Am J Physiol Regulatory Integrative Comp Physiol
279: R1647–R1658, 2000.
Potassium channels in primary cultures of seawater fish
gill cells. I. Stretch-activated K⫹ channels
C. DURANTON, E. MIKULOVIC, M. TAUC, M. AVELLA, AND P. POUJEOL
Unité Mixte de Recherche 6548, Centre National de la Recherche Scientifique,
Université de Nice-Sophia Antipolis, 06108 Nice Cedex 2, France
Received 10 December 1999; accepted in final form 26 May 2000
mechanosensitive K⫹ channels; patch clamp; gill epithelium
Cl⫺ accumulation above its equilibrium leads to Cl⫺
transport across the apical membrane via small conductance Cl⫺ channels sensitive to cAMP. It is now
well established that transepithelial Cl⫺ secretion is
also dependent on K⫹ channel activity. These channels
are required for the control of membrane potential and
also to allow K⫹ ion recycling across the basolateral
membrane. Moreover, K⫹ channels could also be involved in cell volume regulation. It was therefore postulated in the present study that K⫹ channels in gill
cells could be of significant physiological importance in
the control of ionic homeostasis in fish. For these reasons we have undertaken the study of K⫹ channels in
primary cultures of sea bass gill cells in an attempt to
elucidate mechanisms of transepithelial ion transport.
MATERIALS AND METHODS
Animals
Sea bass (Dicentrarchus labrax) weighing 50.5 ⫾ 7.9 g (n ⫽
14 for patch-clamp experiments) were obtained from a local
fish farm (Cannes Aquaculture, Cannes, France). The fish
were kept in 1-m3 tanks containing seawater from the Mediterranean sea (36 g/l salinity) in a semi-open circuit configuration (water completely renewed every 6 h). The fish were
kept in water maintained at ambient temperature (16–17°C
over the course of the year) and were exposed to a natural
photoperiod.
Primary Culture of Gill Cells
that primary cultures
of respiratory cells of the sea bass (Dicentrarchus labrax) gill serve as a good model for studying Cl⫺ secreting epithelia (2, 10). This model could also help to
clarify the mechanism of ion balance regulation in
marine teleosts. Taken together, the results of previous
studies (2, 10) have permitted functional characteristics of this cell model to be more precisely determined.
In this way, the Na⫹-K⫹-ATPase pump, by maintaining low intracellular Na⫹ and Cl⫺ levels, provides the
driving force for Cl⫺ entry into the cell through the
basolateral membrane via Na⫹-K⫹-2Cl⫺ cotransport
and the Cl⫺/HCO3⫺ exchange mechanism. Intracellular
Conditions for the primary culture of gill cells were described previously by Avella et al. (1). Briefly, the following
steps were performed at 18°C in an air-conditioned room.
Before cell culture preparation, fish were held 2 h in a 10-liter
tank of aerated seawater containing the antibiotics and fungicides furaltadone (0.02%; Sigma) and temerol (0.02%; Francodex, Carros, France). Fish were killed by a blow to the
head, then decapitated, and the gills were excised and
weighed.
The following procedures were performed in sterile conditions under a laminar flow hood. The gill arches (cartilaginous part) were removed, and the remaining filaments (primary lamellae) were dipped in the washing medium L15 (see
Solutions and Chemicals). The filaments were washed under
gentle automatic shaking (5 ⫻ 10 min, 100 agitations/min)
Address for reprint requests and other correspondence: P. Poujeol,
UMR CNRS 6548, bâtiment Sciences Naturelles, Université de NiceSophia Antipolis, 06108 Nice Cedex 2, France (E-mail: poujeol
@unice.fr).
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.
IT WAS PREVIOUSLY DEMONSTRATED
http://www.ajpregu.org
0363-6119/00 $5.00 Copyright © 2000 the American Physiological Society
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Duranton, C., E. Mikulovic, M. Tauc, M. Avella, and
P. Poujeol. Potassium channels in primary cultures of
seawater fish gill cells. I. Stretch-activated K⫹ channels.
Am J Physiol Regulatory Integrative Comp Physiol 279:
R1647–R1658, 2000.—Previous studies using the patchclamp technique demonstrated the presence of a small
conductance Cl⫺ channel in the apical membrane of respiratory gill cells in primary culture originating from sea
bass Dicentrarchus labrax. We used the same technique
here to characterize potassium channels in this model. A
K⫹ channel of 123 ⫾ 3 pS was identified in the cellattached configuration with 140 mM KCl in the bath and in
the pipette. The activity of the channel declined rapidly
with time and could be restored by the application of a
negative pressure to the pipette (suction) or by substitution of the bath solution with a hypotonic solution (cell
swelling). In the excised patch inside-out configuration,
ionic substitution demonstrated a high selectivity of this
channel for K⫹ over Na⫹ and Ca2⫹. The mechanosensitivity of this channel to membrane stretching via suction was
also observed in this configuration. Pharmacological studies demonstrated that this channel was inhibited by barium (5 mM), quinidine (500 ␮M), and gadolinium (500 ␮M).
