PHYSIOLOGICAL ROLE OF APICAL POTASSIUM ION CHANNELS IN FROG SKIN BY WILLY VAN ...

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J. Phy8"l. (1984), 356,pp. 79-95 79
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PHYSIOLOGICAL ROLE OF APICAL POTASSIUM ION CHANNELS
IN FROG SKIN
BY WILLY VAN DRIESSCHE
From the Laboratorium voor Fy8iologie, Campus Gasthuisberg,
B-3000 Leuven, Belgium
(Received 13 March 1984)
SUMMARY
1. TheK+permeability oftheapical membraneoffrogskin (Ranatemporaria) was
analysed by recording the short-circuit current and its fluctuations in the presence
ofa mucosa-to-serosa-oriented K+ concentration gradient.
2. Loading of the animals with KCl resulted in an augmentation of the Ba2+-
blockable component of the short-circuit current and the plateau value of the
K+-dependent relaxation noise.
3. Poisoning ofactive transport and exposing both sides ofthe epithelium to KCl
Ringer solution caused an increase ofthe K+ current and its fluctuations recorded
after restoring the inward-oriented K+ gradient.
4. Serosal quinidine (5x 10-4 M), which is thought to increase intracellular Ca2+
activity, depressed the K+ current and the relaxation noise. This effect was
completely reversible.
5. Removal of Na+ from the serosal solution, which is known to result in an
elevation of intracellular Ca2+ by abolishing the driving force for the Na+/Ca2+
exchanger, alsoreducedtheK+currentandtheLorentzianplateau. Bothparameters
returnedtotheircontrolvaluesafterrestoringtheNa+gradientacrossthebasolateral
membranes.
6. It is concluded from these experiments that the apical K+ permeability is
controlled by factors which depend on the intracellular K+ and Ca2+ concentration
and that the apical K+ channels may constitute a pathway for K+ secretion.
INTRODUCTION
Previous investigations showed that the apical membrane of frog skin (Rana
temporaria) has a significant K+ permeability (Zeiske & Van Driessche, 1979). This
was demonstrated by recording the transepithelial short-circuit current and by the
analysis ofits fluctuations: in the presence of-a K+ gradient oriented from mucosa
to serosa, an inward K+ current was recorded and the spectrum ofthe fluctuations
in current contained a Lorentzian component. The latter observation indicates that
the K+ channels open and close randomly (Van Driessche & Zeiske, 1980a). The K+
currents are blockable by mucosal Cs+ and Ba2+ and additional fluctuations in
current were recorded in the presence of the latter cation. The analysis of these
blocker-inducedfluctuationsallowedtheestimationofthesingle-channel currentand
80 W. VAN DRIESSCHE
channel density (Van Driessche &Zeiske, 1980b). The K+ channels permeable for
are
different 'K+-like' cations (Rb+, NH + and Tl+), and the affinity ofthe K+-channel
blocker, Ba2+, strongly depends on the nature of the permeating species (Zeiske &
Van Driessche, 1983). With all these observations we were able to establish a picture
ofthefactorswhich determine the molecular characteristics ofthe apical K+ channel.
However, untilnowwewereignorantabout possiblephysiological functionforthese
a
K+ channels.
Itwaspreviously observed by ourselves (Zeiske & Van Driessche, 1979) and others
(Nagel & Hirschmann, 1980) that not all frogs had a skin with a measurable K+
permeability. SomebatchescontainedveryfewfrogswithaK+-permeable skin, while
in others the majority displayed a significant K+ permeability. The cause for this
difference among frogs could not be resolved until now. In this paper experiments
whichweredesigned toresolvetheseproblems described. Thehypothesis that
are was
the apical K+ channels participate in the K+ homoeostasis ofthe frog by promoting
K+ secretion when the animals experience large K+ uptake, after eating lot
a e.g. a
of insects. The results show that treatments of the preparation such that the
intracellular K+ and/or Ca2+ concentrations can be expected to be altered, give rise
to modifications ofthe K+ permeability.
METHODS
Frogsofthe speciesRanatemporariawereobtainedfrom Stein (Lauingen-Donau, F.R.G.). They
werekeptinplastic containers at an ambient temperature of17 0C. The containers hadareservoir
of101 tapwater, whichwasrefreshedeveryday. Theanimalswerefedmealwormsonadrysurface
ofthe containers. Three days before the experiments, some ofthe frogs which were used for the
K+-adaptationstudiesweretransferredfromthiscontainerintoasmallertank.Thistankcontained
distilledwaterinwhich 15mM-KCl wasdissolved. In this container the animals were continuously
in contactwith the solution. During this period they were not fed.
