Inwardly rectifying K+ channels in esophageal smooth muscle

Junzhi Ji, Anne Marie F. Salapatek, and Nicholas E. Diamant

Departments of Medicine and Physiology, University of Toronto, and Playfair Neuroscience Unit, Toronto Western Hospital, Toronto, Ontario, Canada M5T 2S8


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The whole cell patch-clamp technique was used to investigate whether there were inwardly rectifying K+ (Kir) channels in the longitudinal muscle of cat esophagus. Inward currents were observable on membrane hyperpolarization negative to the K+ equilibrium potential (Ek) in freshly isolated esophageal longitudinal muscle cells. The current-voltage relationship exhibited strong inward rectification with a reversal potential (Erev) of -76.5 mV. Elevation of external K+ increased the inward current amplitude and positively shifted its Erev after the Ek, suggesting that potassium ions carry this current. External Ba2+ and Cs+ inhibited this inward current, with hyperpolarization remarkably increasing the inhibition. The IC50 for Ba2+ and Cs+ at -60 mV was 2.9 and 1.6 mM, respectively. Furthermore, external Ba2+ of 10 µM moderately depolarized the resting membrane potential of the longitudinal muscle cells by 6.3 mV while inhibiting the inward rectification. We conclude that Kir channels are present in the longitudinal muscle of cat esophagus, where they contribute to its resting membrane potential.

esophageal longitudinal muscle; patch clamp; barium; cesium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SMOOTH MUSCLE OF THE LONGITUDINAL layer of the esophageal body differs from the muscle in the circular layer in a number of ways. The esophageal circular muscle demonstrates descending inhibition that precedes its peristaltic contraction and provides for ready propulsion of the bolus along the esophagus. On the other hand, the function of the longitudinal muscle depends solely on its contraction. Its contraction would serve to shorten the esophagus, especially in the region in advance of the peristaltic circular muscle contraction, thereby expanding the lumen and stabilizing the wall in this region and facilitating distal propulsion (22). The mechanical activities of the muscles are dictated by ion channels on the plasma membrane. Several ion channel currents have been identified and characterized in esophageal circular muscle from different species, including delayed-rectifier K+ currents, transient outward K+ currents, Ca2+-activated K+ currents, and L- and T-type Ca2+ channel currents (37, 1, 29). In contrast, much less information is obtained from esophageal longitudinal smooth muscle cells. To our knowledge, only voltage-gated K+ outward currents have been found in human esophageal longitudinal muscle (37).

The resting membrane potential of cat esophageal longitudinal muscle cells is constant and relatively negative along the esophagus, as opposed to that of circular muscle cells, which becomes less negative distally (27). A relative stable resting membrane potential resulting from the activity of ion channels may play a role in modulating physiological function of the esophageal longitudinal smooth muscle. Inwardly rectifying K+ (Kir) channels conduct K+ more readily at membrane potentials negative to the K+ equilibrium potential (Ek) and therefore play a role in the maintenance of resting membrane potential. Kir channels have been identified in a variety of tissue types (13), including cardiac (26), vascular (6), and skeletal muscle (8). In light of these findings, a similar kind of channel that acts to stabilize the resting membrane potential would be expected to exist in the esophageal longitudinal smooth muscle.

In the current study, we applied the whole cell patch-clamp technique to investigate whether there were Kir channels in smooth muscle cells freshly isolated from the longitudinal layer of cat esophagus. We recorded an inward rectifier K+ current in these cells on membrane hyperpolarization negative to the Ek. This inward current was highly K+ selective and was inhibited by extracellular cations such as Ba2+ and Cs+ in a voltage-dependent manner. External Ba2+ depolarized the membrane potential most likely because of inhibition of the outward component of the inward rectification. These results indicate the existence of Kir channels in the esophageal longitudinal muscle and their contribution to the resting membrane potential of the muscle.


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METHODS
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Cell isolation. Adult cats of either sex were killed by intravenous injection of pentobarbital sodium (0.5 mg/kg) according to the protocol approved by The Toronto Hospital Animal Care Committee. Each esophagus was quickly excised and placed in modified Krebs solution composed of (in mM) 115 NaCl, 4.6 KCl, 1.2 NaH2PO4, 1.2 MgSO4, 22 NaHCO3, 2.5 CaCl2, and 11 glucose and bubbled with 95% O2-5% CO2. After the mucosa and the circular muscle were stripped off, the exposed longitudinal muscle was dissected out and cut into squares of ~2 mm2. These squares were placed in a test tube with 1 ml of dissociation solution composed of (in mM) 125 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 2.5 EDTA, and 10 glucose (pH 7.2). Papain (2 mg/ml) as well as collagenase blend F (1.3 mg/ml), 1,4-dithio-L-threitol (154 µg/ml), and BSA (1 mg/ml) were added to the test tube, which was then incubated at 37°C for tissue digestion (28). After 45 min, tissues were washed with enzyme-free dissociation solution three times and gently agitated with a plastic transfer pipette. Spindle-shaped single smooth muscle cells were dispersed and used for patch-clamp study within the following 5 h.

Whole cell patch-clamp recording. Isolated cells in dissociation solution were placed in a 1-ml glass-bottom dish mounted on the stage of an inverted microscope and allowed to adhere to the bottom for 30 min. These cells were perfused with standard external solution containing (in mM) 140 NaCl, 5 KCl, 2.5 CaCl2, 1 MgCl2, 10 HEPES, and 5.5 glucose (pH 7.4). Membrane currents from longitudinal muscle cells were recorded in the voltage-clamp mode. Pipettes were made from borosilicated, thin-walled glass capillary tubings (OD 1.5 mm, ID 1.10 mm; Sutter Instrument, Novato, CA) with a two-stage microelectrode puller (MF-83, Narishige, Tokyo, Japan). The reference electrode made from Ag-AgCl wire was directly connected to the bath. The resistance of a pipette tip was ~3-6 MOmega after being filled with standard pipette solution composed of (in mM) 140 KCl, 0.5 CaCl2, 1 MgCl2, 5 EGTA, 10 HEPES, and 5 Na2ATP (pH 7.2). Recordings were performed using an Axopatch 1D amplifier (Axon Instruments, Foster City, CA). The junction potential between the pipette and the external solution was adjusted to 0 at the beginning of the experiment. A tight seal (>1 GOmega ) was established between the cell membrane and the pipette tip by gentle suction, and then the membrane was ruptured by a further suction. The voltage-clamp protocols were generated by pClamp 6 software (Axon Instruments). Data were filtered at 1 kHz by an on-board eight-pole Bessel filter before digitization with a DigiData 1200 analog-to-digital converter (Axon Instruments). Values for cell capacitance were determined by transient cancellation of capacitive transients. Data were analyzed using pClamp 6-Clampfit software (Axon Instruments). Leak current was subtracted from original current during analysis. The formula applied by Clampfit to estimate leak current is stimulus waveform/(CF × resistance), where CF is the correction factor (0.001 in our experimental situation). Recordings were commenced 5 min after the formation of the whole cell configuration to obtain stable currents. All experiments were performed at a room temperature of 20-22°C.

