SK4/IK1-like channels mediate TEA-insensitive,
Ca2+-activated K+ currents in bovine parotid
acinar cells
T.
Takahata,
M.
Hayashi, and
T.
Ishikawa
Department of Biomedical Sciences, Graduate School of
Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
 |
ABSTRACT |
Although Ca2+-activated
K+ (KCa) channels distinct from
maxi-K+ channels have been suggested to contribute to
muscarinically stimulated K+ currents in salivary acinar
cells, the molecular nature of the channels is unclear. Using
electrophysiological and RT-PCR techniques, we have now investigated
the involvement of SK4/IK1-like channels in native KCa
currents in bovine parotid acinar (BPA) cells.
Ca2+-dependent K+ efflux from perfused bovine
parotid tissues was not inhibited by a maxi-K+ channel
blocker, tetraethylammonium (TEA). Whole cell recordings from BPA cells
showed a TEA-insensitive KCa conductance, which was highly
permeable to Rb+. In inside-out macropatches,
TEA-insensitive Rb+ currents were activated by
Ca2+ with half-maximal values of 0.4 µM.
1-Ethyl-2-benzimidazolinone (1-EBIO) increased the Ca2+
sensitivity of the currents. The calmodulin antagonists
trifluoperazine, calmidazolium, and W-7 inhibited the
Ca2+-activated Rb+ currents. In outside-out
macropatches, Ca2+-activated Rb+ currents were
inhibited by Ba2+, quinine, clotrimazole, and charybdotoxin
but not by d-tubocrarine or apamin. RT-PCR analysis showed
transcripts of SK4/IK1 in BPA cells. These results collectively suggest
that SK4/IK1-like channels mediate the native KCa currents
in BPA cells.
patch clamp; salivary secretion; HCO
transport
 |
INTRODUCTION |
IT IS WELL
ESTABLISHED that Ca2+-activated K+
(KCa) channels play a critical role in muscarinically
stimulated fluid and electrolyte secretion in salivary glands
(7). As in other secretory epithelia, the K+
channels in the basolateral membrane of acinar cells allow
K+ to leave the cell and establish the apical (and
basolateral) cell membrane potential more negative than the Nernst
potential for anions such as Cl
and
HCO
, thereby providing a driving force for the
sustained electrogenic anion efflux across the apical membrane. The
K+ channels responsible for carrying the muscarinically
stimulated outward K+ current during secretion have been
investigated by using patch-clamp methods in salivary acinar cells in
various species. The earliest patch-clamp studies first identified the
presence of tetraethylammonium (TEA)-sensitive, large-conductance,
voltage- and Ca2+-dependent K+ channels
(BKCa or maxi-K+ channels) in the basolateral
membrane of acinar cells (36). In cell-attached patch
studies, maxi-K+ channels have been shown to be activated
by addition of ACh or carbachol to the bathing solution (13, 37,
41, 53).
It has been suggested, however, by a number of other studies
using whole cell patch-clamp techniques or K+ (and
Rb+) efflux measurements that KCa channels,
distinct from maxi-K+ channels, may also contribute
significantly to the outward K+ current in the acinar cells
of various salivary glands including rat parotid (29, 46),
sheep parotid (20, 55), bovine parotid (34),
rat mandibular (26, 27), and mouse mandibular glands (22). These studies have shown that a
maxi-K+ channel blocker, TEA, is largely ineffective in
inhibiting muscarinically or Ca2+-evoked K+
conductance. Available data for the TEA-insensitive,
Ca2+-activated whole cell currents suggest that the
channels are blocked by quinine and Ba2+ and have a
significant conductance for Rb+ (20, 21, 26).
A cell-attached patch study on mouse mandibular acinar cells has also
shown the presence of a TEA-insensitive, 40-pS K+ channel
that is activated by addition of ACh to the bathing solution, conductive for Rb+, and blocked by quinine
(22). However, the molecular nature of the TEA-insensitive
KCa channels responsible for muscarinically stimulated
K+ currents have still remained unknown.
Recent molecular studies have identified four members (SK1-3
and SK4/IK1) of KCa channels that form
Ca2+-activated, small- to intermediate-conductance
K+ channels in various excitable and nonexcitable tissues.
SK1-3 channels are predominantly expressed in excitable tissues
(50), whereas SK4/IK1 channels are expressed in peripheral
nonexcitable cells including secretory epithelia (24, 30, 31,
52), erythrocytes (49), and lymphocytes (32,
35). Intriguingly, Jensen et al. (30) reported that
the highest level of transcripts of hSK4/IK1 was expressed in salivary
glands. These finding prompted us to hypothesize that SK4/IK1 channels
may mediate the native TEA-insensitive KCa currents in
salivary acinar cells. In addition, the heterologously and naturally
expressed SK4/IK1 currents have been shown to be relatively insensitive
to TEA (Kd = 30-40 mM) (15, 31,
35), conductive for Rb+ (15, 30), and
blocked by Ba2+ (15, 31), the properties
compatible with described properties of native salivary KCa
currents mentioned above. However, there is no direct functional and
molecular evidence to suggest that SK4/IK1-like channels contribute to
native KCa currents in salivary acinar cells.
The main aim of the present work thus was to characterize the
biophysical and pharmacological properties of the native
TEA-insensitive KCa currents in bovine parotid acinar (BPA)
cells to provide a basis for the comparison to the described properties
of expressed SK4/IK1 channels. In a K+ efflux study, we
have first confirmed that ACh- and the Ca2+ ionophore
A-23187-evoked K+ efflux from perfused bovine parotid
tissue is not inhibited by TEA. Using the whole cell patch-clamp
technique, we have then shown the presence of a TEA-insensitive
KCa conductance with a significant Rb+
permeability in BPA cells. In inside-out and outside-out macropatches excised from basolateral membrane of BPA cells, we have demonstrated that biophysical and pharmacological properties of TEA-insensitive KCa currents strikingly resemble those of expressed SK4/IK1
currents described to date. Finally, RT-PCR analysis has confirmed the presence of the transcripts of SK4/IK1 in bovine parotid cells. To our
knowledge, this is the first functional evidence for the involvement of
SK4/IK1-like channels in native KCa currents in salivary
acinar cells.
 |
MATERIALS AND METHODS |
K+ efflux study.
Bovine parotid tissue was obtained from a local slaughterhouse
(Hokkaido Ebetsu Meat Inspection Center, Ebetsu, Japan). The animals
were both males and females. The tissue was put in a standard NaCl-rich
solution on ice immediately after removal from the animals and then
transported to the laboratory. Parotid tissue was dissected free of fat
and connective tissue and sliced with a blade. The perfusion method
used in the present study was similar to that described for rat parotid
acinar cells previously (57). In brief, the perfusion
column consisted of a 1-ml micropipette tip sealed with cotton wool.
Approximately 250 mg of sliced bovine parotid tissues were placed
between layers of Bio-Gel P-2 resin (Bio-Rad, Hercules, CA) dissolved
in a standard perfusion solution on the column. The column, the top of
which was covered with silicone cap, was kept in a water bath at
37°C. The sliced tissues were perfused at a flow rate of 1 ml/min
with the standard NaCl-rich solution of the following composition (in
mM): 110 NaCl, 25 NaHCO3, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, and 10 Na-acetate. The
solution was equilibrated with 95% O2-5% CO2,
and pH was 7.4. In a HCO
-free solution,
NaHCO3 (25 mM) was replaced with equimolar Na-glutamate,
and the solution was equilibrated with 100% O2. The
tissues were preperfused for at least 15 min before the start of each experiment.