Channel activity decreased when cytoplasmic pH was decreased from 7.7 to 6.8. The effect of membrane distension
by suction and exposure to hypotonic solutions on K⫹
channel activity is consistent with the hypothesis that
stretch-activated K⫹ channels could mediate an increase
in K⫹ conductance during cell swelling.
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K⫹ CHANNELS IN FISH GILL CELLS
and then teased into small tissue fragments (explants). Each
explant was inoculated in a plastic culture dish (Nunc; 35
mm diameter, multiwell plates). The culture growth medium
L15 was then added gradually to the explants. Explants were
then maintained in a precision-controlled low-temperature
incubator (Jouan) at 17°C in a humidified air atmosphere
(atmospheric PCO2). The growth medium was changed every
second day.
Patch-Clamp Experiments
Prior to the patch-clamp experiments, explant culture cells
were briefly treated (15–30 s) with a solution of trypsin
(0.02%) plus EGTA (0.05%). This slight treatment was performed to isolate electrically each cell from the other without
modifying the monolayer epithelium structure. Single-channel currents were recorded in 7- to 13-day-old cultured cells.
Patch pipettes made from borosilicate glass (1.5 mm OD, 1.1
mm ID; Clay Adams) were pulled in two steps using a vertical
puller (PP-83 Narishige). Pipettes, with resistances from 2 to
5 M⍀ were connected via an Ag-AgCl wire to the headstage of
an RK 300 patch-clamp amplifier (Biologic). Seals were
achieved spontaneously or by applying slight suction to the
patch pipette.
Single-Channel Experiments
Single-channel currents were stored on digital audiotapes
(48 kHz) using a DTR 1200 recorder (Biologic) and later
displayed on a digital oscilloscope for analysis (Gould Electronics, Instrument Systems). In the cell-attached configuration, the potential difference across the patch is equal to the
cell membrane potential (Vm) minus the pipette clamp potential (Vp). Because Vm is not known exactly, values of Vp are
used and are expressed as ⫺Vp, so that the data for potential
changes refer to the extracellular side of the membrane. In
excised inside-out patch recordings, ⫺Vp (mV) indicates the
potential on the cytoplasmic face of the patch membrane
relative to the pipette. In some experiments, the pipette
perfusion technique described by Tauc et al. (33) was used.
The perfusion catheter was made of polyethylene tubing
(PE-10, Clay Adams) and tapered at one end (internal diameter 30–50 ␮m). The perfusion catheter was inserted through
the filled patch pipette and connected to the outlet of a
syringe containing the test solution. After a control period,
test solutions were perfused through the pipette by applying
pressure (⫹10–20 mmH2O) to the syringe. The catheter was
positioned as close as possible to the pipette and electrode
tips to replace quickly (⬍5 s) all the pipette solution and to
avoid excessive background noise.
All experiments were performed at room temperature
(22 ⫾ 2°C).
Data Analysis
Channel current amplitudes were measured from audiotaped data replayed on the digital oscilloscope. Currentvoltage (I-V) relationships of channel activity were established from the average amplitude of well-defined transitions
between closed and open current levels at each potential
(⫺Vp). For the analysis of channel kinetics, data were transferred to an ERN computer at a sampling frequency of 1 kHz.
Channel open probability (Po) was estimated from current
amplitude histograms created and analyzed using Biopatch
software (Biologic). The ratio of the areas of the Gaussianlike distributed current-amplitude histograms corresponding
to the closed and open states of the channel was taken as an
estimate of the Po. When more than one channel was present
in the patch, the level of K⫹ channels activity was quantitated by defining the mean number of open channels (NPo) as
N
NP o ⫽
兺 nPn
n⫽1
where Pn is the probability that n identical channels are
open simultaneously and N is the apparent number of channels in the patch.
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Fig. 1. A: single-channel current recordings of a K⫹ channel at different holding potentials (⫺Vp) in a cell-attached
patch. Pipette and bath contained 140 mM KCl solution. B: single-channel current recordings of K⫹ channels after
replacing the 140 mM KCl solution in the pipette with 140 mM NaCl solution. C: current-voltage (I-V) relationship
of K⫹ channel in cell- attached configuration: F, 140 mM KCl in pipette and bath solutions (n ⫽ 24 cells from 20
monolayers); ■, 140 mM NaCl in pipette and 140 mM KCl in bath solution (n ⫽ 3 cells from 2 monolayers); and Œ,
140 mM RbCl in the pipette and 140 mM KCl in the bath solution (n ⫽ 4 cells from 2 monolayers).
K⫹ CHANNELS IN FISH GILL CELLS
Reversal potentials (Erev) were estimated by interpolation
after a polynomial function of the I-V curve was fitted using
Sigma Plot and Sigma Stat software (Jandel Scientific).
The K⫹-to-Na⫹ permeability ratio was calculated from
Erev in excised patches according to the modified equation of
Goldman-Hodgkin-Katz
P K ⲐP Na ⫽
关Na ⫹兴 pipette ⫺ 关Na ⫹兴 bath exp关⫺E revⲐ共RTⲐF兲兴
关K ⫹兴 bath exp关⫺E revⲐ共RTⲐF兲兴 ⫺ 关K ⫹兴 pipette
where PK and PNa are K⫹ and Na⫹ permeabilities, respectively, and R is the gas constant, T is the absolute temperature, and F is Faraday’s constant. The use of this equation to
evaluate selectivity requires the tacit assumption that the
channel being studied has a negligible selectivity for anions.