Solutions and drugs used. External solutions with 30, 70, and 140 mM K+ were made by increasing the amount of KCl to respective concentrations while decreasing the amount of NaCl to 115, 75, and 0 mM in standard external solution, respectively. Therefore, the total solute osmolarity of external solutions was preserved. Na+-free external solution was prepared by replacing the excessive NaCl with N-methyl-D-glucamine (Sigma Chemical, St. Louis, MO). Mg2+-free pipette solution was made by omitting 1 mM MgCl2 and 0.5 mM CaCl2, and 5 mM EGTA was replaced by 5 mM EDTA in the standard pipette solution. Tetraethylammonium chloride (TEA) (Sigma Chemical) was dissolved in distilled water. Stock solutions of glibenclamide (Sigma Chemical) and 4-aminopyridine (4-AP) (Sigma Chemical) were made in 10% DMSO that was further diluted 100-fold in the final concentration.

Statistical analysis. Data are presented as means ± SE, and n indicates the number of cells, each of which came from a different animal. Unless otherwise stated, unpaired Student's t-test (2-tail) was used to compare data from different groups. P < 0.05 was considered to be significantly different.


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ABSTRACT
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Freshly isolated esophageal longitudinal muscle cells were spindle-shaped and relaxed. Whole cell capacitance was 46.0 ± 1.8 pF averaged from 75 cells. The mean input resistance of these cells was 3.3 ± 0.2 GOmega (n = 30). Reversible contractions could be observed when the cells were exposed to Ca2+-containing external solution, indicating that they have retained contractile function after enzymatic digestion.

Whole cell inward rectifier currents. Esophageal longitudinal muscle cells were bathed in external solution with 5 mM K+ and dialyzed with pipette solution containing 140 mM K+. To investigate the membrane conductance in the inward direction, a series of test pulses from -30 to -150 mV at 20-mV steps were applied for 400 ms from a holding potential of -50 mV. To prevent gigaseal breakdown, the maximal hyperpolarization voltage was limited to -150 mV, and the step voltage protocol was always run from +30 to -150 mV. Figure 1A shows that inward currents were rapidly activated at voltages negative to -70 mV. The inward current amplitudes progressively increased with hyperpolarization, and the current waveforms became noisier at membrane potentials negative to -130 mV. During the 400-ms test pulses, the inward currents did not exhibit obvious inactivation (Fig. 1A). Figure 1B displays the current-voltage (I-V) relationship obtained by plotting current amplitudes at the end of test pulses against membrane potentials. In the I-V curve shown in Fig. 1B, the inward current was rectified at about -70 mV. However, the outward current between -70 and -30 mV was very small, being no more than 3 pA at -70 and -50 mV and 5 pA at -30 mV from that cell. Hence, the inward current exhibited a strong inward rectification. To determine the reversal potential of the inward rectification, tail currents were obtained by voltage steps ranging from -130 to -60 mV (Fig. 1C) after a hyperpolarization voltage to -130 mV for 1 s that fully activated the inward rectifier current. As shown in Fig. 1D, the I-V relationship of tail currents was generated by plotting the instantaneous tail current amplitudes at each test potential. It also demonstrated inward rectification. From this I-V relationship, the extrapolated reversal potential of the inward rectification was -76.5 mV (n = 4, Fig. 1D), which was close to the Ek of -83.9 mV with 140 mM internal K+ and 5 mM external K+. In this situation, the Na+ and Cl- equilibrium potentials were +66.9 and -1.5 mV, respectively. Therefore, the inward rectifier current is primarily K+ selective.


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Fig. 1.   Inward currents in cat esophageal longitudinal muscle cells. A: a series of inward current traces recorded from a longitudinal muscle cell in response to test pulses for 400 ms ranging from -30 to -150 mV in a 20-mV increment (protocol shown at top). The holding potential was -50 mV and the interpulse interval was 10 s. The internal K+ concentration ([K+]i) and external K+ concentration ([K+]o) were 140 and 5 mM, respectively. B: the current-voltage (I-V) relationship was determined by plotting steady-state inward current amplitudes from the same cell against membrane potentials. The I-V relationship exhibits strong inward rectification because outward current between -70 and -30 mV is very small (<5 pA). C: a family of tail currents obtained by voltage steps from -130 to -60 mV for 500 ms after 1-s hyperpolarizing test pulse to -130 mV (protocol shown at top) that fully activated the inward current is shown. D: the I-V relationship of tail currents was plotted by tail current amplitudes against corresponding test potentials. Values are means ± SE averaged from 4 cells. The dotted line was extrapolated from the solid line drawn by a linear least-squares method from -130 to -80 mV and extended to cross the abscissa, thus giving a reversal potential of -76.5 mV as indicated by an arrow. Dotted lines in A and C are zero-current levels.

We next used a voltage ramp of 400 ms from -150 to +50 mV to further identify the properties of the inward rectifier current in esophageal longitudinal muscle cells. This protocol allowed us to continuously record ramp current traces over the voltage range. As shown in Fig. 2, an inward current was detectable at potentials negative to the Ek. At membrane potentials between -80 and -30 mV, there was little outward current. At potentials positive to -10 mV, an outward current was progressively increased with membrane depolarization (Fig. 2).