On the day of the experiment the animals were double pithed and the abdominal skins were
mounted in modified 'Ussing-type' Lucite chambers as described before (Van Driessche & Erlij,
1983). Theareaofskinincontactwiththesolutionwas0-4cm2. Thevoltageandcurrentelectrodes
consisted of Ag/AgC1/1M-NaCl electrodes connected to the solutions by1 M-NaCl-filled agar
bridges. Both chamber compartments were continuously perfused. In order to avoid microphonic
noise, the perfusion was interrupted during the periods where the noise data were collected. The
transepithelial current and voltage were recorded continuously on an X-T recorder. The
transepithelial conductance was measured by recording the current changes caused by the
application ofvoltage pulses of1-5 sduration.
Theelectronic equipment used to record the transepithelial currents, the fluctuation in current
andthemembrane conductance wasthe same asthatdescribedpreviously (Van Driessche & Erlij,
1983). However, the field-effect transistors (2N6451, National Semiconductors) which we used
previously in the input stage ofthe low-noise voltage clamp (Van Driessche & Lindemann, 1978)
were replaced by a newer type (SK146, Toshiba Corp., Tokyo, Japan). The reason for this
replacementwastheimprovednoisecharacteristicsofthelattertype.Especially, theamplifiernoise
atlowerfrequencies was much smaller.
The fluctuations in current were recorded and analysed with an SBC 86/12A and SBC 80/30
(INTEL,SantaClara,U.S.A.)multiprocessorcomputersystem.Theaveragedandfinalspectrawere
transmitted toa PDP11/34 computer andstored on aharddisk. The fluctuations in current were
analysed in one ofthe following frequency ranges: (1) frequency range 1 was identical to the one
usedin apreviouspaper (Van Driessche & Erlij, 1983), and covered the range0-2-670 Hz; (2) the
fundamental and maximal frequency were1 and 900Hz, respectively, forfrequency range 2. The
finalspectraanalysedwithfrequencyrange1 consistedofthreemergedspectrawhichwererecorded
simultaneously,whilethoseanalysedwithfrequencyrange2werethesuperposition oftwospectra.
APICAL K+ CHANNELS INFROGSKIN 81
It has been demonstrated for different membranes and several channels that the spectrum of
the fluctuations of the current through channels which open and close randomly contains a
Lorentziancomponent. ThespectraldensityofsuchaLorentziancomponentreachesaplateau(SO)
inthelowerfrequencylimitanddecreasesby6dB/octaveatthehighestfrequencies.Thefrequency
where the spectral density is 3dB below SO is called the corner frequency (fe). The frequency
dependence ofthe powerdensity, SL(f), fulfils the followingequation:
SLWf) = '+(f/f )2' (1)
wherefrepresents the frequency. The presence ofsuch a Lorentzian component in the spectra
recorded with KCl and NaCl Ringer solution on the mucosal and serosal sides, respectively, was
usedasacriterionfortheexistenceofameasurableamountoffluctuatingK+channelsintheapical
membrane. Themainadvantage ofthiscriterionisthatinoursystem, thefluctuatingK+currents
are selectively recorded in the power spectrum: they give rise to a Lorentzian component with a
fcof80Hz,whilefluctuationsofbackgroundcurrentsproducemostlylow-frequencynoise(G6gelein
& Van Driessche, 1981). Themaindisadvantage ofthiscriterion isthattheplateauvaluedepends
notonlyonthechanneldensity(M)butalsoonthesingle-channelconductance,theelectrochemical
gradient across the apical membrane and on the rate constants ofthe open-close reaction (Van
Driessche & Zeiske, 1980a). So, using the presence ofa Lorentzian in the spectrum as a criterion
forasignificant K+conductance impliesthatweassumethattheelectrochemicalgradientandthe
rateconstantsdoindeedremain constantduringthedifferentphysiological alterationsusedinthe
experiments.Therefore,wealsorecordedtheK+current(IK)throughtheepithelium.Therecording
of these currents is hampered by the presence of shunt currents which might, under certain
circumstances become larger than IK. Therefore, we defined the IK as the amount of current
blockable by2 mM-mucosal Ba2+. Frompreviousexperiments (Van Driessche & Zeiske, 1980b) we
know that 60,uM-mucosal Ba2+ blocks 50% of IK. Thus, assuming simple Michaelis-Menten
kinetics, 97% ofthe K+ channels should be blocked at2mM-mucosal Ba2+. Nevertheless, the K+
currents also depend on the above-mentioned parameters as SO does, although in a different way
(see eqns. (3) and (4) in the Discussion). So the IK data have to be considered as information
complementary to the noise data.