The effluent of the perfusate was collected every 30 s and
subjected to measurement of K+ concentration with a flame
photometer (Corning 480) as described previously (27). Net
efflux of K+ from the tissues (JK)
was calculated by the following equation
where F is flow rate and C is K+
concentration. Subscripts o and i indicate output and input solution of
the column, respectively. Efflux of K+ from the tissues was
assigned a positive value.
Patch-clamp experiments.
The parotid glands were obtained as mentioned in K+
efflux study. Isolated acini and acinar cells were
prepared as described for sheep parotid acinar cells previously
(28). Briefly, small pieces of gland, trimmed of fat and
connective tissue, were minced finely with scissors and incubated in a
Mg2+- and Ca2+-free bathing solution containing
collagenase (type II, 500 U/ml; Worthington, Freehold, NJ) for 30 min
at 37°C in a shaking water bath. After gentle trituration with a
10-ml pipette, the medium was then replaced with a fresh
collagenase-containing solution, and the tissue fragments were
incubated once again for 30 min. The fragments were then sieved through
a 148-µm nylon mesh. The suspension was then washed twice,
resuspended in a standard NaCl-rich bathing solution, and stored at
room temperature or at 4°C until used.
The cell preparations were pipetted on to a coverslip and transferred
to a chamber mounted on an Olympus inverted microscope. Current
recordings were made using the standard whole cell, inside-out, and
outside-out configurations of the patch-clamp technique
(18). The patch-clamp pipettes, which were pulled from
glass capillaries (LG16; Dagan, Minneapolis, MN) using a vertical
puller (model PP-830; Narishige, Tokyo, Japan), had resistances of
about 2-5 M
when filled with a standard K-glutamate-rich
solution. An Axopatch-1D patch-clamp amplifier (Axon Instruments,
Foster City, CA) was used to measure the membrane currents. The
reference electrode was a Ag-AgCl electrode, which was connected to the
bath via an agar bridge filled with a standard NaCl-rich bathing
solution. The amplifier was driven by pCLAMP6 software to allow the
delivery of voltage-step protocols with concomitant digitization of the current. The membrane currents were filtered through an internal four-pole Bessel filter at 1 kHz and sampled at 2 kHz. Current-voltage (I-V) relationships were studied by using 10-mV voltage
pulses, each of 400-ms duration, delivered at voltages ranging between
120 and 50 mV, and voltage pulses were separated by 3 s, during which the membrane potential was held at either
60 or 0 mV. As an
alternative to voltage steps, voltage ramps were applied. Typically, the command voltage was varied from
120 or
80 mV to +50 mV over a
duration of 800 ms every 10 s. The capacitance transient current in experiments where the membrane conductance was not activated was
compensated by using the Axopatch-1D amplifier. In these experiments, the whole cell capacitance and the series resistance
(Rs) were 22.2 ± 2.3 pF (n = 28) and 26.1 ± 2.5 M
(n = 26), respectively. The Rs was not electronically compensated during
the experiments, and the potentials reported here have not been
corrected for the Rs. The whole cell currents
were not corrected for leakage. It was difficult to estimate the
Rs properly in the experiments where the
membrane conductance was activated by an increase in cytosolic Ca2+. In any case, the conductances of currents in the
nanoampere range will be underestimated as a result of the voltage
decrease across the Rs. We therefore performed
excised inside-out or outside-out macropatch experiments to overcome
the errors in the whole cell experiments and to characterize the
currents more accurately. The pipette potential was corrected for the
liquid junction potentials between pipette solution and the bath
solution and between the bath solution and the agar bridge as described
by Barry and Lynch (2) and Neher (38).
The experimental solutions are detailed in Table
1. The free Ca2+
concentrations of the pipette and bath solutions were calculated from
an equation that takes into account the concentrations of Mg2+, Ca2+, EGTA (96% purity), and pH
(40), and the appropriate amount of CaCl2 was
added to the solution.
The relationship between membrane current and intracellular
Ca2+ concentration ([Ca2+]i) at
each membrane voltage was fit to the Hill equation
|
(1)
|
where Y is K+ channel current,
Ymax is the maximum current,
Kd is the apparent dissociation constant, and
nH is the Hill coefficient. In the context of
this equation, the Hill coefficient controls the steepness of the
relationship between K+ channel activation (current) and
[Ca2+]i.
To analyze titration curves for Ba2+ inhibition of the
current, we used the ratio I/I0 of
current measured in the presence of Ba2+ (I) to
that in its absence (I0), described by the
following equation:
|
(2)
|
where Ki(V) and A are the
voltage-dependent inhibitory constant and the concentration of
Ba2+, respectively.
In the case of a voltage-dependent block,
Ki(V) has also been expressed by
Woodhull (54) as a Boltzmann relationship with respect to
the voltage as,
|
(3)
|
where Ki(0) is the
inhibitory constant at 0 mV, z' is a slope parameter,
V is voltage, and F, R, and
T have their conventional meanings. z' is
equal to the product of the actual valence of the blocking ion
z and the fraction of the membrane potential (or electrical
distance)
acting on the ion.
Patch-clamp experiments were performed at room temperature (about
20°C). Bath solution changes were accomplished by gravity feed from
reservoirs. The results were reported as means ± SE of several
independent experiments (n), where n refers to
the number of cells patched.
Statistical significance was evaluated by using the two-tailed paired
and unpaired Student's t-test as appropriate. A value of
P < 0.05 was considered significant.
Chemicals employed were of reagent grade. A-23187, TEA-Cl,
clotrimazole, HEPES, EGTA, W-7-HCl
[N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, HCl], calmidazolium chloride (compound R24571), and trifluoperazine dihydrochloride were obtained from Sigma (Tokyo, Japan). ACh-Cl, atropine sulfate, quinine hydrochloride, and d-tubocrarine
chloride were from Wako Chemicals (Osaka, Japan).
1-Ethyl-2-benzimidazolinone (1-EBIO) was from Tocris (Avonmouth, UK),
and charybdotoxin and apamin were from Peptide Institute (Osaka, Japan).
RT-PCR.
BPA cells were prepared as described in Patch-clamp
experiments. Male Sprague-Dawley rats (200-380 g)
were killed immediately by cervical dislocation, the submandibular
glands were removed rapidly, and acinar cells were isolated as
described for BPA cells. RT-PCR experiments were performed with mRNA
extracted from bovine parotid and rat submandibular acinar cells
prepared by using TRIzol reagent (GIBCO BRL, Tokyo, Japan) and a BioMag
mRNA purification kit (Polysciences, Warrington, PA) following the
manufacturer's instructions. First-strand cDNA was generated from mRNA
using Superscript II RT (GIBCO BRL). The specific oligonucleotide
primers for the PCR were 5'-CCTCCTACCGCAGCATCG-3' (sense) and
5'-TCCATCATGAAGTTGTGCAC-3' (antisense). The size of the expected
fragments was 382 bp. The PCR reactions were performed with Taq
polymerase (GIBCO BRL). The PCR conditioning was as follows: 35 cycles of denaturation at 94°C for 30 s, annealing at 57°C for
30 s, and extension at 72°C for 1 min. As a control,
-actin
cDNA was amplified using the primers 5'-GACTACCTCATGAAGATCCT-3' (sense)
and 5'-CCACATCTGCTGGAAGGTGG-3' (antisense), and a 510-bp product was
obtained. Each PCR reaction was performed at least three times, and
products were visualized by loading a 2-µl sample on a 1% agarose
gel using a 100-bp DNA ladder marker (TOYOBO, Tokyo, Japan) as a
standard. The fragments were first subcloned into pGEM-T Easy vector
(Promega, Tokyo, Japan) and sequenced.
 |
RESULTS |
Characteristics of ACh- or A-23187-evoked
K+ efflux from bovine parotid tissue.