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Fig. 2.   Ba2+-sensitive inward rectifier current. A voltage ramp (top) from -150 to +50 mV lasting for 400 ms elicited membrane currents from a longitudinal muscle cell that displayed inward rectification near the K+ equilibrium potential (Ek) as indicated by the arrow. [K+]i and [K+]o were 140 and 5 mM, respectively. Two current traces were obtained in the absence and presence of external Ba2+ (0.5 mM). Dotted line is the zero-current level.

Ba2+ is a well-known potent blocker of Kir channels. To determine whether the inward current seen in esophageal longitudinal muscle cells was Ba2+ sensitive, we added 0.5 mM Ba2+ to the bath solution. This caused a remarkable reduction of the inward current (Fig. 2). However, Ba2+ did not significantly affect the outward current (Fig. 2). On average, Ba2+ reduced the inward current from 55.0 ± 6.5 to 3.9 ± 0.4 pA (n = 8) at -150 mV with 140 mM K+ in the pipette solution and 5 mM K+ in the bath solution. Therefore, ~93% of the inward current was Ba2+ sensitive. The Ba2+-insensitive outward current was partially blocked by 1 mM TEA, primarily a Ca2+-activated K+ channel blocker at this dose (Fig. 3A). At a membrane potential of +50 mV, 34.8% (n = 4) of the outward current was inhibited by this low dose of TEA. 4-AP (1 mM), a delayed rectifier K+ channel blocker, further reduced the TEA-insensitive outward current by 41.9% (n = 4). However, neither TEA nor 4-AP significantly inhibited the inward rectifier current (Fig. 3A). These results minimized the possibility that this inward current was caused by the weak inward rectification of voltage-gated K+ channels (19). In addition, the inward rectifier current was not affected by 10-5 M glibenclamide (Fig. 3B), an ATP-sensitive K+ channel inhibitor. That is, it is unlikely to be a weak inwardly rectifying ATP-sensitive K+ channel current such as that found in rabbit esophageal muscularis mucosae cells (12). Therefore, the electrophysiological appearance of the Ba2+-sensitive inward current implies the existence of Kir channels in the esophageal longitudinal muscle. The following series of experiments was conducted to further characterize this Ba2+-sensitive inward current.


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Fig. 3.   Effects of K+ channel blockers on the inward rectifier current. Membrane currents were evoked by a voltage ramp from -150 to +50 mV for 400 ms. A schematic protocol is given in A at top. A: tetraethylammonium chloride (TEA) (1 mM) inhibited the outward current but had no effect on the inward current. 4-Aminopyridine (4-AP) (1 mM) further reduced the 1 mM TEA-insensitive outward current but did not block the inward current at all. B: glibenclamide (10 µM) had little effect on both the inward and outward current. [K+]i and [K+]o were 140 and 5 mM, respectively. Arrows indicate the Ek. Dotted lines are zero-current levels.

Effect of external K+. Kir channels are highly selective for K+. An important property of these channels is the positive shift and accentuation of the inward rectification with the elevation of external K+ (6, 25, 23, 34). Therefore, we examined the effects of raising external K+ concentration ([K+]o) on the inward rectifier current. As shown in Fig. 4A, exposure of longitudinal muscle cells to external solutions containing more K+ shifted the reversal potential of the inward rectification in the depolarizing direction and increased the inward current amplitude in a concentration-dependent manner. The values for reversal potential were extrapolated from I-V relationships plotted by the ramp voltage-induced currents against voltages applied. Thus the reversal potentials were actually zero-current membrane potentials. The Ek was calculated according to the Nernst equation: Ek = (RT/F) ln ([K+]o/[K+]i), where R is the gas constant, T is the temperature in Kelvin, F is Faraday's constant, and [K+]i is the internal K+ concentration. The values for reversal potential at 5, 30, 70, and 140 mM [K+]o were -77.8 ± 2.3 (n = 6), -33.8 ± 1.9 (n = 6), -18.5 ± 1.0 (n = 5), and -1.3 ± 0.3 mV (n = 4), respectively, whereas the theoretical Ek values were -83.9, -38.8, -17.5, and 0 mV, respectively. Therefore, the reversal potential of the inward rectification shifted positively following the Ek as external K+ was raised. With a standard [K+]o and [K+]i of 5 and 140 mM, respectively, the reversal potential extrapolated from zero-current membrane potential was -77.8 mV, close to -76.5 mV estimated from tail inward currents (Fig. 1D). The reversal potential was depolarized by 54 mV for each 10-fold increase in external K+ (Fig. 4B), similar to the theoretical value of 58.0 mV calculated from the Nernst equation.


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Fig. 4.   Effects of [K+]o on the inward current. A: the I-V relationships were plotted by ramp voltage-induced inward currents against membrane potentials in the external solutions containing 5, 30, 70, and 140 mM K+ as indicated, respectively. Representative inward current traces from a longitudinal muscle cell were evoked by a ramp voltage from -150 to +50 mV for 400 ms (protocol shown in Fig. 2). The outward current portions were not included. With the elevation of [K+]o, the reversal potential of the inward rectification shifted positively and the linear slope conductance of the inward current increased. Values for the reversal potential and slope conductance at different [K+]o are given in the text. B: reversal potentials measured as zero-current membrane potentials were plotted against each [K+]o with the number of cells studied indicated in parentheses. Abscissa is on logarithmic scale. The solid line indicates the best-fit curve with a slope of 54 mV for 10-fold change in external K+.

Another property of the Kir current is the increase in its slope conductance with the elevation of external K+. Slope conductance was determined from the linear portion of the ramp voltage-induced inward current. As [K+]o was raised, the slope of the inward current became steeper while the current itself became noisier (Fig. 4A). The values for slope conductance at 5, 30, 70, and 140 mM [K+]o were 0.69 ± 0.07 (n = 6), 1.68 ± 0.13 (n = 6), 2.42 ± 0.25 (n = 5), and 3.15 ± 0.34 (n = 4) nS, respectively. The mathematical relationship between the slope conductance of the inward current and external K+ was best fitted by an equation of C × [K+]or (25) in which C was 0.33 nS and r = 0.46. Thus the slope conductance was nearly proportional to the square root of external K+. These results fit with the proposal that the inward current from esophageal longitudinal muscle cells is selective for K+ and shifts its reversal potential with external K+ in a manner quite similar to that previously described for the Kir channel in other cell types.