As described in previous papers (Van Driessche & Zeiske, 1980a, b), most spectra contained,
besides the Lorentzian noise component, a low-frequency noise component (SB) which was
dominant in the low-frequency range. Its dependence on the frequency fulfils the following
equation:
SB(f) A/f', (2)
=
whereArepresentsthepowerdensityatafrequencyoft Hzandatheslopeinadouble-logarithmic
representation. On the other hand, in the high-frequency range the amplifier noise surpassed the
fluctuations of the K+ current. Due to the capacitance of the membrane, the amplifier noise
increasesbecauseofthedecreaseofthe membraneimpedance (VanDriessche& Gullentops, 1982).
AnestimationoftheLorentzianparameters(fcandSO)wasobtainedfromanon-linearregression
(Van Driessche & Zeiske, 1980b) ofthe following equation to the data points: ST = SL+SB. The
datapointsinthehighestfrequencyrangeofthespectrum(wheretheamplifiernoisewasdominant)
were omitted in these calculations.
Solution-. Thetissueswere bathed with Ringersolutionswithdifferentionic compositions. NaCl
Ringer solution contained (in mM): NaCl, 115; KHCO3, 2-5; CaCl2, 1. KCl and choline chloride
Ringersolutionscontained 115 mM-KCland 115 mM-cholinechloriderespectively, insteadofNaCl.
The choline solutions were alwaysfreshly prepared on the day ofthe experiment. To measure the
IK, Ba2+ (2 mM) was added as chloride to the KCI Ringer solution. Quinidine was obtained from
Sigma (St. Louis, U.S.A.) and dissolved to give a concentration of5x 10-4 M in the NaCl Ringer
solution used asserosal medium. Ouabain waspurchasedfrom Sigma (St. Louis, U.S.A.) and used
ataconcentration of2x 10-5 Min the serosal solutions. All studiesweredoneatroomtemperature
with air-saturated solutions at a pH of8-0.
82 W. VAN DRIESSCHE
RESULTS
Four types ofexperiments were done to modify the K+ permeability offrog skin.
Wetriedtocorrelatethesechangeswithalterationsofintracellularionconcentrations.
In the first two parts of the Results section, experiments are described in which
changesinintracellular K+ concentration couldbe considered asprimarytriggersfor
alterations in the apical K+ permeability. On the other hand, in the two following
typesofexperimentintracellularCa2+ concentrationsweremanipulatedandfollowed
by alterations in the K+ permeability, as judged from the alterations in relaxation
noise amplitudes and the Ba2+-blockable K+ currents.
(A) Exposure ofthefrogs to KCI-containing solutions
It has been shown for other K+-transporting epithelia (distal portions of the
nephron and large intestine) that K+ secretion is augmented after chronic dietary
potassium loading (Hayslett & Binder, 1982). It might be that the large variability
in K+ permeability among the frogs is also related to a difference in K+ load on the
animals. Therefore, in analogy to studies byothers inseveral otherepithelia (Fisher,
Binder & Hayslett, 1976; Silva, Brown & Epstein, 1977; Hayslett & Binder, 1982),
we attempted to augment the K+ permeability by exposing the animals to high-
K+-containing solutions and expected a larger K+ intake. Two groups of frogs
composed ofa random selection ofanimals from the same batch from the supplier
were subjected to two different treatments. (1) The first group (ten frogs) was kept
under normal conditions and had access to normal tap water till the day of the
experiment (see Methods). (2) Theexperimental group ofanimals (sixteen frogs) was
transferred into separate containers three days before the experiments. In these
containers the animals were continuously in contact with 15 mM-KCl solution. The
skinsofbothgroupsweremountedinthechambersandcontinuouslyshort-circuited.