Stimulation of salivary glands with muscarinic agonists is well known
to cause a large net K+ efflux due to activation of
KCa channels on the basolateral membrane of the acinar
cells (7). To examine whether TEA-insensitive KCa channels distinct from the maxi-K+ channels
may play a crucial role in muscarinically stimulated K+
efflux in BPA cells, we measured net K+ flux to and from
the perfused segments of the bovine parotid gland tissue using flame
photometry. Figure
1A shows a
typical example of the experiments indicating the dynamic changes of
net K+ movement induced by ACh (10 µM). Stimulation with
ACh for 3 min induced a transient net K+ efflux followed by
a sustained net influx of K+ after the cessation of
stimulation. When ACh was administered during two consecutive
stimulations, with a 15-min period of recovery, the total amounts of
net K+ efflux during the two stimulations were not
different. These ACh-induced responses were completely inhibited by the
muscarinic receptor antagonist atropine (1 µM) (data not shown). The
dose-response relationship of the effect of ACh on net K+
efflux is also shown in Fig. 1A. Because stable and
reproducible responses were obtained at a concentration of 10 µM ACh,
we used this concentration in the remaining experiments.

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Fig. 1.
A: time course of ACh (10 µM)-induced net
K+ efflux from perfused bovine parotid fragments. Fragments
were stimulated twice with ACh for 3 min. Inset:
dose-response relation for the effect of ACh on net K+
efflux. Total net K+ efflux during the 2nd stimulation with
various concentrations of ACh normalized to that during the 1st
stimulation with ACh (10 µM) is plotted as a function of ACh
concentration. Values are means ± SE of 4 experiments.
B: effect of removal of HCO (25 mM)/CO2 (5%) from perfusing solution on ACh-induced net
K+ efflux. Fragments were stimulated 3 times with ACh for 3 min in the presence and absence of
HCO /CO2 in the perfusing solution.
C: effect of removal of
HCO /CO2 from perfusing solution on
A-23187 (3 µM)-induced net K+ efflux. Perfusion of
Ca2+-free solution containing 0.5 mM EGTA was begun before
addition of A-23187. Net K+ efflux was evoked by addition
of CaCl2 (3 mM) to standard perfusing solution in the
presence and absence of HCO /CO2 in
perfusing solution. D: summary of effect of removal of
HCO /CO2 from perfusing solution on ACh
(open bars)- or A-23187 (filled bars)-induced response. Total net
K+ efflux during the 2nd or 3rd stimulation (in the absence
or presence of HCO /CO2, respectively)
with ACh or A-23187 was expressed as a percentage of that of observed
during the 1st stimulation with ACh or A-23187 (control). Values are
means ± SE of 5 experiments. E and F:
effect of tetraethylammonium (TEA; 10 mM) on the ACh (E)- or
A-23187 (F)-induced response. Insets: total net
K+ efflux during the 2nd (in presence of TEA) or 3rd
stimulation with ACh was expressed as a percentage of that of observed
during the 1st stimulation with ACh (control). Values are means ± SE of 7 experiments. F, inset: total net
K+ efflux during the 2nd (in presence of TEA) or 3rd
stimulation with A-23187 was expressed as a percentage of that of
observed during the 1st stimulation with A-23187 (control). Values are
means ± SE of 5 experiments.
|
|
The ACh-induced K+ efflux appeared to be dependent on
cytosolic Ca2+ concentration for the following reasons.
First, in experiments where tissues were stimulated twice with ACh
under conditions in which CaCl2 had been removed from the
perfusing solution (with addition of 0.5 mM EGTA), the first
stimulation with ACh (10 µM) evoked a small and transient net
K+ efflux, whereas the second stimulation with ACh induced
a much smaller net K+ efflux (17.1 ± 2.1%,
n = 5, of the control response). When CaCl2 (1 mM) was reintroduced into the perfusing solution, ACh was again able
to evoke net K+ efflux. Second, the Ca2+
ionophore A-23187 (3 µM) with added Ca2+ (3 mM) was able
to mimic the ACh-induced response (see also Fig. 1F).
We next examined the effect of removal of
HCO
/CO2 from the perfusing solution on
the ACh-induced net K+ efflux. Figure 1B shows
an example of the experiments demonstrating the absolute
HCO
/CO2 requirement for ACh-induced net
K+ efflux in bovine parotid fragments. The
HCO
/CO2 dependency of the net
K+ efflux in bovine parotid fragments was confirmed in
experiments where the effect of removal of
HCO
/CO2 from the perfusing solution was
examined on A-23187-induced net K+ efflux (Fig.
1C). The HCO
/CO2 requirement for ACh- and A-23187-induced net K+ efflux in bovine
parotid fragments is summarized in Fig. 1D. In the control
experiments, replacement of 25 mM Cl
with equimolar
glutamate in the presence of HCO
/CO2 in
the perfusing solution had no effect on the ACh-induced response (data
not shown).
To assess the involvement of maxi-K+ channels in a
HCO
/CO2-dependent,
Ca2+-activated K+ efflux pathway, we next
examined the effect of TEA (10 mM) on the ACh- and A-23187-induced net
K+ efflux. As shown in Fig. 1, E and
F, TEA (10 mM) did not inhibit the ACh- or A-23187-induced
response. Quinine (1 mM) reduced the ACh-induced K+ efflux
to 40.2 ± 4.5% (n = 4; P < 0.002) of the control response. Ba2+ (1 mM) had a tendency
to inhibit the ACh-induced K+ efflux so that it reduced the
efflux to 63.5 ± 11.2% (n = 5; P = 0.06) of the control response.
Characteristics of TEA-insensitive,
Ca2+-activated whole cell
K+ currents in BPA cells.
Using the conventional whole cell patch-clamp technique, we next
examined whether BPA cells would contain a TEA-insensitive KCa conductance. Figure
2A shows
representative recordings of whole cell currents from single BPA cells
dialyzed with the standard K-glutamate-rich pipette solution having
10
7 M free Ca2+. The bath solution was a
standard Na-glutamate-rich solution. Under these experimental
conditions, the steady-state whole cell I-V relationship of
BPA cells had both outwardly and inwardly rectifying components (see
also Fig. 2C). The outwardly rectifying current became
evident at potentials more positive than
22 mV, had a
characteristically noisy time course, and activated over 20-30 ms
following depolarization. The outwardly rectifying conductance was
strongly inhibited by TEA (10 mM), a maxi-K+ channel
blocker. The current was carried by K+, because replacement
of the K+ in the pipette solution by Cs+ or
Na+ abolished the outward current (data not shown). The
inwardly rectifying component was evident at potentials more negative
than
82 mV, was characteristically noise free, and activated rapidly. The inwardly rectifying component was completely blocked by a low
concentration of Ba2+ (0.1 mM) (data not shown). Our
unpublished results suggest that the conductance is mediated by an
inwardly rectifying K+ channel, Kir2.1 (M. Hayashi, S. Komazaki, and T. Ishikawa, unpublished observations).

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Fig. 2.
A and B: representative whole
cell recordings from a single bovine parotid acinar (BPA) cell with
standard K-glutamate [pCa = 7 (A) or pCa = 6 (B)] pipette solution and standard Na-glutamate bath
solution before and after addition of TEA (10 mM) to the bath. The
cells were held at 62 mV and stepped for intervals of 400 ms to
potentials ranging between 122 and +48 mV in 10-mV steps.