Inhibitory effect of Ba2+. The voltage-dependent block of the Kir current by external Ba2+ is an important characteristic of the channel. To observe the Ba2+ action more efficiently, we recorded the inward current at high conductance by increasing [K+]o to 140 mM, which was the same as [K+]i. Under this condition, the inward rectifier currents were evoked by 5-s hyperpolarization potentials from -10 to -110 mV in a 10-mV increment. The holding potential was clamped at 0 mV, which was equal to the resting membrane potential when external and internal K+ were the same. Figure 5A shows representative inward currents at voltage steps to -60 and -100 mV in the presence of external Ba2+ with various concentrations. As expected, Ba2+ caused a dose-dependent inhibition of the inward current. For example, the inward current amplitude at the end of the test pulse was blocked by 24.9% by 1 µM Ba2+ at -60 mV, whereas the block was increased to 51.6% at 3 µM Ba2+ and 78.6% at 10 µM Ba2+, respectively (Fig. 5, A and B). More importantly, the Ba2+-induced inhibition was voltage dependent. At a higher hyperpolarization step, the block was also enhanced. When 3 µM Ba2+ was applied, the inward current was only inhibited by 51.6% at -60 mV but by 77.6% at -100 mV (Fig. 5, A and B). Furthermore, Ba2+ caused faster decay of the inward current when the cell membrane was hyperpolarized to more negative potentials (Fig. 5A). The time constant of the decay was determined by fitting the inward current with a single exponential. With exposure to 10 µM Ba2+, the mean time constants were 396.8 ± 64.4 ms at -30 mV, 168.1 ± 16.0 ms at -60 mV, and 41.3 ± 11.2 ms at -90 mV (n = 5-7). The voltage-dependent inhibition caused by external Ba2+ is theoretically interpreted by assuming that Ba2+ binds in a voltage-dependent manner to a site within the channel pore to prevent K+ movement through the channel (10). If the binding site is within the membrane potential field, the membrane hyperpolarization will facilitate a positively charged Ba2+ to bind to the site. This assumption can be described by the equation
<IT>I</IT>&cjs0823;  <IT>I</IT><SUB>0</SUB><IT>=</IT>1&cjs0823;  (1<IT>+</IT>[Ba<SUP>2+</SUP>]&cjs0823;  <IT>K</IT><SUB>d</SUB>) (1)
in which I and I0 are steady-state inward currents in the presence and absence of Ba2+, respectively, and Kd is the dissociation constant for binding of Ba2+ to its blocking site. When I is half of I0, Kd is equal to [Ba2+] (external Ba2+ concentration). I/I0 from six cells were plotted against external Ba2+ concentration in Fig. 5C. The solid lines were fitted to Eq. 1 and nonlinear least-square optimization. The values for Kd were 7.0 µM at -30 mV, 2.9 µM at -60 mV, and 0.8 µM at -90 mV (n = 6, Fig. 5C). Thus Kd was markedly reduced with membrane hyperpolarization. The relationship between Kd and membrane potential can also be described by the equation
<IT>K</IT><SUB>d</SUB>=<IT>K</IT><SUB>0</SUB> exp(<IT>zF</IT>&mgr;<IT>V</IT>&cjs0823;  <IT>RT</IT>) (2)
where K0 is the dissociation constant at 0 mV, µ represents the fraction of the potential difference across the membrane experienced at the level of the binding site, R is the gas constant, T is the temperature in Kelvin, F is Faraday's constant, and z is the valence of the ion (2 for Ba2+). The K0 for Ba2+ inhibition calculated from Eq. 2 was 18.1 µM with µ of 0.4. 


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Fig. 5.   Voltage-dependent block of the inward current by external Ba2+. A: the inward currents were evoked by hyperpolarization steps from -10 to -110 mV for 5 s from a holding potential of 0 mV. [K+]o was raised as high as [K+]i of 140 mM to enhance the inward rectification conductance. Representative inward current traces at voltage steps to -60 and -100 mV from a longitudinal muscle cell are shown here to compare the voltage-dependent inhibition of the inward currents in the presence of external Ba2+ at 0 (control), 1, 3, and 10 µM. Dotted lines are zero-current levels. B: I-V relationships of the inward currents were obtained from the same cell by the same protocol as used in A. Both [K+]i and [K+]o were 140 mM. The inward currents were measured at the end of 5-s voltage steps in the presence of 0 (control), 0.3, 1, 3, and 10 µM external Ba2+. C: relationship between fractional inhibition of the inward currents and external Ba2+ at membrane potentials of -30, -60, and -90 mV is shown. I0 represented the control inward current, whereas I was the current in the presence of Ba2+ from 0.3 to 10 µM. Values are means ± SE (n = 6). The solid line is the best-fit curve determined by Eq. 1 given in RESULTS.

Inhibitory effect of Cs+. External Cs+ is also a voltage-dependent blocker of Kir channels in skeletal muscle, vascular smooth muscle, and blood cells, although a higher concentration of Cs+ is required for this inhibition compared with Ba2+ inhibition (8, 25, 34). We therefore examined the effect of extracellular application of Cs+ on the inward rectifier current in esophageal longitudinal muscle cells bathed in external solution with 140 mM K+. The inward currents were evoked by 5-s hyperpolarizing test pulses from a holding potential of 0 mV. As shown in Fig. 6A, external Cs+ (300 µM) inhibited the inward current, with more hyperpolarization increasing this inhibition. For instance, when exposed to 300 µM Cs+, the inward current amplitude measured at the end of the test pulse was blocked by 14% at -60 mV and by 57.5% at -80 mV (Fig. 6, A and B). In contrast to Ba2+ (Fig. 5B), Cs+ demonstrated a steeper block of the inward current with membrane hyperpolarization in the I-V relationship (Fig. 6B). As external Cs+ was raised, the block of the inward current began to appear at less negative membrane potentials (Fig. 6B). In addition, Cs+-induced inhibition was also concentration dependent. Figure 6C summarizes the concentration-dependent Cs+ inhibition from six cells at different membrane potentials. The solid lines were best fitted with Eq. 1 and nonlinear least-squares optimization. At -90 mV, the inward current was inhibited by 14.2%, 49.0%, and 74.2% at 30, 100, and 300 µM external Cs+, respectively (n = 6, Fig. 6C). Using the same equations, the Kd values for Cs+ block were calculated at -30, -60, and -90 mV to be 2,800, 1,570, and 142 µM, respectively (n = 6). Therefore, the Kd was greatly decreased with membrane hyperpolarization. The estimated value for K0 from Eq. 2 was 192 mM with µ of 2.0. 


