Science Research Council (grant 11-0971) and the Carlsberg Foundation.
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Abbreviations used in this paper |
---|
ANOVA, analysis of variance; CFTR, cystic fibrosis transmembrane conductance regulator; GHK, Goldman-Hodgkin-Katz; PDS, power density spectrum.
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