C: steady-state current-voltage (I-V)
relationships for TEA-insensitive whole cell currents from cells
dialyzed with pipette solutions having pCa = 7 ( ;
n = 4) and pCa = 6 ( ;
n = 20), respectively. Vm,
membrane potential.
|
|
Figure 2B shows representative recordings of the whole cell
currents before and after addition of TEA (10 mM) to the bath solution
for a single BPA cell dialyzed with the standard pipette solution with
10
6 M free Ca2+. In the presence of TEA (10 mM), depolarizing and hyperpolarizing voltage steps from
62 mV
rapidly elicited whole cell currents with a reversal potential
(
67.2 ± 1.4 mV, n = 14) close to the Nernst
potential for K+ (
87.3 mV). Under these experimental
conditions, the average current amplitude at
2 mV was 1,452.2 ± 288.2 pA (n = 13). The I-V relationships
corresponding to the two different free Ca2+ concentrations
(i.e., 10
7 and 10
6 M) are shown in Fig.
2C. In contrast to the cells dialyzed with pipette solutions
with 10
7 free Ca2+, cells dialyzed with
pipette solutions containing 10
6 M free Ca2+
exhibited a large whole cell outward current whose I-V
relationship was nearly linear over a wide voltage range under a
physiological Na+/K+ gradient. The reversal
potential of the TEA-insensitive current was shifted from
80.1 ± 5.8 mV (n = 4) to +1.4 ± 0.7 mV
(n = 12) with a slope of 44.2 mV per e-fold
change when external K+ concentration was varied
between 2 and 150 mM (Fig.
3A). We then performed
experiments where K+ (150 mM) in the bath solution was
replaced with equimolar Rb+ and found little, if any,
change in reversal potential (0.0 ± 0.9 mV, n = 4) (Fig. 3B), suggesting that Rb+ had an equal
permeability relative to K+. We also performed additional
experiments in which cells were dialyzed in the standard
Na-glutamate-rich bathing solution containing TEA (10 mM) with a
pipette solution in which K+ was totally replaced with
Rb+, Cs+, or Na+. In independent
experiments for Cs+ or Na+, the average current
amplitude at a holding potential of
3 or 0 mV was 16.6 ± 11.2 pA (n = 7) or
20.0 ± 70.0 pA (n = 7), respectively. In contrast with Cs+ or Na+
substitution experiments, when the cells were dialyzed with a Rb-glutamate-rich pipette solution, a large outward conductance was
observed (Fig. 3C). In these experiments, the current
amplitude at
2 mV and the reversal potential were 3,442.9 ± 917.7 pA (n = 7) and
66.6 ± 2.8 mV
(n = 7), respectively. Taken together, these results
suggest that the sequence of the relative monovalent cation
permeabilities for the TEA-insensitive KCa conductance is
K+ = Rb+
Na+ = Cs+.

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Fig. 3.
A: semilogarithmic plot of the reversal
potential of TEA (10 mM)-insensitive whole cell currents as a function
of extracellular K+ concentration
([K+]o). The cells were dialyzed with pipette
solutions having pCa = 6. The continuous line shows the linear
regression fit to the data. Values are means ± SE of 4-14
experiments. B: steady-state I-V relationships
for TEA-insensitive whole cell currents from cells bathed in 150 mM
K-glutamate-rich ( ) or 150 mM Rb-glutamate-rich
( ) solution. The cells were dialyzed with pipette
solutions having pCa = 6. Values are means ± SE of 4 experiments. C: steady-state I-V relationships
for whole cell currents from cells dialyzed with Rb-glutamate-rich
( ; mean of 7 experiments) or Cs-glutamate-rich
( ; mean of 7 experiments) pipette solution having
pCa = 6. The bath solution was the standard Na-glutamate solution.
Error bars representing SE were omitted when so small as to lie within
symbols. D: dependency of TEA-insensitive whole cell current
activation on free Ca2+ concentrations
([Ca2+]i) in the pipette solution. Whole cell
current amplitudes at 2 mV are plotted against
[Ca2+]i in the pipette solution. Values are
means ± SE of 4-20 experiments.
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|
We next examined the Ca2+ dependency of the TEA-insensitive
K+ currents in experiments in which cells were dialyzed
with pipette solutions having different, buffered free Ca2+
concentrations (Fig. 3D). In contrast to the cells dialyzed
with pipette solutions having 10
6 or 3 × 10
7 M free Ca2+ concentrations, cells
dialyzed with pipette solutions containing either 10
7 or
10
8 M free Ca2+ concentrations exhibited only
small whole cell outward currents. The average current amplitudes at
2 mV obtained from independent experiments with 10
7 and
10
8 M free Ca2+ were 184.5 ± 52.9 pA
(n = 4) and 96.1 ± 26.6 pA (n = 6), respectively.
Ca2+-dependent activation of
Rb+ currents in excised inside-out
macropatches obtained from basolateral membrane of BPA cells.
We further characterized the TEA-insensitive KCa
conductance in inside-out or outside-out macropatches excised from
basolateral membrane of acinar cells. In preparing dispersed cells from
the bovine parotid gland, we could easily collect intact acini in which
the cell orientation was undisturbed and routinely obtain excised
patches. In these experiments, we used Rb+ as a charge
carrier because the contribution of the conductance through inwardly
rectifying K+ channels can be minimized (M. Hayashi, S. Komazaki, and T. Ishikawa, unpublished observations). We also included
TEA (10 mM) in the pipette or the bath solution to block the
maxi-K+ channels in inside-out or outside-out patch
experiments, respectively. Figure
4A shows
the representative tracings of currents elicited by 400-ms voltage
steps from inside-out macropatches excised from basolateral membrane of
acinar cells. Voltage-step commands evoked large, time-independent
currents only when Ca2+ was included in the (bath) internal
solution. Under these conditions, average current amplitudes of
Ca2+ (1 µM)-activated Rb+ currents at
75
and
55 mV were
481.5 ± 48.4 pA (n = 41)
and
371.2 ± 40.2 pA (n = 41), respectively. In
separate experiments, as shown in Fig. 4B, we confirmed the
monovalent cation selectivity of the Ca2+ (1 µM)-activated currents under these conditions. When the bathing solution was replaced from an
N-methyl-D-glucamine (NMDG)-glutamate-rich to a
Na-glutamate-rich solution, the reversal potential of the currents was
not changed. However, K+ substitution for Na+
caused a shift of the reversal potential to close to zero. Figure 4C shows an example of inside-out macropatch experiments
where free Ca2+ concentration in the fluid bathing the
cytosolic surface of the patch was varied between <1 nM and 10 µM.
Figure 4D plots Rb+ inward current amplitude at
55 mV against free Ca2+ concentration in the fluid
bathing the cytosolic surface of the patch, demonstrating a steep
Ca2+-dependent activation of the current. To provide a
quantitative description of the Ca2+ dependence, we fit the
Hill equation (see MATERIALS AND METHODS) to these data and
obtained an apparent Kd of 0.43 ± 0.05 µM and a Hill coefficient of 2.54 ± 0.29 (n = 5) at
55 mV. Figure 4E also shows the plots of
Kd and Hill coefficient against membrane potential and demonstrates that Ca2+ activation of the
currents is independent of membrane potential. The mean
Kd values and Hill coefficients were 0.46 ± 0.06 µM and 3.43 ± 0.51 at
95 mV, 0.47 ± 0.05 µM
and 2.96 ± 0.41 at
75 mV, 0.47 ± 0.06 µM and 2.71 ± 0.34 at
35 mV, and 0.45 ± 0.06 µM and 2.52 ± 0.21 at
15 mV, respectively (n = 5).