Both sides ofthe epithelium were exposed to NaCl Ringer solution for45 min. After
this equilibration period, all mucosal Na+ was isotonically replaced by K+. In the
presence of this mucosa-to-serosa-oriented K+ concentration gradient, the K+
permeabilitycanbemonitoredbyrecordingthenoisespectrumandtheBa2+-blockable
component of the short-circuit current (see Methods). From the ten frogs in the
control group, only one skin showed a measurable K+ conductance, i.e. only for this
skin could we fit a Lorentzian curve to the data points. For this experiment, the
Lorentzian parameters were: SO = 1-08 x 10-21A2s/cm2 andf, = 76-2 Hz. Moreover,
this was the only skin ofthe control group in which a significant K+ current could
berecorded: IK = 2-8#sA/cm2. InthenineotherskinsaLorentziancomponentmight
have been present in the spectrum, but the plateau would have been so small that
the low-frequency noise and amplifier noise dominated over the entire frequency
range. Also, the Ba2+-blockable K+ currents were small. The mean value calculated
from these nine skins was: IK = 1 200+±69#uA/cm2. The experiments with the
animals ofthe experimental group showed that skin offourteen out ofthe sixteen
frogs had ameasurable K+permeability. The mean valuesforIK and the Lorentzian
parameters calculatedfrom these fourteenexperimentswere: IK = 7-3+1F1 ,A/cm2,
SO = 3-9+14x 10-21 A2s/cm2 andf, = 76-7+3-8 Hz.
These results suggest that the in vivo load of the frogs with KCl elicits an
APICAL K+ CHANNELS INFROG SKIN 83
augmentationofthe K+ permeability oftheapical membrane. Whetherthis K+ load
causesanincreaseoftheintracellularK+concentrationisstillunknown.Consequently,
the final mechanism responsible for the increase ofapical K+ permeability could be
anincreaseoftheintracellularK+ concentration, themodification oftheintracellular
concentrationofotherionslikeCa2+,alterationoftheintracellularATPconcentration,
or the modification ofsome other metabolic or hormonal factor.
(B) In vitro ouabain-KCI treatment
Previously, we reported (Van Driessche & Zeiske, 1980a) that the apical K+
permeability was depressed after abolishing active transport with ouabain. In the
present series ofexperiments the same observation was made in many experiments,
although in others an augmentation of the Lorentzian plateau and IK could be
observed. The short-circuit current recorded during this treatment is shown in
Fig. 1A. After an equilibration period of45 min, during which the skin was exposed
to NaCl Ringer solution on both sides, the K+ permeability was tested by recording
thenoisespectraandIKinthepresenceofamucosa-to-serosa-oriented K+gradient. In
theexperiment shown in Fig. 1 a Lorentzian component was present inthe spectrum
(So= 0-75 x 10-21 A2s/cm2), and the Ba2+-blockable current was only 0-4#A/cm2
when the skin was exposed to KCl and NaCl Ringer solution on the mucosal and
serosal sides, respectively. In the presence ofthese solutions, 2 x 10-5 M-ouabain was
added to the serosal medium. After the addition of ouabain, the transepithelial
current increased slowly and reached a peak after about 30-40 min and decreased
again. Concomitantly, the transepithelial conductance increased considerably. After
50 min exposure to ouabain, the K+ permeability was tested by recording theIK and
noise. In five experiments out often, the IK as well as the Lorentzian plateau were
considerably augmented (see Fig. 1). In the five other experiments the Lorentzian
component remained below background noise levels and theIK did not increase (not
shown). After ouabain treatment we should expect a decrease ofIK and So, ifthe K+
permeabilityremainedunchanged. Becauseoftheelimination oftheelectrogenic Na+
transport across the basolateral border, the electrical driving force for K+ across the
apical border should be reduced (Helman, Nagel & Fisher, 1981). On the other hand,
the intracellular K+ concentration could be smaller after ouabain treatment, due to
K+ loss in exchange for Na+. In the present experiments the mucosal compartment
wasNa+free, sotheNa+entryshouldoccurthroughthebasolateral membranewhich
has only a small Na+ permeability. Consequently the loss ofintracellular K+ should
be hampered by the limited Na+ entry. On the other hand, intracellular K+ should
be kept high because ofK+ entry through the apical border. So, it is likely that the
increase inIK and So is not only a consequence of an increased driving force but is
at least partly due to an augmented K+ permeability ofthe apical membrane. After
60 min of ouabain treatment, all serosal Na+ was replaced by K+ for 30 min. The
purpose of this exposure of both sides of the skin to high K+ was to increase the
intracellular K+ concentration. After switching back to NaCl Ringer solution on the
serosal side we always recorded largerIK and So values than before basolateral K+
depolarization (see Fig. 1). Also, in the five skins in which ouabain alone did not
increase theIK and the relaxation noise, the exposure to serosal KCl raised IK to
4-1+ 1-7,sA/cm2 and.So to 2-1+ 1-3 x 10-21 A2S/cm2. The result ofan experiment in
84 W. VANDRIESSCHE
._
A 9 Ba2+ K+
IBa2+, v X -IT
Na+ Ba2+
-6
0 40 80 120
Time (min)
B
10o-16-
10-17
10-18
~~~4
'V
E i'
2
0~
10-21
*.