Solutions and Chemicals
dence, etc.) than the Cl⫺ channel described in our last
paper (10). Because the interest of this small conductance Cl⫺ channel was minor in this study dealing with
K⫹ conductance present in the gill cells in primary
culture, we eliminated their recordings from the data
described here. Despite our search to find other types of
K⫹ channels, we never recorded, out of 200 single
patch-clamp experiments, any other K⫹ channel than
the stretch-activated one shown in this study.
Single-Channel K⫹ Current Recorded in
Cell-Attached Patch
Typical records of channel activity in a cell-attached
patch can be seen in Fig. 1A. Under these conditions,
the pipette and bath contained the standard KCl solution (see MATERIALS AND METHODS). Overall, channel
openings appeared in bursts of activity alternating
with closed periods lasting from a few milliseconds to
several seconds. At 0 mV, no current variations were
measured. Depolarizing or hyperpolarizing holding potentials (Vh) increased current amplitudes without significantly affecting channel Po. The I-V relationship
corresponding to the channel presented in Fig. 1A is
given in Fig. 1C.
The maximal channel conductance, calculated as the
slope of the straight line between ⫺60 and ⫹60 mV,
RESULTS
Gigaseals were obtained in 20.5% of the cells tested
(in all, 521 cells from 56 monolayers). A total of 107
patches exhibited channel activity. However, this activity depended on the age of the culture in that singlechannel activity was never recorded from cells aged ⬍7
days, whereas the maximum activity was found in cells
from monolayers aged 10 ⫾ 3 days.
Patch-clamp studies performed with 140 KCl in the
pipette and bath solutions on the same cell preparation
showed a Cl⫺ channel with same biophysical characteristics (small conductance ⬍10 pS, potential depen-
Fig. 2. A: inactivation of K⫹ channel activity in cell-attached configuration. Pipette and bath contained 140 mM KCl solution. The
pipette potential was held at Vp ⫽ ⫺40 mV. C and the dotted line
indicate the closed state of the channel. B: mean number of open
channel (NPo) plotted against time (NPo was calculated by computer
analysis of periods of 5-s duration). Values are means ⫾ SE for 6
experiments.
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Primary cultures. All solutions were prepared using tissueculture quality chemicals.
WASHING MEDIUM. Leibovitz L15 medium (GIBCO-BRL) was
supplemented with 20 mM NaCl, 0.1 mg/ml fungizone, 200
UI/ml penicillin, 200 mg/ml streptomycin, and 400 mg/ml
gentamicin. All antibiotics were supplied by Sigma. The final
pH was adjusted to 7.8 with 1N NaOH.
CULTURE GROWTH MEDIUM. Leibovitz L15 medium was supplemented with 10% fetal bovine serum (Multiser, Cytosystems), 20 mM NaCl, 100 UI/ml penicillin, 100 mg/ml streptomycin, and 200 mg/ml gentamicin. The final pH was
adjusted to 7.8 with 1N NaOH.
Solutions for electrophysiological studies. The standard
bath and pipette solutions contained (in mM) 140 KCl, 5
HEPES, and 0.1 CaCl2 (pH 7.8 adjusted with 1M KOH) for
control conditions. In some experiments, cells were also
bathed in an NaCl solution containing (in mM) 140 NaCl, 5
HEPES, and 0.1 CaCl2 (pH 7.8 adjusted with 1N NaOH). For
determination of the channel ionic selectivity, solutions containing (in mM) 60 KCl, 80 NaCl, 5 HEPES, and 0.1 CaCl2 or
with 100 mM CaCl2 and 5 HEPES were also used.
For pH studies, the standard 140 mM KCl bath solution
described above was used; pH was adjusted to 6.8 or 8.2 with
stock 1M KOH solution.
For calcium studies, the bath solution contained (in mM)
140 KCl, 5 HEPES, and 0.5 EGTA. The Ca2⫹-free concentration was lowered to 10⫺9 M by buffering calcium ions with
EGTA.
Barium and gadolinium (Gd3⫹) solutions were prepared
from a 1 M BaCl2 or GdCl3 stock solution (Sigma) and added
directly to the 140 mM KCl solution. Tetraethylammonium
(TEA) and quinidine were each obtained as chloride and
sulfate salts (Sigma) and were added to the 140 mM KCl
solution at final concentrations of 20 mM for TEA and 50–
500 ␮M for quinidine.
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K⫹ CHANNELS IN FISH GILL CELLS
spontaneous run-down phenomenon is illustrated in
the recording shown in Fig. 2A. In this experiment, the
pipette contained the standard KCl solution and the Vh
was maintained at ⫺40 mV. One minute after seal
formation, the channel activity was so low that it was
often very difficult to obtain a reliable estimate of the
mean NPo. In fact, the time necessary to obtain complete inactivation of channel activity varied from 1 to 3
min. The curve in Fig. 2B shows average results for six
cell-attached patches in which inactivation occurred
within 1 min.
The mean NPo decreased with time. After channel
activity had been lost, it was possible to restore it by
application of a negative pressure to the patch pipette.
