Voltage-dependent K+ currents in smooth muscle cells from mouse gallbladder

J. H. Jaggar1, G. M. Mawe1, and M. T. Nelson2

Departments of 1 Anatomy and Neurobiology and of 2 Pharmacology, The University of Vermont, Burlington, Vermont 05401-2500

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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 MOmega . 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
<IT>I</IT><SUB>act</SUB> = <FR><NU><IT>I</IT><SUB>max</SUB></NU><DE>1 + <IT>e</IT><SUP>(<IT>V</IT><SUB>0.5</SUB>−<IT>V</IT><SUB>m</SUB>)/<IT>k</IT></SUP></DE></FR>
where Iact is the outward current (I) at a test potential Vm, V0.5 is the membrane potential where one-half the maximal current will be activated (or inactivated), Imax is the maximal current that can be activated, and k is the steepness factor.

To determine the exponential decay of currents, data were fit with Microcal Origin software using an exponential decay function
<IT>I</IT><SUB>act</SUB> = <IT>Ae</IT><SUP>−<IT>t</IT>/&tgr;</SUP>
where Iact is the outward current at time t, A is the amplitude, and tau  is the time constant.

Kd values were estimated assuming a 1:1 binding ratio using the following equation
<IT>I</IT>/<IT>I</IT><SUB>max</SUB> = [1 + ([X]/<IT>K</IT><SUB>d</SUB>)]<SUP>−1</SUP>
where X is the concentration of the drug for which Kd (apparent dissociation constant) is being calculated, and I/Imax is the relative drug effect compared with control.

Results are expressed as means ± SE of n cells.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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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Fig. 1.   Inhibition of K+ currents by tetraethylammonium (TEA+) (1 mM) and iberiotoxin (IBTX; 100 nM). A: whole cell currents elicited by a step from a holding potential of -70 to +50 mV, during control conditions, with 1 mM TEA+, or with 1 mM TEA+ + 100 nM IBTX. In all traces, dotted lines refer to 0 current level. B: mean whole cell current data (n = 4). Inhibition of control current (black-square) by 1 mM TEA+ (black-triangle) or 1 mM TEA+ + 100 nM IBTX (×) was observed at potentials positive to +10 mV. C: typical outward currents observed in the presence of 1 mM TEA+, elicited from a holding potential of -70 mV, by depolarizing in 10-mV steps (from -60 to +50 mV) for 1.8 s, every 10 s. Bath and pipette K+ were 5 and 140 mM, respectively.

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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Fig. 2.   Voltage dependence of K+ currents. A: whole cell currents elicited from a holding potential of -70, -10, and +10 mV, with symmetrical 140 mM K+. Cells were held at test potentials for 400 ms, with steps applied every 10 s. At the end of each pulse, holding potential was returned to -70 mV, evoking a large inward K+ tail current. B: mean tail currents, plotted against test holding potentials, fit with a Boltzmann function (n = 17). Current was 50% activated (V0.5) at a potential of +0.7 mV, activated e-fold per 9.1 mV depolarization, and maximal tail current at -70 mV was shown to be 1,034.3 pA. TEA+ (1 mM) was present in bathing solution.

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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Fig. 3.   Voltage dependence of K+ channels. A: single-channel recordings of voltage-activated K+ channels recorded in the same on-cell patch at -20, 0, and +20 mV, elicited from a holding potential of -70 mV. Capacitative transients have been erased by subtracting a null trace in which no channel openings were elicited. Dashed line indicates closed level. Unitary currents for this recording were 0.33, 0.22, and 0.15 pA for +20, 0, and -20 mV, respectively. Pipette and bathing K+ were 5 and 140 mM, respectively, with 1 mM TEA+ present in the pipette solution. B: current-voltage plot for mean unitary currents. Nos. in parentheses indicate no. of on-cell patches from which unitary current measurements were obtained. Data were fit with a Goldman-Hodgkin-Katz (GHK) function. C: peak whole cell outward current (I) (n = 10) divided by single-channel conductance (i) obtained from the GHK fit (B), plotted as a function of voltage. Data were fit with a Boltzmann function indicating V0.5 of -4.2 mV, a steepness factor (k) of 13.3 mV, and a maximal I/i of 1,531.6.

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 -100 and +20 mV, with a 10-s, -70-mV holding potential applied between steps. The evoked current at +20 mV decreased with an increase in the test (inactivation) potentials (Fig. 4, A and B). Plotting the available current (in pA) at +20 mV against holding potential (in mV) revealed that data could be fit by a Boltzmann function (Fig. 4B). Inactivation of the current was half maximal at -24 mV (V0.5) and increased e-fold per 7.7 mV (k). At +20 mV current was maximal at 407 pA, and a noninactivating component was 79 pA (n = 8).


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Fig. 4.   Voltage dependence of inactivation. A: whole cell current records depicting effect of a test potential of -60 and -20 mV, on current elicited at +20 mV. Cells were held at -70 mV for 10 s, stepped to a test potential for 60 s, then to -70 mV for 30 ms (to close any open channels), and to +20 mV for 2 s to evoke an outward current. Evoked current at +20 mV was 567.5 pA after a test potential of -60 mV and 228.7 pA after -20 mV. Dashed line indicates 0 current level. B: inactivation relationship plotted against voltage was fit with a Boltzmann function with V0.5 of -24 mV and k of 7.7 mV. Current was maximal at 407 pA, and a noninactivating component was 79 pA (n = 8). C: exponential decay of currents. Original record of the current elicited at +20 mV, after holding at -70 mV (for 10 s), fit by an exponential decay function. In this record, the time constant (tau ) for decay was 5.9 s, the noninactivating component was 111.3 pA, and the current elicited at +20 mV, after the 60-s test potential, was 101.1 pA. Bath (external) and pipette (internal) K+ were 5 and 140 mM, respectively. TEA+ (1 mM) was present in bathing solution.

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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Fig. 5.   TEA+ inhibition of voltage-activated K+ currents. A: original records of whole cell currents evoked by a step from a holding potential of -70 to +50 mV, and the inhibitory effect of 10 mM TEA+ (1 mM TEA+ is present in control conditions). In this example application of 10 mM TEA+ reduced the outward current by 42.4%. B: mean effects of 10 mM TEA+ (×) compared with control (black-square) (n = 6). Bath (external) and pipette (internal) K+ were 5 and 140 mM, respectively.

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).