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Fig. 4.
A: representative tracings of currents
elicited by voltage steps from an inside-out macropatch excised from
basolateral membrane of a BPA cell in the presence (left) or
absence (right) of internal Ca2+ (1 µM). The
membrane patch was held at +5 mV and stepped for intervals of 400 ms to
potentials ranging between 95 and +55 mV in 10-mV steps. The pipette
contained a Rb-glutamate-rich solution having 10 mM TEA, and the bath
contained the standard NMDG-glutamate solution. B: current
traces elicited by 800-ms voltage ramps from 75 to +55 mV from an
inside-out macropatch excised from basolateral membrane of a BPA cell.
The traces were obtained in the bathing solutions containing the
indicated monovalent cations (K+, Na+, and
NMDG+) in the presence of 1 µM free Ca2+. The
pipette contained a Rb-glutamate-rich solution having 10 mM TEA.
C: representative I-V relationships for currents
in the presence of different [Ca2+]i
( , <1 nM; , 0.1 µM;
, 0.3 µM; , 1 µM; ,
10 µM). The pipette contained a Rb-glutamate-rich solution having 10 mM TEA, and the bath solution was NMDG-glutamate rich. D:
Ca2+ concentration response for TEA-insensitive
Rb+ currents measured at 55 mV normalized by the response
to Ca2+ (1 µM)
[I/ICa2+ (1 µM)]
plotted as a function of [Ca2+]i. Data were
fit with the Hill equation (Eq. 1), yielding a
Kd = 0.43 µM and a Hill coefficient = 2.54. Values are means ± SE of 5 experiments. E:
voltage independence of Kd.
Kd values (top) and Hill coefficient
(bottom) plotted as functions of the membrane potential.
Values are means ± SE of 5 experiments.
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Pharmacological characteristics of TEA-insensitive,
Ca2+-activated
Rb+ currents in excised basolateral
membrane macropatches.
We next investigated the effect of K+ channel blockers on
the TEA-insensitive, Ca2+-activated Rb+
currents in outside-out macropatches. The pipette contained a Rb-glutamate-rich solution with a 1 µM free Ca2+
concentration, and the bath contained a Rb-Cl-rich solution with TEA
(10 mM). The addition of quinine (1 mM) to the bath inhibited Ca2+-activated Rb+ currents (Fig.
5A), and the inhibition was
reversible and voltage independent (data not shown). Extracellular
Ba2+ (1 and 10 mM) also reduced the currents in a
concentration-dependent manner. In contrast to quinine,
Ba2+ inhibited the currents in a voltage-dependent manner
with the block increasing with hyperpolarization (Fig. 5B).
The addition of Ba2+ (1 and 10 mM) to the bathing solution
reduced the current amplitude at
65 mV to 79.2 ± 7.6%
(P = 0.07) and 50.3 ± 7.2% (P < 0.01) of the control level (n = 5), respectively (Fig.
5B). We next analyzed the voltage dependence of the
Ba2+ block (Fig. 5C). The data for 10 mM
Ba2+ were fitted by using Eq. 2 (see
MATERIALS AND METHODS) to determine the
Kd at each potential. The following
Kd values were estimated: 1.06 ± 0.12 mM
at
125 mV, 2.05 ± 0.43 mM at
115 mV, 3.13 ± 0.62 mM at
105 mV, 4.75 ± 0.98 mM at
95 mV, 6.30 ± 1.18 mM at
85 mV, and 9.67 ± 1.86 mM at
75 mV (n = 3). These
data were fitted by using Eq. 3 (see MATERIALS AND
METHODS) with a Kd(0) of
266.71 ± 95.79 mM and a slope (
) of 0.53 ± 0.03 (n = 3).

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Fig. 5.
A: effect of quinine on Ca2+ (1 µM)-activated Rb+ currents in outside-out macropatches.
Representative I-V relationships in the absence
( , control) and presence of quinine ( ,
0.1 mM; , 1 mM) obtained from an outside-out macropatch
are shown (left). The pipette contained a Rb-glutamate-rich
solution having 1 µM free Ca2+, and the bath
contained a RbCl-rich solution containing 10 mM TEA. Quinine was
applied to the bathing solution. , Data obtained in a
Rb+-free, NMDG-Cl-rich bath solution containing 10 mM TEA.
Bar graph shows normalized current in the presence of quinine (0.1 and
1 mM) (right). Normalized current was determined by
comparing the current amplitude at 65 mV. In each experiment, current
was recorded in a Rb+-free, NMDG-Cl-rich solution and
subtracted from each total current to calculate the
Ca2+-activated Rb+ currents. *P < 0.05, **P < 0.01. B: effect of
Ba2+ ( , 0.1 mM; , 1 mM; and
, 10 mM) on Ca2+ (1 µM)-activated
Rb+ currents in outside-out macropatches. Representative
I-V relationships in the absence ( , control)
and presence of blocker obtained from an outside-out macropatch are
shown (left). The pipette and the bath contained a
Rb-glutamate-rich solution having 1 µM free Ca2+ and a
RbCl-rich solution containing 10 mM TEA, respectively.
, Data obtained in a Rb+-free, NMDG-Cl-rich
bath solution containing 10 mM TEA. Bar graph shows normalized current
in the presence of Ba2+ (1 and 10 mM) (right).
Normalized current was determined by comparing the current amplitude at
65 mV. In each experiment, current was recorded in a
Rb+-free, NMDG-Cl-rich solution and subtracted from each
total current to calculate the Ca2+-activated
Rb+ currents. **P < 0.01. C:
voltage dependence of Kd values for
Ba2+ (10 mM) block of Ca2+ (1 µM)-activated
Rb+ currents. Data were fitted with an equation derived
from Eqs. 2 and 3 (see MATERIALS AND
METHODS). Kd values were plotted on a
semilogarithmic scale. In each experiment, current was recorded in a
Rb+-free, NaCl-rich solution containing 10 mM TEA and
subtracted from each total current to calculate the
Ca2+-activated Rb+ currents. The continuous
line shows the linear regression fit to the data. Values are means ± SE of 3 experiments.
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We also examined several other compounds for their ability to
block TEA-insensitive, Ca2+-activated Rb+
currents in outside-out macropatches. Figure
6, A and B, shows an example of experiments using clotrimazole or charybdotoxin, a known
blocker of SK4/IK1 channel, respectively. Extracellular application of
clotrimazole (0.1 and 1 µM) reduced the Rb+ currents in a
dose-dependent manner (Fig. 6A). We also found that
charybdotoxin (100 nM) strongly blocked the Rb+ currents
(Fig. 6B). Clotrimazole- or charybdotoxin-induced inhibition of the currents was not voltage dependent (data not shown). In contrast
to these inhibitors, neither apamin (100 nM) nor
d-tubocrarine (100 µM), a known SK(1-3)
channel blocker did reduce the currents (Fig. 6, C and
D).

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Fig. 6.
A: effect of external clotrimazole (0.1 and 1 µM) (A), charybdotoxin (100 nM) (B), apamin
(100 nM) (C), or d-tubocrarine (100 µM)
(D) on Ca2+ (1 µM)-activated Rb+
currents in outside-out macropatches. Representative instantaneous
I-V relationships in the absence and presence of the
blockers are shown. Insets: bar graphs showing normalized
current in the presence of each blocker. Normalized current was
determined by comparing the current amplitude at 65 mV in the absence
(C) or presence of each blocker. In each experiment, current was
recorded in a Rb+-free, NMDG-Cl-rich solution and
subtracted from each total current to calculate the
Ca2+-activated Rb+ currents. Values are
means ± SE of 5 (A, D) or 6 (B,
C) experiments. *P < 0.05, **P < 0.01.