A typical experiment is shown in Fig. 3A. Increasing
the negative pressure ⬎20 mmH2O produced a rapid
and reversible increase in NPo (Fig. 3C). This stretch
activation of the channel was effective only up to a
negative pressure of 40 mmH2O, after which patches
usually ruptured. The I-V curve of the reactivated
channel (with a negative pressure ranging between 30
and 40 mmH2O) was linear, with a slope conductance
of 132 ⫾ 6 pS (n ⫽ 7) and a Erev unchanged from 0 mV
(Fig. 3B). Finally, the main characteristics of the channel before and after stretch activation were very similar.
To investigate whether these stretch-activated channels could also be activated by osmotic pressure, the
effect of cell swelling, induced by exposure of cells to a
hypotonic solution, was then studied. The perfusion of
six different cell-attached patches with a 200 mosmol/
kgH2O solution brought about an increase in channel
activity. A typical recording obtained at a Vh of ⫺20
mV and with the KCl solution in the pipette, is given in
Fig. 4A. The mean NPo increased from 0.05 ⫾ 0.01 to
0.53 ⫾ 0.06, n ⫽ 6 (Fig. 4B) within ⬃25 s of the onset
of perfusion of the hypotonic solution. This effect was
reversible after washout with an isotonic solution. The
Fig. 3. A: negative pressure dependence of K⫹ channel activity for cell-attached configuration. Up to 3 channels are
activated simultaneously by increasing negative hydrostatic pressure (applied as suction to the patch pipette) as
indicated on left. The pipette potential was held at ⫺Vp ⫽ ⫺20 mV. Pipette and bath contained 140 mM KCl
solution. B: I-V relationship of K⫹ channel in cell-attached configuration during negative pressure stimulation of
between 30 and 40 mmH2O. C: mean NPo as a function of pipette pressure (suction).
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was 123 ⫾ 3 pS (n ⫽ 24). The zero-current potential
(1.6 ⫾ 0.5 mV, n ⫽ 24) was close to the expected Erev for
K⫹ (assuming an intracellular K⫹ concentration ranging between 120 and 140 mM). This indicates that the
observed current was carried by K⫹. The analysis of
four independent seals presenting only one open state
revealed that the probability of the channel being in
the open-state was independent of the membrane potential (Po at ⫺40 mV ⫽ 0.24 ⫾ 0.03; Po at ⫹40 mV ⫽
0.25 ⫾ 0.01; n ⫽ 4). The kinetic analysis of recordings
made at ⫺40 mV revealed that the channel showed one
open state with a time constant of 27 ⫾ 5 ms (n ⫽ 4).
Cell-attached recordings were also performed with
the NaCl solution in the pipette and the standard KCl
solution in the bath (Fig. 1B). Under these conditions,
positive currents were observed from ⫺60 to ⫹40 mV.
The corresponding I-V curve for these experiments is
shown in Fig. 1C. The relationship was nonlinear over
the range ⫺60 to ⫹40 mV, whereas the extrapolated
Erev (⫺78 mV) was close to the expected Erev for K⫹.
The Goldman-Hodgkin-Katz constant field equation
was applied to the data on the assumption that the
channel was selective for K⫹. As shown in Fig. 1C, the
experimental data are described accurately by the theoretical curve. Assuming the cell-attached configuration, internal potassium concentration is 140 mM and
the calculated mean permeability is 2.24 ⫾ 0.07 ⫻
10⫺13 cm3/s (n ⫽ 3). The maximal channel conductance, calculated as the slope of the curve between 0
and ⫹40 mV, was 96 ⫾ 12 pS (n ⫽ 3). The I-V curve in
Fig. 1C also illustrates the effect of Rb⫹ when the
pipette was filled with an RbCl solution. The substitution of KCl with RbCl did not modify the slope conductance (114 ⫾ 7 pS, n ⫽ 4) nor the Erev (1.3 ⫾ 5.4 mV,
n ⫽ 4), indicating that the channel was equally permeable to K⫹ and Rb⫹ ions.
Channel activity rapidly declined with time in 100%
of the cell-attached recordings, irrespective of the ionic
composition of the bath and pipette solutions. This
K⫹ CHANNELS IN FISH GILL CELLS
NPo returned to control levels with a similar delay
(NPo ⫽ 0.08 ⫾ 0.03, n ⫽ 6).
Single Channel K⫹ Current Recorded in Excised
Inside-Out Configuration
Channel characteristics were then studied in excised
patch experiments. Such a study was complicated by
the inactivation properties of the channel observed in
the cell-attached configuration. For this reason, channel activity was first tested in the cell-attached mode at
a Vh of ⫺40 mV. The patch was then excised, and the
pulse protocol was rapidly applied between Vh of ⫺40
mV and ⫹40 mV. Figure 5A illustrates the singlechannel currents recorded in an excised inside-out
patch when the pipette and bath solutions both contained 140 mM standard KCl. In 13 patches, upward
and downward currents were observed at positive and
negative potentials, respectively. The I-V relationship
(Fig. 5E) was linear, with a zero-current potential at
0.47 ⫾ 0.46 mV and a channel conductance of 121 ⫾ 4
pS (n ⫽ 13).
