Departments of 1 Anatomy and Neurobiology and of 2 Pharmacology, The University of Vermont, Burlington, Vermont 05401-2500
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ABSTRACT |
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The ionic
mechanisms associated with the control of gallbladder contractility are
incompletely understood. One type of
K+ current, the voltage-dependent
K+
(KV) current, is relatively
uncharacterized in gallbladder cells and may contribute to muscular
excitability. The main focus of this study was therefore to determine
the voltage dependence and pharmacological nature of this
K+ current in isolated myocytes
from mouse gallbladder, using the patch-clamp technique. Currents
through Ca2+-activated
K+ channels were minimized by
buffering of intracellular Ca2+
(20 nM free Ca2+) and by
inclusion of 1 mM tetraethylammonium
(TEA+) in the bathing solution.
With 140 mM symmetrical K+,
membrane depolarization increased
K+ currents, independent of
driving force, as assessed by tail current analysis. Half-maximal
activation of K+ currents occurred
at ~1 mV and increased e-fold per 9 mV. Inactivation also increased on depolarization, with a midpoint of
24 mV. Single KV channels
were recorded in the cell-attached configuration, exhibiting a
single-channel conductance of 4.9 pS.
TEA+ at 10 mM reduced
KV currents by 36%. At +50 mV, 1 mM and 10 mM 4-aminopyridine inhibited currents by 18% and 35%,
respectively, whereas 1 and 10 mM 3,4-diaminopyridine inhibited
currents by 11% and 21%, respectively. Quinine inhibited
KV currents (at +50 mV, 100 µM
and 1 mM quinine inhibited current by 24% and 70%, respectively). In
summary, we describe voltage-activated
K+ currents from the mouse
gallbladder that are likely to contribute to the control of muscular
excitability.
excitability; aminopyridine; tetraethylammonium; quinine
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INTRODUCTION |
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VOLTAGE-DEPENDENT K+ (KV) currents play an important role in the regulation of gallbladder smooth muscle excitability (16), which modulates Ca2+ entry and ultimately motility. KV channels have been described in all smooth muscle types (10) and regulate both membrane potential and action potential repolarization, as in the case of gallbladder smooth muscle (16).
KV currents, Ca2+-sensitive K+ (KCa) channels, and ATP-sensitive K+ (KATP) channels have been identified in gallbladder smooth muscle (15-17). Only KATP channels in gallbladder smooth muscle have been extensively characterized with respect to their voltage dependence, pharmacology, ATP dependence, and modulation by calcitonin gene-related peptide (via cAMP and protein kinase A) (15, 17).
Although KV channels regulate gallbladder smooth muscle excitability, little is known about their fundamental properties. The goals of this study were to provide the first characterization of the voltage dependence of KV currents in gallbladder smooth muscle, because this is key to their physiological function, and to provide a pharmacological fingerprint of this current for future dissection of its physiological role. We chose to study gallbladder smooth muscle from the mouse, since this would set the stage for future studies with transgenic animals.
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METHODS |
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Tissue Preparation
Mice (CD1, B6++, or SVJ) of either sex, weighing ~20 g, were anesthetized with pentobarbitone solution (0.5 ml) and killed by exsanguination. The abdominal cavity was opened, and the gallbladder was removed and transferred to cold, aerated (95% O2-5% CO2) Krebs solution. Under a dissecting microscope, the gallbladder was incised from the end of the cystic duct to the base and washed twice with an isolation medium (to remove any bile salts) containing (in mM) 80 L-glutamic acid (monosodium salt), 55 NaCl, 6 KCl, 10 HEPES, 2 MgCl2, and 10 glucose.Cell Isolation
The whole gallbladder was bisected longitudinally and initially placed into isolation medium containing 0.5 mg/ml papain (Worthington), 1 mg/ml dithioerythritol, and 1 mg/ml BSA (Sigma Chemical). The tissue pieces were incubated at 37°C for 30 min, after which they were transferred to a solution containing 0.5 mg/ml collagenase (Fluka), 1 mg/ml BSA, and 100 µM Ca2+ for 5 min. The tissue was washed with ice-cold isolation medium and then transferred to a vial containing isolation medium (with 1 mg/ml BSA) and triturated to yield single smooth muscle cells. When required, 2-3 drops of the cell suspension were diluted with isolation medium and placed in a recording chamber. Cells were allowed to adhere to a coverslip in the recording chamber for 30 min before recording.Solutions