lo-22
100 101 102
Frequency (Hz)
Fig. 1A andB. Forlegend see opposite.
APICAL K+ CHANNELS INFROG SKIN 85
C
10-18
10-21
;l7o-211~-~ 4~ ~ ~ v\
E~~~~~~~~
1001°0 102 103
Frequency (Hz)
Fig. 1. Response offrog skin to KCl-ouabain treatment. Under control conditions, the
skinwasexposedtoNaCiRingersolution (serosalside)andKClRingersolutionmucosall
side). The current deflexions in A are caused by transepithelial voltage pulses of 1-5s
duration and 3mV amplitude. A (current recording) and B (noise spectra) are data
obtained from the same experiment. The spectra were recorded with frequency range 1
(see Methods). In this experiment the skin had a small, though measurable, K+
permeability: asmall Ba2+-blockable current (A) and a Lorentzian component (B) were
recorded. AftertestingtheK+permeability (spectrum 1),ouabain (2x 10-6M)wasadded
to the serosal solution. The transepithelial current generally reached a maximum after
about 50min. The irregularities in the recording of the current before and after the
maximumaregenerallypresent.Intheexperimentshown,theBa2+-blockablecurrentwas
largerafter 1 hofouabainpoisoning thanundercontrol conditions. Also, the Lorentzian
plateau was higher after ouabain (spectrum 2). After exposing the serosal side to KCl
Ringer solution the K+ permeability was assessed again (spectrum 4). With KCl Ringer
solutiononbothsides,thecurrentfluctuationsareveryclosetotheinstrumentationnoise
(spectrum 3). Cshows the spectra recorded in another experiment before and after the
same ouabain-KCl treatment. For clarity, the intermediately recorded spectra are
omitted.InthiscasenosignificantK+permeabilitywasrecordedundercontrolconditions.
Aftertheouabain-KCItreatmentaLorentziannoisecomponentaswellasaBa2+-blockable
current of8-5 sA/cm2 wasrecorded.
which no Lorentzian component was recorded under control conditions is displayed
in Fig. 1C. After the ouabain-KCl treatment the relaxation noise clearly emerged
above the background noise.
As pointed out above, it is not clear how the intracellular K+ will change after
inhibition of active transport under the conditions used. Nevertheless, it can be
expected that the intracellular K+ concentration will increase when the skin is
exposedtoKClRingersolutiononbothsides. Asthistreatmentcauses (afterouabain
poisoning) an increase of the relaxation noise amplitude, we suggest that in these
experiments, the intracellular K+ concentration acts as the primary trigger for the
increase ofapical K+ permeability. Nevertheless, it cannot be excluded that other
86 W. VANDRIESSCHE
intracellularionsalsomodulatetheapicalK+permeability.Ithasbeenshowninother
preparationsthatsomeK+-channeltypesareactivatedbyCa2+.However,asweknow
fromprevious studies (Zeiske & Van Driessche, 1983), the apical K+ channels infrog
skin do not resemble the Ca2+-activated K+ channels, e.g. they are not blockable by
tetraethylammonium (TEA) (Van Driessche & Zeiske, 1980a). In spite of this
negativeargument,weperformedtheexperiments, describedinthenexttwosections,
in which intracellular Ca2+ was altered.