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We next examined the effect of 1-EBIO on TEA-insensitive,
Ca2+-activated Rb+ currents in excised
inside-out patches. 1-EBIO has been shown to activate heterologously
expressed SK4/IK1 channels (30, 43, 51). Figure
7A illustrates the response of
the Rb+ currents to cytoplasmic application of 1-EBIO (100 µM) at 0.3 µM free Ca2+. 1-EBIO induced robust
activation of Rb+ currents at 0.3 µM free
Ca2+. However, 1-EBIO did not induce currents in a
Ca2+-free solution (<1 nM) and was not able to further
increase the amplitude of the Rb+ currents when applied at
a saturating free Ca2+ concentration of 1 µM (Fig. 7,
B and C). These results suggest that 1-EBIO
shifted the Ca2+ dependency of channel activation without
changing the maximum level.

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Fig. 7.
A and B: effect of
1-ethyl-2-benzimidazolinone (1-EBIO; 100 µM) on TEA (10 mM)-insensitive Rb+ currents activated by 0.3 (A) or 1 µM Ca2+ (B).
Representative traces of the currents elicited by 800-ms voltage ramps
from 75 to +55 mV obtained from an inside-out macropatch are shown.
The pipette contained a Rb-glutamate-rich solution having 10 mM TEA,
and the bath contained a NMDG-glutamate-rich solution. C:
Ca2+ concentration response for the native TEA-insensitive
Rb+ currents in the absence ( ) and presence
of 1-EBIO ( ). The current measured at 55 mV
normalized by the response to Ca2+ (1 µM) in the absence
of 1-EBIO is plotted as a function of
[Ca2+]i. Values are means ± SE of
3-6 experiments, except for data point at 0.1 µM free
Ca2+ obtained from a single experiment.
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Calmodulin antagonists inhibit TEA-insensitive,
Ca2+-activated
Rb+ currents in BPA cells.
To examine the role of calmodulin (CaM) in the Ca2+
activation of the currents, we next investigated the effects of
structurally unrelated Ca2+/CaM antagonists,
trifluoperazine (TFP), calmidazolium, and W-7, on the TEA-insensitive,
Ca2+-activated Rb+ currents in inside-out
macropatches. Representative current traces are shown in Fig.
8,
A-C. In each experiment,
Ca2+-independent current was recorded and subtracted from
each total current to calculate the Ca2+-activated
Rb+ currents. As shown, these compounds inhibited
TEA-insensitive, Ca2+(1 µM)-activated Rb+
currents. When bath-applied during a recording, TFP (100 µM), calmidazolium (10 µM), and W-7 (50 µM) reduced the current to 43.0 ± 20.9% (n = 9; P < 0.0001), 4.7 ± 5.5% (n = 4; P < 0.0001), and 64.8 ± 25.3% (n = 6;
P < 0.02) of the control currents at
55 mV, respectively.
In contrast to the effects of calmidazolium, TFP and W-7 partially
blocked the currents, although one experiment (of 9 experiments for TFP
and of 6 experiments for W-7) showed a complete inhibition by these
blockers. Inhibitory effects of TFP and W-7 were reversible, but the
calmidazolium-induced inhibition was not. We also analyzed the voltage
dependence of the inhibition, which is summarized in Fig. 8,
D-F, by plotting the current amplitude as a function of
membrane potential. None of these CaM antagonists showed a
voltage-dependent block.

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Fig. 8.
Effects of the calmodulin antagonists
trifluoperazine (TFP; 100 µM), calmidazolium (10 µM), and W-7 (50 µM) on TEA (10 mM)-insensitive Rb+ currents activated by
1 µM Ca2+. A-C: representative
instantaneous I-V relationships of currents in inside-out
macropatches in the absence and presence of TFP (A),
calmidazolium (B), or W-7 (C). Ramp command
voltages were applied from 75 to +55 mV. D-F: average
current in the presence of TFP (D), calmidazolium
(E), or W-7 (F) expressed as a percentage of
control current at each membrane potential between 75 and +25 mV.
Values are means ± SE of 4-9 experiments.
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RT-PCR study.
Because the above-described electrophysiological properties of native
TEA-insensitive KCa currents identified in BPA cells are
strikingly similar to those of expressed the SK4/IK1 channels, we next
performed RT-PCR with mRNA of freshly isolated BPA cells to examine
whether the cells contain the molecular candidate of the K+
channels. Because complete sequences for SK4/IK1 for Rattus
norvegicus and Homo sapiens (but not for Bos
taurus) have been published, we performed RT-PCR with specific
primers for hSK4/IK1 and rSK4/IK1 (described in detail in
MATERIALS AND METHODS). Under the experimental conditions
chosen, we could detect the expected 382-bp amplicon. The results are
summarized in Fig. 9A. The
sequence analysis of the PCR fragment showed that the sequence was
almost identical to those reported in human (GenBank accession nos.
NM_002250 and AF000972), rat (NM_023021), and mouse SK4/IK1 (NM_008433)
so that it shared 94, 87, and 90% nucleotide identity with them and 99, 96, and 98% similarity at the amino acid level, respectively. Because it has been shown that rat submandibular acinar cells exhibit
the TEA-insensitive KCa currents, we used these same
primers and performed RT-PCR with mRNA of freshly isolated rat
submandibular acinar cells, also detecting the expected 382-bp amplicon
(Fig. 9B), which was confirmed by sequence analysis to be
the described rSK4/IK1 (NM_023021).

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Fig. 9.
RT-PCR studies of BPA cells (A) and rat submandibular
acinar cells (B). Amplified PCR products generated using
gene-specific primers (described in MATERIALS AND METHODS)
for SK4/IK1 and -actin were fractionated on 1% agarose gels, and
size markers were used to indicate the size of the experimental
fragments. Lane M, 100-bp DNA ladder size standard. RT,
reverse transcriptase.
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 |
DISCUSSION |
Muscarinically and Ca2+-evoked
K+ efflux in BPA cells are mediated by
TEA-insensitive, Ca2+-activated
K+ channels distinct from
maxi-K+ channels.
To our knowledge, there is only one study of muscarinic agonist,
carbachol-activated Ca2+-dependent K+ efflux
from bovine parotid tissue using 86Rb+ as a
K+ marker (34). The
86Rb+ efflux study showed that
carbachol-induced 86Rb+ loss from bovine
parotid was not blocked by 5 mM TEA. However, because described
properties of maxi-K+ channels in salivary acinar cells do
have a very low conductance for Rb+ (12), the
involvement of maxi-K+ channels in mediating
Ca2+-dependent K+ efflux might not have been
assessed correctly. Therefore, we performed the present K+
flux measurements by using tissue fragments to ascertain whether maxi-K+ channels play an important role in
Ca2+-dependent K+ efflux during muscarinically
stimulated fluid secretion in bovine parotid as shown in other salivary
glands (27). Two observations provided consistently
negative answers. Neither ACh (10 µM)- nor Ca2+ ionophore
A-23187 (3 µM)-induced net K+ efflux from perfused bovine
parotid tissues was inhibited by TEA (10 mM) (Fig. 1, E and
F). Because the concentration of TEA used in the present
study is shown to completely block the maxi-K+ channels in
BPA cells (data not shown), as also shown in various tissues, these
results should reveal that a major part of the Ca2+-dependent K+ efflux, most probably across
the basolateral membrane, is largely independent of maxi-K+
channels. This conclusion was further supported by the present whole
cell patch-clamp studies demonstrating a TEA-insensitive KCa conductance in BPA cells (Fig. 2B). The view
that the TEA (10 mM)-insensitive whole cell currents described in this
study are mediated by KCa channels is strongly supported by
the following observations: 1) the current was
K+ selective over Na+ and Cs+
(Figs. 2C and 3, A and C), and
2) the current was activated by cytosolic Ca2+
(Fig. 3D). Although the localization of the TEA-insensitive
KCa channels to the basolateral membrane or the apical
membrane of the acinar cells cannot be decided from the whole cell
patch-clamp experiments, the present study in macropatches excised from
the basolateral membrane provides direct evidence for the basolateral localization of the channels. Our results do not exclude the
possibility that the channels are also functionally expressed in apical
membrane as well, however.