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Fig. 6.   Voltage-dependent block of the inward current by external Cs+. A: the inward currents were evoked by hyperpolarization steps from -10 to -110 mV for 5 s from a holding potential of 0 mV. [K+]o was raised as high as [K+]i of 140 mM to enhance the inward rectification conductance. Representative inward current traces from a longitudinal muscle cell were evoked by voltage steps to -40, -60, -80, and -100 mV before (control) and after the external solution containing 300 µM Cs+. Inhibition of the inward current by external Cs+ was greater, with higher hyperpolarization. Dotted lines are zero-current levels. B: I-V relationships of the inward currents were obtained from the same cell by the same protocol used in A. Both [K+]i and [K+]o were 140 mM. The inward currents were measured at the end of 5-s voltage steps in the presence of 0 (control), 30, 100, 300, and 1,000 µM Cs+. C: relationship between fractional inhibition of the inward currents and external Cs+ at membrane potentials of -30, -60, and -90 mV is shown. I0 represented the control inward current, whereas I was the current in the presence of Cs+ from 30 to 1,000 µM. Values are means ± SE (n = 6). The solid line is the best-fit curve calculated from Eq. 1 described in RESULTS.

Effect of external Na+ on inward current. In those experiments with higher [K+]o, external K+ was increased in the solutions by equimolar replacement of Na+. External Na+ is known to cause inactivation of Kir channels. Therefore, we next tested whether the inward rectifier current was affected by Na+-free external solution. Na+-free external solution was made by replacing equimolar Na+ with N-methyl-D-glucamine. The step voltage protocol was initially applied when the cells were bathed in normal solution (external Na+ concentration = 140 mM) and then repeated in Na+-free solution. As seen in Fig. 7, Na+-free solution only slightly increased the inward current by 9.3% at -150 mV and by 12.9% at -130 mV (n = 5) with no statistical significance, suggesting that external Na+ is not an effective regulator of the inward current in the voltage range used and furthermore that this current is not carried by sodium ions.


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Fig. 7.   Effect of external Na+ on the inward current. I-V relationships were obtained from longitudinal muscle cells by plotting the inward currents against test pulse voltages ranging from -30 to -150 mV with a 20-mV increment. The holding potential was -50 mV. The test pulses lasted for 400 ms, with a 10-s interpulse interval. The control data were obtained in the presence of standard external solution with 140 mM Na+. After that, the bath solution was replaced by Na+-free/N-methyl-D-glucamine-containing external solution. Values are means ± SE (n = 5).

Effect of internal Mg2+ on inward current. Intracellular cations such as Mg2+ and polyamines have been found to modify gating properties of the Kir channel by decreasing the outward component of the Kir channel current (32, 18). We next examined whether intracellular Mg2+ could contribute to the inward rectification seen in esophageal longitudinal muscle cells. The pipette solution contained 140 mM K+ and 5 mM EDTA but was free of Mg2+ and Ca2+. Under these conditions, internal Mg2+ was reduced to a minimal level. As shown in Fig. 8, dialysis of the cytoplasm with Mg2+-free pipette solution did not significantly alter the outward current. Although the inward current at -150 mV was increased by 11% (n = 5) in Mg2+-free pipette solution, this change failed to reach statistical significance. This lack of a significant effect of low internal Mg2+ concentration on Kir current has also been reported in vascular smooth muscle cells (25).


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Fig. 8.   Effect of intracellular Mg2+ on the inward currents. I-V relationships were obtained from longitudinal muscle cells by plotting the inward currents against test pulse voltages ranging from -30 to -150 mV with a 20-mV increment. The holding potential was -50 mV. The test pulses lasted for 400 ms with a 10-s interpulse interval. Data were collected from cells treated with Mg2+-containing pipette solution (control, n = 6) or with Mg2+-free pipette solution with 5 mM EDTA (n = 6). Values are means ± SE.

Contribution of Kir channel to resting membrane potential. To study whether Kir channels could play a role in stabilizing the resting membrane potential of the esophageal longitudinal muscle, low-dose Ba2+ (10 µM) was added to the standard bath solution with 5 mM K+. The experiments were designed to alternatively record membrane currents and membrane potentials in the voltage- and current-clamp modes, respectively. Figure 9B shows that external Ba2+ (10 µM) induced a depolarizing change in membrane potential, which reached a plateau in 20 s. In eight longitudinal muscle cells, the resting membrane potential was -51.3 ± 3.0 mV, in agreement with our (27) previous results. In the presence of 10 µM Ba2+, the cell membrane was depolarized by 6.3 ± 0.8 mV (n = 8). In the voltage-clamp mode, the inward rectifier current evoked by a ramp voltage was shown to be greatly inhibited by 10 µM Ba2+ (Fig. 9, A and C), indicating that the Ba2+-induced depolarization is related to the block of the Kir channel. These results suggest that the Kir channel is involved in the maintenance of the resting membrane potential of the longitudinal muscle and the mechanism is likely mediated by a small outward K+ current of the channel at rest.