Under these experimental conditions, the ion selectivity of this channel could not be accurately determined because K⫹ or Cl⫺ was able to induce the same
current. To verify the K⫹ selectivity of the channel,
inside-out excised patches were perfused on the cytoplasmic side with a K⫹-free solution or a solution
containing only 60 mM K⫹. Figure 5B illustrates the
single-channel currents recorded when the pipette solution contained 140 mM KCl and the bath medium
140 mM NaCl. At a Vh of 0 mV, channel openings
showed downward deflections with an average unitary
current amplitude of ⫺2.3 ⫾ 0.1 pA (n ⫽ 8). By definition, this had to correspond to the passage of K⫹ ions,
because Na⫹ currents would have produced upward
deflections and Cl⫺ is at electrochemical equilibrium.
The mean I-V relationship of eight experiments performed under these conditions is shown in Fig. 5E.
From the current Erev estimated by extrapolation
(⫹74 ⫾ 6 mV), a K⫹-to-Na⫹ permeability ratio (PK/PNa)
equal to 19 was calculated. The maximal slope conductance was 84 ⫾ 6 pS.
Further experiments were performed by replacing
the 140 mM NaCl bathing solution with a solution
containing 60 mM KCl and 80 mM NaCl. Figure 5C
illustrates single-channel currents recorded under
these conditions. The corresponding I-V relationship
reported in Fig. 5E displayed an inward rectification
over the applied potential range from ⫺50 to ⫹40 mV.
In these experimental conditions, the K⫹ Nernst Erev
was calculated at ⫹21.3 mV. The experimental Erev
was determined at ⫹19.8 ⫾ 1.2 mV (n ⫽ 7), leading to
a PK/PNa ⫽ 20.8. The average conductance of the channel was 76 ⫾ 4 pS (n ⫽ 7).
To examine the permeability of the channel to Ca2⫹
ions, experiments were performed with 100 mM CaCl2
solution in the pipette and 140 mM standard KCl
solution in the bath, respectively. Figure 5D illustrates
the kinetic properties of the channel, while the I-V
curve in Fig. 5E shows that the currents were always
positive for Vh ranging from ⫹20 to ⫺40 mV. The Erev
tended toward that of K⫹; however, it was not possible
to record channel activity at potentials more negative
than ⫺40 mV because larger hyperpolarizations ruptured the patch seal. Nevertheless, the recorded current was mainly due to K⫹, indicating an absence of
Ca2⫹ permeation through the channel. Under these
conditions, the maximal slope conductance of the channel was 104 ⫾ 11 pS (n ⫽ 7).
As observed in cell-attached experiments, channel
activity decreased within 1 to 3 min after the patch
was excised. In 17 of the 28 inside-out excised
patches, channel activity was completely lost. The
application of negative pressure to the patch pipette
produced an increase in channel activity that was
rapid and reversible on removal of the suction. Such
stretch-activation was observed in 11 of 15 patches
studied. Figure 6A illustrates this phenomenon for a
Vh of ⫺40 mV, when the bath and the pipette solutions contained standard 140 mM KCl. In this typical
recording, the application of suction (46 mmH2O) to
the pipette induced the opening of three channels. As
illustrated in Fig. 6B, this stretch activation did not
depend on the Ca2⫹ concentration at the inside surface of the membrane. In fact, lowering Ca2⫹ to 10⫺9
M in the bath (see MATERIALS AND METHODS) did not
suppress the stretch activation of K⫹ channels in
these cultured cells.
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Fig. 4. A: activation of K⫹ channel in cell-attached patch during
change of external hypertonic saline solution to hypotonic solution.
Fifty seconds before time zero, the external hypertonic bath medium
(350 osmol/kgH2O NaCl) was replaced by a hypotonic medium (200
osmol/kgH2O NaCl). Note the increase in channel activity during the
“hypotonic” period. B: mean NPo during hypotonic shock (NPo was
calculated by computer analysis of periods of 5-s duration) (n ⫽ 6).
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K⫹ CHANNELS IN FISH GILL CELLS
Pharmacological Properties
To identify the nature of the channels under investigation here in cultured seawater fish gill cells, the
effect of 5 mM Ba2⫹ was studied on five excised
patches, with standard KCl solution in the pipette and
in the bath. As shown in Fig. 7A, the application of
Ba2⫹ to the cytoplasmic side of the channel considerably modified the current recordings. Three channels
were opened simultaneously under control conditions
at a Vh of ⫹40 mV. The addition of Ba2⫹ to the bath
reduced the channel openings and the current amplitude. The mean NPo was estimated from the amplitude
histograms of Fig. 7B, the surface of the Gaussian
curves corresponding to the NPo of the three channels
measured under control conditions (NPo ⫽ 0.70). In the
presence of Ba2⫹, two of the three open states completely disappeared (NPo ⫽ 0.14).
Gd3⫹ is a blocker of stretch-activated channels in a
variety of tissues, acting principally at the extracellular surface of the channels. To investigate whether K⫹
channel activity could be blocked by Gd3⫹, the perfusing pipette technique of Tauc et al. (33) was used.