ACh- or A-23187-evoked net K+ efflux from the bovine
parotid tissue was totally dependent on the presence of
HCO
/CO2 in the perfusate (Fig. 1,
B and C). These results are in good agreement
with the previous 86Rb+ flux study with bovine
parotid (34), which showed that carbachol-induced 86Rb+ loss in a nominally
HCO
/CO2-free solution was significantly
less than in the presence of HCO
/CO2. Interestingly, that study also showed that the
86Rb+ loss was inhibited by the anion channel
blocker diphenylamino-2-carboxylate and unaffected by the complete
replacement of extracellular Cl
with gluconate
(34). Furthermore, a previous study with sheep parotid
acinar cells also demonstrated that ACh caused a large, transient
decrease in cytosolic pH, which was largely reduced in the
absence of extracellular HCO
/CO2, blocked by the anion channel blocker
5-nitro-2-(3-phenylpropylamino)-benzoate, and unaffected when
extracellular Cl
was replaced by gluconate
(47). Taken together with these previous data, assuming
that the currently accepted model of salivary secretion would be
applicable to bovine parotid secretion, the
HCO
/CO2-dependent K+ efflux
could be interpreted as a model showing that the secretion is driven by
electrogenic apical HCO
efflux via anion channels
that is electrically balanced by a simultaneous basolateral
K+ efflux via Ca2+-activated K+
channels distinct from maxi-K+ channels. Although there is
no direct electrophysiological evidence for the presence of a
HCO
-permeable anion channel in BPA cells at this
time, it is interesting to note that a Ca2+-activated anion
conductance in rat submandibular acinar cells has been shown to be
permeable to HCO
(25). Further
patch-clamp studies are indeed required to answer the question of
whether such a HCO
-permeable anion conductance is
also present in BPA cells.
The inhibition of ACh- or A23187-evoked net K+ efflux upon
removal of HCO
/CO2 could also be, at least in part, due to an inhibition of the TEA-insensitive
KCa channels by changes in intracellular pH, because a
transient intracellular acidification induced by ACh is known to be
inhibited in a nominally HCO
/CO2-free
buffer in various salivary acinar cells including ruminant parotid
acinar cells (7, 47). In fact, endogenously expressed
SK4/IK1-like channels in T84 cells, human colonic adenocarcinoma cells,
have been reported to be highly sensitive to cytosolic pH
(9).
Comparison of native bovine parotid KCa currents with
heterologously expressed SK4/IK1 currents and other native SK4/IK1-like
currents.
Biophysical properties of the native TEA-insensitive,
Ca2+-activated K+ (Rb+) currents in
BPA cells had the following features: 1) weakly inward
rectifying I-V relationships in symmetrical Rb+
conditions and nearly linear I-V relationships in
physiological Na+/K+ (Rb+) gradient
(Figs. 2C, 3C, and 5A), 2)
time- and voltage-independent activation (Fig. 4A),
3) a permeability
(PX/PK) sequence
estimated from reversal potentials indicating K+ = Rb+
Na+, NMDG+ (Figs. 3,
B and C, and 4B) activation by
submicromolar free Ca2+ (Figs. 3D and
4D). These characteristics of the native currents are in
common with those of the naturally and heterologously expressed SK4/IK1
currents characterized at the single-channel and whole cell level
(15, 24, 30, 31, 32, 35, 51) as follows: 1)
inwardly rectified single channel (10-54 pS) and whole cell I-V relationships in symmetrical K+ conditions
and nearly linear I-V relationships in physiological Na+/K+ gradient, 2) time- and
voltage-independent activation of the whole cell currents and
single-channel gating, 3) a permeability (PX/PK) sequence for
whole cell current indicating K+ = Rb+ > Cs+
Na+,
Li+, NMDG+, and 4) activation by
submicromolar free Ca2+ concentration with an
EC50 of 0.1-0.5 µM. Although we have not provided
evidence for a similarity of the native channels to the expressed
SK4/IK1 at the single-channel level in the present study, our
preliminary inside-out patch experiments have shown an inwardly rectifying, Rb+-permeable, Ca2+-activated,
intermediate-conductance (40-50 pS) K+ channel.
Further studies are indeed required to identify a single-channel conductance that mediates the currents identified in the present study.
The pharmacological properties of the native TEA-insensitive,
Ca2+-activated Rb+ currents in BPA cells had
the following features: the native currents were blocked by
clotrimazole (100 nM) (Fig. 6A) and charybdotoxin (100 nM)
(Fig. 6B), but not by apamin (100 nM) and
d-tubocrarine (0.1 mM) (Fig. 6, C and
D). Clotrimazole has been reported to block expressed
SK4/IK1 and native SK4/IK1-like currents (1, 4, 24, 30, 32, 35,
44) but not SK3-like currents (5). Charybdotoxin is
shown to block with high affinity expressed SK4/IK1 currents (24,
30, 31, 32, 35) and SK4/IK1-like currents in the native human T
lymphocyte and in red blood cells (Gardos channel) (4, 15, 32,
39, 44), whereas other SK channels are not affected (14,
19, 48). Conversely, apamin and d-tubocrarine have
been shown to block the cloned SK1-3 channels with different
affinities (16, 17, 24, 33, 45, 48), whereas there is no
effect on heterologously expressed SK4/IK1 (31, 32, 35)
and native SK4/IK1-like currents (3, 15, 32). Thus the
pharmacological profiles of the native KCa currents in BPA
cells are also similar to those of expressed SK4/IK1 channels but not
to those of SK1-3 channels.
Among the blockers tested in the present study, only extracellular
Ba2+ induced a voltage-dependent block of the
TEA-insensitive, Ca2+-activated Rb+ currents in
excised outside-out macropatches (Fig. 5, B and
C). As expected for an ion binding within the membrane
electrical field (54), the Kd (0 mV) for Ba2+ and the slope (
) of Ba2+ block
were estimated to be 267 mM and
= 0.53, respectively. These
results suggest that the Ba2+ binding site is located at an
electrical distance half the way across the ion conductive pathway from
the outside. A similar voltage-dependent block of Ba2+ has
been reported for naturally expressed SK4/IK1-like whole cell currents
in human T lymphocytes. The block of the currents by extracellular
Ba2+ was also as steep as expected from the movement of a
single divalent cation about 75% into the membrane field (
= 0.74) (15). To our knowledge, voltage-dependent block of
Ba2+ has not been reported for the heterologously expressed
SK4/IK1 channels, although there is a report showing that extracellular application of 1 mM Ba2+ reduced whole cell outward
K+ currents by 88% elicited by membrane depolarization to
+80 mV from the holding potential of
80 mV in Chinese hamster ovary (CHO) cells stably expressing hSK4/IK1 (31).