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Fig. 9.   Ba2+ depolarized the membrane potential while inhibiting the inward rectifier current. Membrane currents and membrane potentials recorded from a longitudinal muscle cell in alternative voltage- and current-clamp modes. A and C: in the voltage-clamp configuration, the inward currents were obtained by a voltage ramp protocol (shown in A at top) in the absence (A) and presence (C) of 10 µM Ba2+. B: the voltage-clamp recordings were separated by a period in which the current-clamp configuration was applied to observe the membrane potential alteration in the presence of 10 µM Ba2+. Arrows indicate the Ek. Dotted lines are zero-current levels in A and C and membrane potentials in B.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study provides, for the first time, strong evidence that Kir channels exist in the longitudinal muscle of cat esophagus. This evidence is based on the following major findings: 1) strong inward rectification near the Ek; 2) the shift of the reversal potential with the Ek and the increase in current magnitude when external K+ was raised; and 3) the voltage-dependent block of the inward rectifier current by external Ba2+ of low concentration and Cs+. Furthermore, the inward rectifier current is insensitive to blockers of Ca2+-activated K+ channels, delayed rectifier K+ channels, and ATP-sensitive K+ channels. Together, these data suggest that the inward rectifier current seen in the esophageal longitudinal muscle results from K+ ion flow through a Kir channel.

An ATP-sensitive K+ channel current has been found in rabbit esophageal smooth muscle cells from muscularis mucosae (12). This channel current shows weak inward rectification and is blocked by glibenclamide. The Kir current observed in our study can be distinguished from the ATP-sensitive K+ channel current because it displayed strong inward rectification and was not inhibited by 10-5 M glibenclamide (Fig. 3B). In addition, 5 mM ATP was routinely included in the pipette solution to minimize the contamination of our recordings from ATP-sensitive K+ channel currents. A hyperpolarization-activated cation inward current has been reported in smooth muscle cells from different tissues (3, 9). This current is slowly activated, nonselectively permeable to potassium and sodium ions, and reversed at approximately -25 to -30 mV. These characteristics are quite different from the Kir current in our preparation. Voltage-gated K+ channels that show weak inward rectification (19) have been found to exist in the longitudinal muscle of cat (27) and human esophagus (37). However, TEA and 4-AP did not inhibit the Kir current from esophageal longitudinal muscle cells, ruling out its origin from voltage-gated K+ channels (Fig. 3A). Human eag-related gene (HERG) also encodes an inwardly rectifying channel that exhibits rapid inactivation and slow deactivation (35, 30). The HERG current is voltage-independently blocked by Ba2+ with an IC50 of 0.6 mM (35). All of these electrophysiological and pharmacological characteristics are absent for the Kir current recorded in our study.

The I-V relationship of the Kir current from esophageal longitudinal muscle cells exhibited strong inward rectification with a reversal potential of -76.5 mV, which is close to the Ek (Fig. 1). The reversal potential of the inward rectification shifted to more depolarizing potentials following the Ek as external K+ increased (Fig. 4A). The reversal potential changed by 54 mV for a 10-fold alteration in external K+, near the estimated value of 58.0 mV from the Nernst equation. Therefore, the channel responsible for the Kir current acts as a K+ electrode when external K+ is altered. The slope conductance of the Kir current increased with external K+ in a nonlinear manner. It was proportional to [K+]or with r of 0.46, close to values for r between 0.42 and 0.47 in other cell types (25, 34).

A voltage-dependent block of the Kir channel by external Ba2+ of low concentration and Cs+ is the most prominent property of this channel. Ba2+ is a potent blocker of the Kir channel with relative high affinity. The Kd for Ba2+ at -60 mV is 2.1 µM in vascular smooth muscle cells (25), 14 µM in skeletal muscle (-65 mV) (31), and 13.7 µM in starfish oocytes (11). In our preparation, the Kd for Ba2+ was 2.9 µM at -60 mV (Fig. 5C), comparable with the Kd values in other cell types mentioned above. This voltage-dependent block caused by Ba2+ can be explained by a model in which the fraction of the applied electric field (µ) acting on the Ba2+ binding site is 0.4. This value is comparable with that of chromaffin cells (0.25) (15), smooth muscle cells (0.51) (25), and oocytes (0.6-0.7) (10). In addition, the Ba2+ inhibition developed in a time-dependent manner, with more hyperpolarization causing faster decay of the Kir current (Fig. 5A). This phenomenon has also been noted for the Kir current in egg cells (11), skeletal muscle (31), cardiac Purkinje fibers (4), and smooth muscle from resistance-sized cerebral arteries (23). In contrast to its highly sensitive block of the Kir channel, external Ba2+ was less effective in blocking ATP-sensitive K+ channels in skeletal muscle (Kd 100 µM at -60 mV) (24), Ca2+-activated K+ channels in rabbit skeletal muscle (Kd 335 µM at -60 mV) (36), and voltage-dependent K+ channels (Kd > 1 mM) in smooth muscle (20). Thus Ba2+ can be employed as a selective blocker of the Kir channel in the esophageal longitudinal muscle.

Cs+ is also an effective blocker of the Kir channel but less potent than Ba2+ in our preparation and other cell types. In contrast to Ba2+, the Cs+ block displayed greater voltage dependence because the I-V relationship in the presence of Cs+ was steeper (Fig. 6B) than that in the presence of Ba2+ (Fig. 5B). The mean value of µ for Cs+ was 2.0, >0.4 for Ba2+, indicating higher sensitivity of Cs+ to the applied electric field, which was consistent with the stronger voltage dependence of the Cs+ block. On the other hand, the Cs+ block of the Kir current was instantaneous (Fig. 6A), unlike the time-dependent decay of the Kir current seen in the Ba2+ block (Fig. 5A). Although Cs+ and Ba2+ are assumed to share a common mode of action by blocking the Kir channel, the discrepancy shown in our preparation and other cell types reflects some slight difference in the molecular mechanisms by which these ions affect the Kir channel.

It is well established that the gating property of the Kir channel is regulated by intracellular Mg2+ and polyamines (21). Using Mg2+-free pipette solution with 5 mM EDTA to minimize the internal Mg2+, we saw Mg2+ produce a small but insignificant alteration of the inward rectification (Fig. 8). A similar result has also been reported elsewhere in coronary artery muscle cells (25). Presumably, in esophageal longitudinal smooth muscle internal polyamines are sufficient to regulate the gating property of the Kir channel without a major role for Mg2+. Polyamines are metabolites of amino acids and found in almost all cells (33). Application of polyamines to inside-out patches containing IRK1 (Kir2) channels in the absence of Mg2+ restores all of the essential features of intrinsic rectification, suggesting that they may function as physiological blockers of the Kir channel (7). We did not assess the role of polyamines in our study.