For the majority of the experiments performed in this study, the whole cell bathing solution contained (in mM) 135 NaCl, 5 KCl, 1 MgCl2, 10 HEPES, and 0.1 CaCl2, adjusted to pH 7.4 with NaOH. The solution contained a low concentration of Ca2+ to decrease Ca2+ influx, and therefore the activity of KCa channels, and unless otherwise stated contained 1 mM tetraethylammonium (TEA+), also to block KCa channels (10). For tail current experiments, the whole cell bathing solution contained (in mM) 140 KCl, 1 MgCl2, 10 HEPES, 0.1 CaCl2, and 1 TEA+, adjusted to pH 7.4 with KOH. The bathing solution for the cell-attached single-channel experiments was (in mM) 140 KCl, 5 NaCl, 1 MgCl2, 10 HEPES, and 0.1 CaCl2, pH 7.4. For all whole cell experiments, the pipette solution was (in mM) 107 KCl, 33 KOH, 1 NaCl, 2 MgCl2, 10 EGTA, 10 HEPES, 1 CaCl2, 1 Na2ATP, and 1 Na2GTP, pH 7.2. The free Ca2+ concentration of this solution was estimated to be 20 nM, which should also limit the activity of KCa channels. The cell-attached pipette solution contained (in mM) 135 NaCl, 5 KCl, 1 MgCl2, 10 HEPES, 2 CaCl2, and 1 TEA+, pH 7.4.All experiments were performed at room temperature (22°C). Pipettes
were pulled from borosilicate glass (Sutter Instruments) with a
vertical puller (Narishige PP-83), coated with sticky dental wax, and
polished with a fire polisher (Narishige MF-83) to give a final tip
resistance of ~3-6 M. Currents were measured as previously described (7), using an Axopatch 200 amplifier (Axon Instruments), and
filtered using an eight-pole Bessel filter. Data acquisition rates were
as follows. For whole cell outward currents, experiments were filtered
at 500 Hz and digitized at 1 kHz; tail current experiments were
filtered at 1 kHz and digitized according to a two-step protocol, initially at 5.6 kHz and then at 16.7 kHz immediately before the step
to
70 mV; and cell-attached experiments were filtered at 1 kHz
and digitized at 4 kHz. Data were acquired and analyzed using pCLAMP
software (Axon Instruments) on an IBM 386/20 personal computer. Mean
whole cell capacitance was 45.0 ± 1.3 pF
(n = 61).
Materials
EGTA, HEPES, TEA+, 4-aminopyridine (4-AP), 3,4-diaminopyridine (3,4-DAP), and quinine were obtained from Sigma Chemical.Data Analysis
Pulsed whole cell currents and tail currents were measured using pCLAMP software. All currents described refer to peak current. Activation and inactivation curves were fit using Microcal Origin software on a Pentium personal computer according to the following Boltzmann distribution function
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To determine the exponential decay of currents, data were fit with Microcal Origin software using an exponential decay function
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Kd values were estimated assuming a 1:1 binding ratio using the following equation
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Results are expressed as means ± SE of n cells.
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RESULTS |
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Separation of KV Current from KCa Current
Membrane potential depolarization will activate KCa channels and KV currents in smooth muscles, including gallbladder (16). To minimize the contribution of KCa currents to the outward currents, the bathing and pipette solutions contained low levels of Ca2+ (external, 100 µM Ca2+; internal, buffered to 20 nM). TEA+ (1 mM) [~5 times the half-block constant for KCa channels (4, 9)] reduced the mean outward current at +50 mV by 7.8 ± 2.6% (n = 16) (Fig. 1, A and B). To determine whether 1 mM TEA+ was inhibiting all KCa channels in the cell membrane, 100 nM iberiotoxin [~10-100 times the half-block constant for KCa channels (4, 5)] was applied. Iberiotoxin (100 nM) reduced currents by a further 2.9 ± 0.9% (n = 4) at +50 mV (Fig. 1, A and B). These results suggest that control of Ca2+ (both intracellular and extracellular) and inclusion of 1 mM TEA+ in the bathing solution are successful at eliminating the majority of KCa current present in smooth muscle cells from mouse gallbladder.
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The outward currents increased steeply with membrane depolarization in
10-mV increments from a holding potential of 70 mV (Fig. 1,
B and
C). The activation component of the
currents (1 mM TEA+ in the bathing
solution) was fit with a single exponential and demonstrated that the
activation time constants decreased with depolarization
(t1 for
10, 0, +10, +20, +30, +40, and +50 mV was 161.4, 117.4, 81.5, 57.6, 41.8, 34.4, and 28.1 ms, respectively; n = 15). Under these
conditions, the K+,
Na+, and
Cl+ equilibrium potentials
(EK,
ENa, and
ECl,
respectively) were
85, +191, and
5 mV, respectively. An
increase in outward current could be observed at potentials more
positive than
40 mV, consistent with the current being
K+ selective. Also, under
conditions in which external K+
was 140 mM (i.e., symmetrical K+,
EK = 0 mV), the
reversal potential of the current was 0 mV (see Fig.
2A),
providing further evidence of the
K+-selective nature of the
currents.
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Voltage Dependence of K+ Current
Estimated from whole cell tail currents.
To determine the voltage dependence of the current, independent of
changes in unitary current, the activity of
KV channels was assessed at a
common potential (70 mV), after channels were activated to test
potentials between
50 and +50 mV, in 10-mV increments (400-ms
voltage steps were applied every 10 s). Pipette and bathing
K+ were both 140 mM
(EK = 0 mV).