Figure 7C illustrates the effect of Gd3⫹ on K⫹ channel
activity recorded from an inside-out patch. Under control conditions, two channels were open simultaneously. The perfusion of 500 ␮M Gd3⫹ through the
patch pipette strongly blocked channel openings, thus
reducing the mean NPo from 0.75 ⫾ 0.20 to 0.04 ⫾ 0.02
(n ⫽ 5, Fig. 7D). The unitary conductance of the channel, however, remained unchanged.
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Fig. 5. A: single-channel current recordings
of a K⫹ channel at different holding potentials (⫺Vp) in excised inside-out configuration. Pipette and bath contained the 140 mM
KCl solution. C on the right of the recordings
indicates the closed state of the channel.
Same excised patch after replacing the bath
solution with the 140 mM NaCl solution (B)
or by the 60 mM KCl ⫹ 80 mM NaCl solution
(C). D: single K⫹ channel current trace in
excised configuration with 100 mM CaCl2 solution in the pipette and 140 mM KCl solution in the bath. E: I-V relationship of the
channel in excised inside-out configuration
under symmetrical 140 mM KCl solution in
the bath and in the pipette (F, n ⫽ 13), after
replacing the bath solution with 140 mM
NaCl (■, n ⫽ 8) or 60 mM KCl ⫹ 80 mM NaCl
(䊐, n ⫽ 7 ) or after replacing the pipette
solution with 100 mM CaCl2 (E, n ⫽ 7).
K⫹ CHANNELS IN FISH GILL CELLS
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lar (added to the pipette solution) or the intracellular
surface (added to the bath solution) of the patches. The
use of TEA concentration as high as 20 mM did not
significantly modify the channel activities recorded under either experimental condition (Fig. 8C).
Dependence on pH
DISCUSSION
Fig. 6. Activation of K⫹ channels by application of negative pressure
to the patch in excised inside-out configuration for 2 different concentrations of free Ca2⫹ in the bath solution. A: pipette contained the
140 mM KCl solution with 0.1 mM CaCl2 and bath contained the 140
mM KCl solution with 1 mM CaCl2. The holding potential was held
constant at ⫺Vp ⫽ ⫺40 mV. B: pipette was filled with the same 140
mM KCl solution but bath contained 140 mM KCl solution with low
concentration of free Ca2⫹ (1 nM) by addition of EGTA. The holding
potential was held constant at ⫺Vp ⫽ ⫺20 mV. Note that stretch
activation of the K⫹ channel was independent of the cytosolic Ca2⫹
concentration.
Quinidine applied to the internal surface of five inside-out patches also induced strong inhibition of channel activity. One example of a recording obtained with
50 and 500 ␮M quinidine is given in Fig. 8A. Quinidine
(500 ␮M) decreased the current amplitude from 2.3 ⫾
0.2 to 0.30 ⫾ 0.03 pA (n ⫽ 3) and reduced the mean NPo
from 0.41 ⫾ 0.05 to 0.020 ⫾ 0.001 (Fig. 8B). The effect
of quinidine was partially reversible when the drug
was washed out. The current amplitude was not significantly reduced at a low quinidine concentration (50
␮M; Fig. 8, A and B).
The effect of TEA on the K⫹ channel was studied
when the blocker was applied to either the extracellu-
The present study focused on the identification of
potassium channels present on gill cells in primary
culture. Patch-clamp experiments were performed on
the apical membrane of these cells. This single-channel
analysis revealed the existence of a channel that was
selective to potassium over sodium ions. In the cellattached mode and under control conditions (KCl standard solution), the channel was spontaneously active
at the resting membrane potential and could permit
K⫹ efflux from the cell. The unitary conductance of the
K⫹ channel under these conditions ranged between
100 (physiological conditions) and 120 pS (symmetrical
K⫹ solutions).
The kinetic properties of the channel in excised configuration were not distinguishable from those seen in
cell-attached patches. For both recording configurations and in the presence of identical K⫹ concentrations on both sides of the patch, the inward and outward K⫹ currents were similar. The rectification
observed in the presence of asymmetrical solutions was
the consequence of a Goldman-type rectification. Moreover, the open state probability of the channel did not
exhibit voltage-dependence characteristics over the
range of imposed potentials.
The channel was blocked by Ba2⫹ ions, with the
kinetics of this inhibition being characteristic of the
action of slow blocking agents. Thus Ba2⫹ ions reduced
the length of the burst duration and induced long
periods of inactivity between any two bursts. Moreover,
Ba2⫹ reduced not only the open probability of the
channel, but also the amplitude of the current flowing
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To examine the pH dependence of K⫹ channel activity, the pH of solutions was varied at the cytoplasmic
surface of inside-out patches. In this way, pipettes
were filled with the standard KCl solution, whereas the
bath contained the same solution at one of three different pH values. Figure 9A shows single-channel current trace for one patch held at ⫺20 mV and in the
presence of bath solutions at pH 7.7, 6.8, and 8.2. It can
be clearly seen that a change in pH from 7.7 to 6.8
reduced channel activity, corresponding to a large decrease in the mean NPo from 0.71 ⫾ 0.21 to 0.03 ⫾ 0.01
(n ⫽ 7; Fig. 9B). The activity of the patch was partially
recovered when the pH was increased secondarily to
8.2. To test the reversibility of the pH effect, we performed additional experiments with repetitive decrease and increase of pH over the time in the same
recording. In that case, we observe a total reversible
effect of acidification (Fig. 9C). The correlated mean
number of open channels followed the same inhibition
and stimulation profile (Fig. 9D).