Several types of Kir channels have been cloned and found to be expressed in different tissues, including the strongly rectifying Kir2.1 (IRK1) (17) and Kir3.1 (GIRK1) (5) and the weakly rectifying Kir1.1a (ROMK1) (14) and Kir6 (rcKATP-1) (2). Unlike voltage-gated K+ channels, Kir channels have only two transmembrane domains (17). The Kir current observed in our study likely falls into the strong rectification group because little outward current was seen at positive potentials to the Ek between -70 and -30 mV (Figs. 1A and 2). Moreover, the cloned IRK1 increases its conductance in a proportion to 0.47 power of external K+ and is also highly sensitive to Ba2+ (Kd < 30 µM) (17). These data are comparable with the observations for the Kir current from the esophageal longitudinal muscle. Further study such as RT-PCR is needed to determine the Kir channel subtype in our preparation.

Although some important features of the Kir current in the esophageal longitudinal muscle are similar to those of the classic Kir current (e.g., strong inward rectification, K+-mediated current, and high sensitivity to Ba2+ and Cs+), there are also some distinct differences. First, inactivation of the classical Kir current is usually seen at high hyperpolarization levels (e.g., -150 mV). However, this was not seen in the Kir current of longitudinal muscle cells at hyperpolarization of -150 mV. Second, Na+ removal does not significantly increase the Kir current at -150 mV in our preparation. Third, the Kir current in longitudinal muscle cells is noisy at hyperpolarization levels from -130 to -150 mV, which is usually not observed in other preparations. A possible explanation for the first and second differences may be that the hyperpolarization level was not negative enough to show the inactivation characteristics (34). However, these differences require further study.

The Kir channel conducts more efficiently at membrane potentials negative to the Ek, thus permitting K+ to pass more readily into the cell than out of it. However, the small outward current through the Kir channel should also be considered because it is likely to play a physiological role over the normal range of membrane potentials. The average resting membrane potential for esophageal longitudinal muscle cells was -51.3 mV, positive to the Ek. Low-dose Ba2+ (10 µM) caused a moderate membrane depolarization with an average level of 6.3 mV and inhibited the Kir current, suggesting that Ba2+-induced depolarization is associated with the block of the Kir channel. Because the outward current component through the Kir channel was only 1 or 2 pA (25), it was difficult to accurately and consistently measure it in macroscopic recording. However, at a concentration as low as 10 µM, Ba2+ has not been found to efficiently affect any known K+ channels except the Kir channel. Therefore, the Ba2+-induced mild membrane depolarization indicates that a small outward current through the Kir channel may contribute to the membrane potential at rest. A similar result has been obtained by a previous study performed on vascular smooth muscle (6). The inward current component, the major portion of the Kir channel, is assumed to maintain a stable resting membrane potential during membrane hyperpolarization. It may modulate the degree of membrane hyperpolarization of esophageal smooth muscle as in the situation of the inhibitory junction potential. Direct evidence for this assumption should be further investigated.

In conclusion, the longitudinal muscle cells of cat esophagus have Kir channels. External Ba2+ is an efficient and selective blocker of this channel. One physiological role of the Kir channel is likely to maintain more negative resting membrane potential, which results from outflow of K+ through this channel at rest. Its role in modulating the hyperpolarization of the inhibitory junction potential is unknown.


    ACKNOWLEDGEMENTS

This study was supported by a grant from Astra Pharmaceutical and Medical Research Council of Canada Grant PA-13527.


    FOOTNOTES

Portions of this work have been published previously in abstract form (16).

Address for reprint requests and other correspondence: N. E. Diamant, 12-419 Playfair Neuroscience Unit, Toronto Western Hospital, 399 Bathurst St., Toronto, ON, Canada M5T 2S8.

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.

Received 12 January 2000; accepted in final form 24 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Akbarali, HI. K+ currents in rabbit esophageal muscularis mucosae. Am J Physiol Gastrointest Liver Physiol 264: G1001-G1007, 1993[Abstract/Free Full Text].

2.   Ashford, ML, Bond CT, Blair TA, and Adelman JP. Cloning and functional expression of a rat heart KATP channel. Nature 370: 456-459, 1994[ISI][Medline].

3.   Benham, CD, Bolton TB, Denbigh JS, and Lang RJ. Inward rectification in freshly isolated single smooth cells of the rabbit jejunum. J Physiol (Lond) 383: 461-476, 1987[Abstract].

4.   Carmeliet, E, and Mubagwa K. Characterization of the acetylcholine-induced potassium current in rabbit cardiac Purkinje fibres. J Physiol (Lond) 371: 219-237, 1986[Abstract].

5.   Dascal, N, Schreibmayer W, Lim NF, Wang W, Chavkin C, DiMagno L, Labarca C, Kieffer BL, Gaveriaux-Ruff C, Trollinger D, Lester HA, and Davidson N. Atrial G protein-activated K+ channel: expression cloning and molecular properties. Proc Natl Acad Sci USA 90: 10235-10239, 1993[Abstract].

6.   Edwards, FR, Hirst GDS, and Silverberg GD. Inward rectification in rat cerebral arterioles: involvement of potassium ions in autoregulation. J Physiol (Lond) 404: 455-466, 1988[Abstract].

7.   Ficker, E, Tagliaatela M, Wible BA, Henley CM, and Brown AM. Spermine and spermidine as gating molecules for inward rectifier K+ channels. Science 266: 1068-1072, 1994[ISI][Medline].

8.   Gay, LA, and Stanfield PR. Cs+ causes a voltage-dependent block of inward K currents in resting skeletal muscle fibres. Nature 267: 169-170, 1977[ISI].

9.   Green, ME, Edwards G, Kirkup AJ, Miller M, and Weston AH. Pharmacological characterization of the inwardly-rectifying current in the smooth muscle cells of the rat bladder. Br J Pharmacol 119: 1509-1518, 1996[Abstract].

10.   Hagiwara, S, Miyazaki S, Moody M, and Patlak J. Blocking effects of barium and hydrogen ions on the potassium current during anomalous rectification in the starfish egg. J Physiol (Lond) 279: 167-185, 1978[Abstract].