Therefore the size of the inward current recorded at the step to
70 mV is a function of the activity of voltage-activated
K+ channels at the test potential
and the driving force at
70 mV. Figure
2A demonstrates that at a step from
70 to
10 mV, current is activated, but since
EK is 0 mV the
current recorded is inward. Immediately on the step back to
70
mV a tail current is evoked, the size of which is a function of the
activity of the channels activated at
10 mV and the driving
force at
70 mV. A step from
70 to +10 mV induces an
outward current, which is followed by a larger tail current at
70 mV, indicating that channel activity was higher at +10 mV
(Fig. 2A). Data were fit with a
Boltzmann function, revealing that the current was 50% activated
(V0.5) at a
membrane potential of +0.7 mV, and the maximal tail current at
70 mV was 1,034.3 pA (n = 17)
(Fig. 2B). The steepness factor (k), which provides an indication of
the sensitivity of the current to voltage, showed that the channels
were activated e-fold per 9.1 mV
depolarization.
Estimated from whole cell KV currents
using measurements of single-channel current amplitude.
To estimate the voltage dependence of these currents in
physiological external K+
(EK = 85
mV), whole cell currents (I)
(n = 10) were divided by the unitary
current (i) measured in Fig.
3B, which
should be equal to the channel activity
NPo, where
N is the number of channels and
Po is the open
probability (11). Single-channel currents were measured in
on-cell patches, with 1 mM TEA+ in
the pipette solution to block KCa
channels, and the cell membrane potential was zeroed by 140 mM
K+ bathing solution. With a
holding potential of
70 mV, patches were stepped to test
potentials (for 1,500 ms) to elicit channel openings. Unitary current
recordings were obtained over a range of membrane potentials between
40 and +60 mV, with a channel of similar conductance size
activated on membrane depolarization (Fig. 3,
A and
B). The mean unitary current of the
channel at 0 mV was 0.23 ± 0.02 pA
(n = 6), with a single-channel
conductance of 4.9 pS over the range
20 to +20 mV. The
relationship between I/i
and membrane potential was fit by a Boltzmann function, with V0.5 of
4.2 mV, k of 13.3 mV, and
maximal NPo
(I/i)
of 1,531.6 (Fig. 3C).
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Current Inactivation
KV channels demonstrate not only voltage-dependent activation but also voltage-dependent inactivation. To assess the degree of inactivation, test potential duration was 60 s to inactivate the current, followed by a step to +20 mV to induce outward currents at a standard potential. Test potentials ranged between
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Over a range of test (inactivation) potentials (0 to +20 mV) the currents elicited over the 60-s step were fit to determine their exponential decay relating to time. The currents could be fit with a single exponential and demonstrated a time constant of 6.1 ± 0.9, 6.4 ± 0.6, and 7.0 ± 0.6 s at 0, +10, and +20 mV, respectively (n = 7) (Fig. 4C).
Pharmacological Characterization of Voltage-Activated K+ Current
TEA+.
In general, KV channels from
smooth muscle are relatively insensitive to block by
TEA+, with millimolar
concentrations of TEA+ required for half block
(11-14). Therefore, the TEA+
sensitivity of voltage-activated
K+ currents from mouse gallbladder
myocytes was investigated. Experiments were performed as previously
described with 140 mM K+ in the
pipette and 5 mM K+ in the bath.
Test pulses were applied every 10 s in 10-mV increments, from a holding
potential of 70 mV. Under control conditions 1 mM
TEA+ was present in the bathing
solution to block KCa channels.
Application of 10 mM external TEA+
reduced mean outward current from 878.7 ± 77.6 to 561.0 ± 45.0 pA at +50 mV, a reduction of 35.7 ± 0.0%
(n = 6) (Fig.
5). Over a range of voltages from 0 to +50
mV, the percentage reduction in current by 10 mM
TEA+ was the same (e.g., 35.2% at
0 mV), indicating that TEA+ did
not demonstrate voltage-dependent block. Assuming a 1:1 binding ratio
of TEA+, the calculated
Kd for
TEA+ at +50 mV is 17.7 mM.
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Aminopyridines. KV channels from other types of smooth muscle are known to be inhibited by aminopyridine compounds, demonstrating a wide range of sensitivities (11-13). We have previously shown that 4-AP inhibits outward currents in isolated cells from guinea pig gallbladder (16). Aminopyridine compounds were superfused into the recording chamber and allowed to equilibrate for 2 min before recordings were made.
When applied to mouse gallbladder smooth muscle cells, 1 mM 4-AP inhibited voltage-activated K+ currents by 17.4 ± 3.3% (at +50 mV), and 10 mM 4-AP inhibited currents by 35.4 ± 2.3% (at +50 mV) (n = 8) (Fig. 6, A and B).