Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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We investigated the effects of
clinically relevant ethanol concentrations (5-20 mM) on the
single-channel kinetics of bovine aortic smooth muscle maxi-K channels
reconstituted in lipid bilayers (1:1
palmitoyl-oleoyl-phosphatidylethanolamine:
palmitoyl-oleoyl-phosphatidylcholine). Ethanol at 10 and 20 mM
decreased the channel open probability (Po) by
75 ± 20.3% mainly by increasing the mean closed time (+82 to
+960%, n = 7). In some instances, ethanol also
decreased the mean open time (40.8 ± 22.5%). The
Po-voltage relation in the presence of 20 mM
ethanol exhibited a rightward shift in the midpoint of voltage
activation (
V1/2
17 mV), a slightly
steeper relationship (change in slope factor,
k,
2.5 mV), and a decreased maximum Po (from
~0.82 to ~0.47). Interestingly, channels inhibited by ethanol at
low Ca2+ concentrations (2.5 µM) were very
resistant to ethanol in the presence of increased Ca2+ (
20 µM). Alcohol consumption in clinically relevant amounts may alter
the contribution of maxi-K channels to the regulation of arterial tone.
calcium dependent; voltage-gated potassium channels; planar lipid bilayer; arterial tone; alcohol
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INTRODUCTION |
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LARGE CONDUCTANCE Ca2+-activated voltage-gated K+ channels (also called big K, BK, or maxi-K channels) are activated by both membrane depolarization and an elevation of intracellular Ca2+ (56). The ensuing K+ current hyperpolarizes the cell, thereby decreasing the entrance of Ca2+ into the cell by closing the voltage-dependent Ca2+ channels (VDCCs). Smooth muscle maxi-K channels colocalize with VDCCs (29, 35, 40), and when the activity of the VDCCs are activated by membrane depolarization, the maxi-K channels are activated by the increase in intracellular Ca2+ concentration and the depolarization. Maxi-K channels regulate several physiological processes, including repolarization of the action potential (1), neurotransmitter release (40), and arterial tone (3, 22, 27, 28, 35, 41). Consequently, pharmacological modulation of maxi-K channel activity may affect several biological processes.
It is challenging to investigate the pharmacology of agents acting on
maxi-K channels because these channels exist as related types with
diverse electrophysiological properties (25, 31) specifically adjusted to carry out the functions of a particular cell.
This diversity is achieved partly by differential expression of maxi-K
channels with different primary structures among and within tissues and
cells. The maxi-K channel is believed to be a tetrameric complex of
-subunits, derived from the highly conserved slo gene
(4, 58), containing the pore structure responsible for
K+ selectivity and the sites conferring sensitivity to
voltage and Ca2+ concentration. Variation in channel
activity can arise from various spliced variants of the slo
gene (4, 17, 37, 51) whose transcripts have different
electrophysiological properties (2, 24, 51). Differential
expression of regulatory
-subunits among tissues (19, 50,
52) is known to modulate maxi-K channel activity (30,
32). Furthermore, maxi-K channel activity is regulated by
conditions that differ among and within tissues, including interaction
with G proteins (45), phosphorylation status (26,
39), redox potential (11, 57), and the membrane lipid environment (5, 6, 20, 33).
Ethanol (EtOH) exerts different effects on the activities of
maxi-K channels from different tissues, and even within the same cell.
EtOH stimulates the activity of maxi-K channels from a diversity of
mammalian cells and tissues (7, 13, 23). Clinically relevant concentrations of 10-100 mM EtOH (for reference, the maximum legal intoxication concentration of EtOH is 22 mM) stimulate maxi-K channels in isolated neurohypophysial terminals using
patch-clamp techniques (13), an effect believed to
contribute to the reduced release of vasopressin and oxytocin caused by
EtOH ingestion (12). EtOH also stimulates maxi-K channels
isolated from rat skeletal muscle T tubule membranes reconstituted into
planar lipid bilayers (7) and the cloned maxi-K channel
from mouse brain (mSlo) expressed in Xenopus
oocytes (15). The transient stimulation of maxi-K channels
in pituitary tumor cells is apparently mediated by its phosphorylation
state (23), which may correlate with its effects on growth
hormone secretion (18, 53). A similar stimulation of
maxi-K channel activity by EtOH treatment has, therefore, been found in
a diverse variety of tissues regardless of the technique used to
measure ion channel activity. Although this would suggest that EtOH
potentiates all maxi-K channels, the information in one report
(14) indicates that not all maxi-K channels are stimulated by EtOH. Dopico and Treistman (14) reported that
50-100 mM EtOH inhibited the activity of the cloned maxi-K channel
-subunit from bovine aortic smooth muscle [BASM (bSlo)]
expressed in Xenopus oocytes. The observation that EtOH can
differentially affect maxi-K channels in different regions of
supraoptic neuronal cells, stimulating the channels in the nerve
endings but exerting no effect on those in the cell body
(16), also is not in keeping with a universal EtOH
potentiation of maxi-K channels.