11.   Hagiwara, S, Miyazaki S, Moody M, and Rosenthal NP. Potassium current and the effect of cesium on this current during anomalous rectification of the egg cell membrane of a starfish. J Gen Physiol 67: 621-638, 1976[Abstract].

12.   Hatakeyama, N, Wang Q, Goyal RK, and Akbarali HI. Muscarinic suppression of ATP-sensitive K+ channel in rabbit esophageal smooth muscle. Am J Physiol Cell Physiol 268: C877-C885, 1995[Abstract/Free Full Text].

13.   Hille, B. Ionic Channels of Excitable Membranes (2nd ed.). Sunderland, MA: Sinauer, 1992.

14.   Ho, K, Nichols CG, Lederer WJ, Lytton J, Vassilev PM, Kanazirska MV, and Herbert SC. Cloning and expression of an inwardly rectifying ATP-regulated potassium channel. Nature 362: 31-38, 1993[ISI][Medline].

15.   Inoue, M, and Imanaga I. G protein-mediated inhibition of inwardly rectifying K+ channels in guinea pig chromaffin cells. Am J Physiol Cell Physiol 265: C946-C956, 1993[Abstract/Free Full Text].

16.   Ji, J, Salapatek AMF, and Diamant NE. Characterization of inwardly rectifying K+ channels in esophageal longitudinal smooth muscle cells (SMCs) (Abstract). Gastroenterology 116: A968, 1999.

17.   Kubo, Y, Baldwin TJ, Jan YN, and Jan LY. Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature 362: 127-133, 1993[ISI][Medline].

18.   Lopatin, AN, Makhina EN, and Nichols CG. Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification. Nature 372: 366-369, 1994[ISI][Medline].

19.   Lopatin, AN, and Nichols CG. Internal Na+ and Mg2+ blockade of DRK1 (Kv2.1) potassium channels expressed in Xenopus oocytes. Inward rectification of a delayed rectifier. J Gen Physiol 103: 203-216, 1994[Abstract].

20.   Nelson, MT, and Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol Cell Physiol 268: C799-C822, 1995[Abstract/Free Full Text].

21.   Nichols, CG, and Loptin AN. Inward rectifier potassium channels. Annu Rev Physiol 59: 171-191, 1997[ISI][Medline].

22.   Paterson, WG. Studies on opossum esophageal longitudinal muscle function. Can J Phsiol Pharmacol 75: 65-73, 1997[ISI][Medline].

23.   Quayle, JM, McCarron JG, Brayden JE, and Nelson MT. Inward rectifier K+ currents in smooth muscle cells from rat resistance-sized cerebral arteries. Am J Physiol Cell Physiol 265: C1363-C1370, 1993[Abstract/Free Full Text].

24.   Quayle, JM, Standen NB, and Stanfield PR. The voltage-dependent block of ATP-sensitive potassium channels of frog skeletal muscle by cesium and barium ions. J Physiol (Lond) 405: 677-698, 1988[Abstract].

25.   Robertson, BE, Bonew AD, and Nelson MT. Inward rectifier K+ currents in smooth muscle cells from rat coronary arteries: block by Mg2+, Ca2+, and Ba2+. Am J Physiol Heart Circ Physiol 271: H696-H705, 1996[Abstract/Free Full Text].

26.   Sakmann, B, and Trube G. Conductance properties of single inwardly rectifying potassium channels in ventricular cells from guinea-pig heart. J Physiol (Lond) 347: 641-657, 1984[Abstract].

27.   Salapatek, AMF, and Diamant NE. Electrophysiologic diversity along the feline smooth muscle of esophagus (Abstract). Neurogastroenterol Motil 10: 68, 1998.

28.   Salapatek, AMF, Wang Y-F, Mao Y-K, Mori M, and Daniel EE. Myogenic NOS in canine lower esophageal sphincter: enzyme activation, substrate recycling, and product actions. Am J Physiol Cell Physiol 274: C1145-C1157, 1998[Abstract/Free Full Text].

29.   Sims, SM, Vivaudou MB, Hillemeier C, Biancani P, Walsh JV, Jr, and Singer JJ. Membrane currents and cholinergic regulation of K+ current in esophageal smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 258: G794-G802, 1990[Abstract/Free Full Text].

30.   Smith, PL, Baukrowitz T, and Yellen G. The inward rectification mechanism of the HERG cardiac potassium channel. Nature 379: 833-836, 1996[ISI][Medline].

31.   Standen, NB, and Stanfield PR. A potential- and time-dependent blockade of inward rectification frog skeletal muscle fibres by barium and strontium ions. J Physiol (Lond) 280: 169-191, 1978[Abstract].

32.   Stanfield, PR, Davies NW, Shelton PA, Khan IA, Brammar WJ, Standen NB, and Conley EC. The intrinsic gating of inward rectifier K+ channels expressed from the murine IRK1 gene depends on voltage, K+ and Mg2+. J Physiol (Lond) 475: 1-7, 1994[Abstract].

33.   Tabor, CW, and Tabor H. Polyamines. Annu Rev Biochem 53: 749-790, 1984[ISI][Medline].

34.   Tare, M, Prestwich SA, Gordienko DV, Parveen S, Carver JE, Robinson C, and Bolton TB. Inwardly rectifying whole cell potassium current in human blood eosinophils. J Physiol (Lond) 506: 303-318, 1998[Abstract/Free Full Text].

35.   Trudeau, MC, Warmke JW, Ganetzky B, and Robertson GA. HERG, a human inward rectifier in the voltage-gated potassium channel family. Science 269: 92-95, 1995[ISI][Medline].

36.   Vergara, C, and Latorre R. Kinetics of Ca2+-activated K+ channels from rabbit muscle incorporated into planar bilayers. Evidence for a Ca2+ and Ba2+ blockade. J Gen Physiol 82: 243-368, 1983.

37.   Wade, GR, Laurier LG, Preiksaitis HG, and Sims SM. Delayed rectifier and Ca2+-dependent K+ currents in human esophagus: roles in regulating muscle contraction. Am J Physiol Gastrointest Liver Physiol 277: G885-G895, 1999[Abstract/Free Full Text].


Am J Physiol Gastrointest Liver Physiol 279(5):G951-G960
0193-1857/00 $5.00 Copyright © 2000 the American Physiological Society




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