Ca2+ (1 µM)-activated hSK4/IK1-like K+
currents in T84 cells, in which transcripts of hSK4/IK1 are expressed, have been shown to be blocked by intracellular Ba2+ but not
by extracellular Ba2+ (9, 23).
We also extended the pharmacological study to include 1-EBIO, which has
been shown to increase both the heterologously expressed SK4/IK1
(30, 32, 35, 43, 51, 52) and other native SK4/IK1-like
currents (10, 32, 52). As expected from the striking
resemblance between native bovine KCa and expressed SK4/IK1 currents, the native Ca2+-activated Rb+
currents were modulated by 1-EBIO (Fig. 7). We also showed in excised
inside-out patches that 1-EBIO (100 µM) enhanced the currents induced
by a low Ca2+ activity (0.3 µM), but it had no effect at
a lower Ca2+ activity (<0.1 µM), and it did not further
increase the currents at a higher Ca2+ activity (1 µM)
(Fig. 7, B and C), suggesting that the activation of the native TEA-insensitive, Ca2+-activated
Rb+ currents by 1-EBIO is Ca2+ dependent and
likely due to a shift in Ca2+ sensitivity. Similar 1-EBIO
modulations have been reported for the cloned SK4/IK1 channels
(43, 51).
There is strong evidence that Ca2+ sensitivity of SK4/IK1
channels is mediated by CaM (11, 32, 56). It has also been
proposed that 1-EBIO interacts with the intracellular COOH terminus of SK4/IK1 channels, most likely the CaM binding domain, and stabilizes the Ca2+-CaM channel interaction that drives channel
opening (42). Therefore, 1-EBIO modulation and the
steepness of intracellular Ca2+ dependence of native
KCa currents in BPA cells may together suggest a
cooperative mechanism of channel activation via a Ca2+-CaM
channel interaction. In support of this view, all three CaM antagonists
consistently induced a reduction of the native KCa currents
in a voltage-independent manner (Fig. 8). We cannot exclude the
possibility, however, that these drugs inhibited the currents via a
Ca2+/CaM-independent mechanism. In fact, the published
information on the effect of CaM inhibitors on SK4/IK1 channels is
contradictory. Khanna et al. (32) found in conventional
whole cell configuration that both expressed hSK4/IK1 currents in CHO
cells and native human T-lymphocyte SK4/IK1-like currents were
sensitive to the CaM antagonists W7, calmidazolium, and TFP.
Conversely, Fanger et al. (11) showed that these
antagonists were ineffective in blocking whole cell currents at
physiological membrane potentials recorded from native T lymphocytes
and hIK1-transfected COS-7 cells. Von Hahn et al. (51)
found in inside-out patches that TFP and W-7 reversibly inhibited
expressed rSK4 currents in Xenopus oocytes but that
calmidazolium had no effect even at 10 µM. However, native
IKCa channels (Gardos channels) of human erythrocytes have been shown not to be inhibited by these CaM antagonists in
cell-attached and in excised inside-out patches (8).
Further studies are thus needed to elucidate the role of CaM in
Ca2+-dependent activation of the native SK4/IK1-like currents.
Physiological role of SK4/IK1-like channels in BPA and other
salivary acinar cells.
Unlike other well-studied salivary glands, little is known about the
secretory mechanism in bovine parotid by which large volumes of
HCO
-rich saliva are produced not only in response to
a secretomotor stimulus but also at rest (6). Because it
is well established that muscarinic stimulation of salivary gland to
drive fluid and electrolyte secretion is tightly coupled to
[Ca2+]i (7), it is reasonable to
conclude that the SK4/IK1-like channels identified in the present work
may be responsible for muscarinically stimulated secretion but not for
resting secretion by bovine parotid gland. This view is supported by
several lines of evidence. First, the SK4/IK1-like channels described
here exhibited the sensitivity needed to become active during a rise in
cytosolic free Ca2+ concentration above 10
7 M
(Figs. 3D and 4D). On the other hand, when BPA
cells were dialyzed with the standard K-glutamate-rich pipette solution
containing 10
7 M free Ca2+, or when the
cytosolic surfaces of excised inside-out patches were exposed to the
bathing solution containing 10
7 M free Ca2+,
the SK4/IK1-like channels were almost inactive (Figs. 3D and 4D). Under these conditions, the whole cell K+
conductance in these cells was dominated by outwardly and inwardly rectifying components, attributable to maxi-K+ channels and
inwardly rectifying K+ channels, respectively (Fig.
2A) (M. Hayashi, S. Komazaki, and T. Ishikawa, unpublished
observations). Second, the high Rb+ conductivity (Fig. 3,
B and C) and charybdotoxin sensitivity (Fig.
6B) of the channels are also in keeping with previous
86Rb+ efflux data in a bovine parotid mince
preparation (34), where muscarinic agonist
(carbachol)-induced 86Rb+ loss was inhibited by
Leiurus quinquestriatus venom, which contains charybdotoxin
(34). Finally, quinine and Ba2+ sensitivities
of the channels are in line with the present K+ efflux data
indicating that quinine (1 mM) and Ba2+ (1 mM) reduced the
ACh-induced K+ efflux. In the present study, we found that
Ba2+ (1 mM) had a tendency to inhibit the ACh-induced
K+ efflux, but the effect was not statistically significant
(P = 0.06). Given that extracellular Ba2+
blocks the SK4/IK1-like currents in a voltage-dependent manner (Fig. 5,
B and C), Ba2+ may not necessarily
block the K+ efflux significantly at physiological membrane
potentials in intact tissues. In fact, extracellular Ba2+
(1 mM) only reduced the current amplitude at
65 mV to 79.2 ± 7.6% of the control level (P = 0.07) (Fig.
5B).
An important question arising from the present study concerns the role
of SK4/IK1-like channels in mediating Ca2+-activated
K+ currents in other salivary acinar cells. As mentioned
previously, electrophysiological profiles of the SK4/IK1-like currents
in BPA cells have properties similar to those of Ca2+- or
ACh-activated whole cell K+ conductance reported in other
native salivary cells such as rat and mouse submandibular acinar cells
(21, 26) and in sheep parotid acinar cells
(20). Furthermore, our RT-PCR analysis has also shown
transcripts of rSK4/IK1 in rat submandibular acinar cells (Fig.
9B). The recent finding that human parotid acinar cells
exhibit charybdotoxin-sensitive, carbachol-induced whole cell
K+ currents (41) is also tempting to postulate
for a possible role of SK4/IK1 channels in mediating muscarinically
stimulated K+ currents in human as well. Therefore, it is
highly possible that SK4/IK1-like channels are commonly involved in
mediating K+ currents during muscarinically evoked
secretion by salivary glands in various species.
 |
ACKNOWLEDGEMENTS |
We thank Dr. K. Yoshimura for valuable advice on the perfusion
method and A. Inagaki for help in analyzing data and for comments on
the manuscript.
 |
FOOTNOTES |
This study was supported by grants from the Ito Foundation, Northern
Advancement Center for Science & Technology Foundation, and in part by
grants-in-aid for Scientific Research from the Ministry of Education,
Science, Sport and Culture of Japan. M. Hayashi was supported by a
Research Fellowship of the Japan Society for the Promotion of Science
for Young Scientists.
Address for reprint requests and other correspondence: T. Ishikawa, Laboratory of Physiology, Dept. of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido Univ., Sapporo 060-0818, Japan (E-mail:
torui{at}vetmed.hokudai.ac.jp).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published October 9, 2002;10.1152/ajpcell.00250.2002
Received 30 May 2002; accepted in final form 11 September 2002.
 |
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