(C) The effect ofquinidine
Quinidine has beenusedextensively to study iontransport indifferent tissues. Its
effect on muscle contraction has been known for many years (Isaacson & Sandow,
1967), this effect having been attributed to an increase of intracellular Ca2+
concentration. Quinidine is thought to inhibit mitochondrial Ca2+ uptake, thus
causinganincreaseofcytosolicCa2+activity(Carvalho, 1968;Batra, 1976). Recently,
it has been shown that the addition ofquinidine to the serosal medium (4x 10-4 M)
oftheisolatedurinarybladderimpairsNa+transportthroughthisepithelium (Taylor
&Windhager, 1979; Windhager& Taylor, 1983;Arruda, 1983).ThereductionofNa+
transport is associated with an increase ofCa2+ release (Arruda & Sabatini, 1980).
It was therefore suggested that quinidine causes a release ofCa2+ from intracellular
organelles, thus increasing cytosolic Ca2+ concentration which is responsible for the
reduction ofNa+ transport.
We did six experiments in which quinidine (5x 10-4 M) was added to the serosal
bathingsolutionwhich consistedofNaCl Ringersolution, whilethe mucosalsidewas
bathed with KCl Ringer solution. The results ofa typical experiment are shown in
Fig. 2. In the experiment shown, the Ba2+-blockable short-circuit current was
3 ,uA/cm2 under control conditions and the spectrum of the current fluctuations
contained a Lorentzian component. After addition ofquinidine, the Ba2+-blockable
currentdecreasedwithinabout 16 mintolessthan30% ofitscontrolvalue (Fig. 2A),
and the plateau ofthe Lorentzian componentwasdepressedto the backgroundnoise
level (Fig. 2B). After washing out quinidine from the serosal medium, the Ba2+-
blockable current increased slowly and reached the control level after 60 min. Also,
the Lorentzian noise increased after the removal of quinidine and the plateau
increased to the control value in about 48 min. As can be seen from the current
deflexionscausedbythevoltagepulses,thetransepithelialconductanceisirreversibly
increased by quinidine, an observation which remains inexplicable.
From these experiments we conclude that quinidine impairs the apical K+
permeabilityoftheskin.WhetherquinidinedirectlyaffectstheapicalK+conductance
or acts via the augmentation ofintracellular Ca2+ concentration cannot be decided
fromtheseexperiments.Therefore,weattemptedtomanipulatetheintracellularCa2+
concentration in a different way, by removal ofNa+ from the serosal solution.
Fig.2.EffectofserosalquinidineontheapicalK+permeability.A,transepithelialcurrent
recordedbefore, duringandafterwashingwithquinidine. B, spectrarecordedbeforeand
after addition of quinidine. C, spectra (frequency range 2) recorded after wash-out of
quinidine. Mucosal solution, KCl Ringersolution; serosalsolution, NaCI Ringersolution.
The transepithelial conductance was measured with voltage pulses of 3 mV. The K+
current was recorded by adding 2 mm-Ba2+ to the mucosal solution.
APICAL K+ CHANNELS INFROG SKIN 87
A ±5x101 M-quinidine
*+2mM-Ba21
m m mr-_
5.
E
U
2 2-5-
:1
0 25 50 75 100
Time(min)
B
10-20
E
ce
10-21
Frequency (Hz)
10-19
C
,min
10-20
A
E
min
C a
coo
0min
10-21
I I A.I.A1 .A .a A aA IA . . A . . A.tA.....
100 101 U02 103
Frequency (Hz)
Fig. 2. Forlegend see opposite.
88 W. VANDRIESSCHE
A Na+ choline Choline Na+ Ba2+
- -
8
1- I 1
E
Q- 4,
_S
0 0 1 I I
0 15 30 45 60
Time(min)
B
a
a
a
a
g;- io-'9
E
Control
c 10-20 After
During
4
4+44+-+
10-21
N.
N040:4+1,
... l11i00 ..a..Ia1II0*.i. a a I a1..1-.8.102 I.-;-;- ..sAA103
Frequency (Hz)
Fig. 3. Effect ofreplacing all serosal Na+ by choline. A, transepithelial current. B, noise
spectra (frequency range 1) recorded before, during and after replacing Na+ by choline.
Mucosalsolution, KCl Ringersolutiontowhich2mM-Ba2+wasaddedtomeasuretheK+
current. The transepithelial conductance was measured with voltage pulsesof3 mV.