R1654
K⫹ CHANNELS IN FISH GILL CELLS
through the channel. Quinidine and gadolinium were
also potent inhibitors of the channel, whereas TEA did
not influence its activity.
Cytosolic acidification has been shown strongly to
reduce open-time probability in many K⫹ channels (8,
12). The maximal channel activity observed in the
present study was with an intracellular pH ranging
from 7.5 to 7.8. It is possible that a culture medium
maintained at pH 7.8 represents physiological pH values in seawater fish gill cells (considering that this pH
value is close to that of sea bass plasma). In the present
study, the effect of acidification was partially or totally
reversible. Repetitive decrease and increase of pH over
the time in the same recording induced a reversible
effect of acidification showing that the inhibition of the
activity of the channel was a real effect and not a
run-down phenomenon.
Some interesting findings made here concerned the
fact that channel activity exhibited a spontaneous rundown phenomenon that could be reactivated by the
application of a negative pressure via the patch pipette. The reactivated channel exhibited kinetic and
conductance characteristics very similar to those determined before the inactivation process taking place.
This suggests that the pipette suction probably reactivated the same channel and that the K⫹ channel in the
present study was a stretch-activated channel. Ion
channels activated by membrane stretch have been
detected in several epithelial cells (7, 35, 36). Many
of these channels are nonselective stretch-activated
channels with single conductances ranging between 25
and 35 pS. In primary cultures of gill cells, it appears
that the channel was at least 19 times more permeable
to K⫹ than to Na⫹ and relatively impermeable to Ca2⫹.
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Fig. 7. Effect of 5 mM BaCl2 on the K⫹ channel
activity in an excised inside-out patch. A: in the
control recording, pipette and bath contained the
140 mM KCl solution. C on the right of the
recordings indicates the closed state of the channel; “1, 2, 3” and the dotted lines indicate the
different open states. In the lower recording, 5
mM BaCl2 was added to the bath solution. B:
amplitude histograms of the 2 recordings shown
in A. C: effect of 500 ␮M GdCl3 on the K⫹ channel
activity in an excised inside-out patch. The pipette contained the 140 mM KCl solution and
after a control period was perfused with the 140
mM KCl containing 500 ␮M GdCl3. The bath
solution contained 140 mM KCl. D: mean NPo
calculated for control conditions, after 140 mM
KCl perfusion, and after 140 mM KCl ⫹ 500 ␮M
gadolinium perfusion. Values are means ⫾ SE of
5 experiments.
K⫹ CHANNELS IN FISH GILL CELLS
R1655
Stretch-activated K⫹ channels with small conductances and insensitive to calcium have been identified
in amphibian proximal tubules (15, 16, 27–29). However, none of these channels exhibit characteristics
such as those identified here in gill cell K⫹ channels.
Maxi K⫹ channels have also been shown to exhibit
mechanosensitive properties in different cell preparations. For example, Pacha et al. (23) described a highconductance K⫹ channel in the apical membrane of
intercalated cells of the rabbit collecting duct that
shares some of its properties with the K⫹ channel
examined in the present study. In particular, the conductance was close to 100 pS and the channel could be
activated by pipette suction in the cell-attached as well
as inside-out patch-clamp configurations. Moreover,
this mechanical activation was not dependent on cytosolic calcium. Some important differences between the
two channel types, however, also exist. Notably, the
activity of K⫹ channels in gill cells was not gated by
voltage and was not sensitive to TEA. Therefore, the
mechanical activation could definitely not be attributed to an increase in cytosolic Ca2⫹. This observation
led to the conclusion that the channel was activated
directly by membrane stretching. It is therefore evident that the channel is considerably different from the
large conductance K⫹ channel (maxi K⫹ channels) typically found in other epithelial tissues (4, 22, 24).
Finally, because very little information exists concerning K⫹ channels in fish gill cells, it is not possible
to directly compare the K⫹ channel in the present
investigation with K⫹ channels described in other reports. The most relevant studies concerning our findings are those of Gögelein et al. (12) and Greger et al.
(13) on the rectal gland of the dogfish and of Chang and
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Fig. 8. A: effect of quinidine on K⫹ channel activity in an excised inside-out patch.
Under control conditions, the pipette and
bath contained the 140 mM KCl solution.
The quinidine concentration was increased from 50 to 500 ␮M in the bath
solution. The presence of 500 ␮M quinidine abolished channel activity, which
was partially reversible. B: representation
of unitary current (I) and mean NPo under
control conditions or in the presence of 50
or 500 ␮M quinidine. Values are means ⫾
SE for 5 experiments. Statistics: Student’s
unpaired t-test: data at rinse are significantly different from values after quinidine addition (500 ␮M) with P ⬍ 0.05 (*).
C: K⫹ channel activity for control conditions (left) and with 20 mM tetraethylammonium (TEA) added to the pipette solution (right) for cell-attached conditions.
Pipette and bath contained 140 mM KCl
solution. No inhibition was observed with
20 mM TEA.
R1656
K⫹ CHANNELS IN FISH GILL CELLS
Loretz (5, 6), who reported on goby enterocytes. The
former work revealed that the basolateral membrane
possesses a K⫹ channel with a unitary conductance,
kinetic characteristics, and rectifying properties very
different from the K⫹ channel in cultured gill cells. The
latter study described a mechanosensitive cation channel of 70 pS that poorly discriminated between Na⫹
and K⫹ and which may operate rather as an Na⫹
channel. Therefore, to our knowledge, the present work
is the first study describing stretch-activated K⫹ channels in epithelial gill cells.
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Fig. 9. A: effect of pH variations on K⫹ channel activity in an excised
inside-out patch. Note the inhibition of K⫹ channels by acidification
of the bath solution from 7.7 to 6.8 and a partial reactivation by
increasing pH to 8.2. Pipette and bath contained 140 mM KCl
solution. The pipette potential was held constant at ⫺Vp ⫽ ⫺20 mV.
B: representation of the mean NPo for 3 different values of pH: 7.7,
6.8, and 8.2. Values are means ⫾ SE for 7 experiments. C: repetitive
decrease (from 7.7 to 6.8) and increase (from 6.8 to 7.7) of pH over the
time showing a total reversible effect of acidification. D: correlated
mean NPo after the same inhibition and stimulation profile.
To understand the physiological role of the K⫹ channels in the apical membrane of gill cells, it is necessary
to know whether this channel is active at the resting
membrane potential. This question is important because the channel inactivated rapidly after seal formation in the cell-attached and inside-out patch-clamp
configurations. The inactivation process could be interpreted in light of two different hypotheses. 1) The
channel was inactive at rest before seal formation and
the application of suction to seal the membrane-induced stretching that activated the channel. Subsequently, when the application of negative pressure was
terminated, the patch returned to its inactive state. 2)
The channel was active at rest, and its spontaneous
activity was, therefore, representative of the cell conductance under normal conditions. After the seal formation, the channel run down observed could have
been due to mechanical constraints on the cell membrane induced by the patch pipette.
The importance of the membrane geometry on mechanosensitive K⫹ channels was recently studied by Patel et al. (25). They identified a cloned mammalian two
P-domain mechanogated S-like K⫹ channel and demonstrated that the opening of TREK 1 (tandem of P
domains in a weak inward rectifying related K⫹ channel) was highly sensitive to the curvature of the membrane. We favor this second hypothesis over the first
because, in whole cell experiments (see companion
paper, Ref. 9a), a spontaneous decrease of the ionic
conductance was never observed. According to the data
of Patel et al. (25), it is reasonable to postulate that the
cell membrane exhibits a crenated form at rest, which
is compatible with channel activity. Soon after the
formation of a gigaseal, this notched configuration of
the membrane progressively disappears because the
membrane beneath the patch becomes more rigid. The
crenated form is thus replaced by a cup form (which is
not conductive to channel activity). Afterward, the application of a negative pressure reinduces the crenated
form and channel activity is restored. The fact that a
positive pressure was unable to induce channel activity
would tend to favor this explanation.
Considering that the K⫹ channel could be active in
resting cells, it may be involved in K⫹ fluxes across the
apical membrane of gill cells. We previously demonstrated that primary cultures of respiratory cells of the
sea bass gill represent a good model for studying Cl⫺secreting epithelia (2, 10). In accordance with classical
models for electrogenic chloride secretion, the secretion
of K⫹ takes place across the basolateral membrane and
serves to recycle K⫹ ions accumulated in the cytosol by
the Na⫹-K⫹-ATPase and the Na⫹-K⫹-2Cl⫺ cotransporter mechanisms (13). However, K⫹ channel activity
has been recorded in the apical membrane of some
chloride-secreting epithelia, including lacrimal acinar
cells (31) and vas deferens epithelial cells (30). Moreover, K⫹ fluxes have been also measured across the
isolated skin of the marine teleost Gillichthys mirabilis
(20) and seawater opercular epithelia (21).
Using an equivalent circuit model, Cook and Young
(9) examined the effect of an apical K⫹ conductance on
K⫹ CHANNELS IN FISH GILL CELLS
Perspectives
The sea bass is considered euryhaline and capable of
surviving in an estuarine environment where seawater
salinity is drastically reduced (3). Therefore, the fish
could be naturally subjected to significant changes in
osmotic gradients that would initially affect the gill
epithelia. It is thus reasonable to postulate that the gill
cells have developed efficient mechanisms of cell volume regulation. The large conductance stretch-activated K⫹ channel described here could form part of
these mechanisms.
In a companion paper (9a), we investigate potassium
conductances and fluxes in both apical and basolateral
membranes and their behavior in response to exposure
of the membranes to a hypotonic medium. For this
purpose, whole cell patch-clamp and 86Rb⫹ efflux experiments on primary cultures of gill cell were employed.
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the secretion of fluid by an epithelium in which secretion was driven by the secondary active transport of
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K⫹ CHANNELS IN FISH GILL CELLS
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