Previously, the effects of EtOH on maxi-K channel activity have been
examined with cloned -subunits or channels from brain neuroendocrine
tissues where mainly the
-subunit alone may have been expressed
(52). In this study, we examined the EtOH effects on the
maxi-K channel activity in BASM preparations, where it most likely
exists as a complex containing
- and
-subunits (19, 20,
50). Although the functions of maxi-K channels in aortic smooth
muscle are not precisely defined, they apparently limit myogenic tone
(3). In this study, we investigated the EtOH effects on
BASM sarcolemmal membrane maxi-K channel activity from membrane
vesicles reconstituted into planar lipid bilayers. We show that EtOH,
at clinically relevant concentrations of 20 mM or lower, inhibited the
channel activity by affecting channel gating.
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METHODS |
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Chemicals. Salts, decane, cholesterol, agar, and HEPES were purchased from Sigma (St. Louis, MO) and were of the highest grade. Synthetic 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and synthetic 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) were obtained from Avanti Polar Lipids (Pelham, AL).
Planar lipid bilayers. Planar lipid bilayers were formed in aqueous solution by painting lipids (35 mg/ml in decane) across a 0.15- to 0.25-mm aperture in a Delrin plastic bilayer chamber (cups and chambers purchased from Warner Instrument). Bilayer thinning was monitored visually with a ×30 zoom stereo microscope (Bausch & Lomb) and through membrane capacitance measurements (3910 expander module, Dagan). Bovine aortic sarcolemmal membranes prepared as described by Vazquez et al. (55) were fused with the bilayer in the presence of an osmotic gradient of 150 mM KCl on the cis side and 50 mM KCl on the trans side. The bilayer buffer typically consisted of 10 mM HEPES and 2.5 µM CaCl2, pH 7.4 cis and trans; free [Ca2+] in the buffer was confirmed with a Ca2+ electrode prepared as in Sigel and Affolter (47). Ca2+ standards were from World Precision Instruments (Sarasota, FL). Vesicles that were directed toward the bilayer from the cis side with a micropipette and oscillations of ±30 to ±60 mV (1 Hz) were often used to assist with fusion. After single-channel currents were observed, channel orientation was confirmed by voltage and/or Ca2+ sensitivity, then the trans side was elevated to 150 mM KCl for recording. Additions of EtOH or Ca2+ were generally accompanied by 10-30 s of stirring and a 2-min delay in recording to allow for equilibration with the channel.
Electrophysiology.
An Ag/AgCl electrode was connected via an agar bridge (1% in 0.2 M
KCl) to each side of a Delrin plastic cup, and bilayer membranes were
then voltage clamped using a Dagan 3900A integrating patch-clamp
amplifier (Dagan, Minneapolis, MN). The trans side served as
the ground with current and voltage expressed in the normal
electrophysiological convention. Single-channel currents were low-pass
Bessel filtered at 2 kHz (3db, Dagan 3900A) and sampled at 5 kHz. A
500-Hz low-pass digital filter setting in Clampex 7 (Axon Instruments,
Foster City, CA) via a Digidata 1200B Interface (Axon Instruments) was
imposed during acquisition for an effective cutoff frequency of 485 Hz.
Recordings of 1-4 min were typically used to acquire sufficient
events for reliable analysis of channel open probability
(Po) and dwell-time distributions. Single-channel currents were analyzed with the aid of pCLAMP 7 (Axon
Instruments); no additional filtering was imposed during analysis.
Data analysis.
The majority of experiments described (20 out of 26) consisted of
records from a single channel inserted into the bilayer; only six
records of multichannel recordings (2 or 3 channels) were used. The
threshold for closed-to-open transition was set at 50% of the open
level amplitude according to the Fetchan software (Axon Instruments),
and open- or closed-channel events with durations of <0.6 ms were
eliminated. On occasion, a channel exhibited a marked change in its
Po either up or down >75% compared with the rest of the surrounding record; these relatively rare observations (in
only 4 experiments) were only of 10-20 s in duration and were generally omitted from the events list for Po
analyses and estimates of dwell-time distributions. Current-voltage
relationships (I-V) were formed from mean current amplitude
data of recordings typically in the range of 20 to +60 mV; slope
conductance was then calculated via linear regression analysis.
Po was calculated in pStat (Axon Instruments)
according to the specified formula applied to events lists:
Po = (to/ti)/N
where to is the total open time for the level,
ti is the time interval over which
Po is measured, and N is the number
of channels in the patch. Intervals of 1 s were used over the
entire 1- to 4-min data record for these analyses.
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RESULTS |
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The effects of EtOH on the activity of the maxi-K channel.
Single-channel recordings demonstrated outward unitary currents
fluctuating between two levels that corresponded to the closed and open
channels (Figs.
1A and
2A). A decrease in
the time the channels spent in the open state coupled with an increase
in the time spent in the closed state was apparent after the addition of EtOH (Figs. 1A and 2A). The inhibitory effect
of EtOH on the channel activity was analyzed further by examining the
Po of the channels before and after the addition
of EtOH. Alterations of the Po were found to be
statistically significant (Table 1). Of
all channels examined, 84% (22/31) exhibited inhibition of the
Po by 10 or 20 mM EtOH. An additional 16%
(5/31) were not inhibited at 20 mM but were inhibited when the EtOH
concentration was increased to 40 mM. Overall, 90% of channels (28/31)
showed inhibition by EtOH concentrations of 60 mM or lower (3/31
channels were insensitive to EtOH at 60 mM). Inhibition of channels by 10 or 20 mM EtOH was typically quite pronounced in its effects on the
average open and closed times (Table 1). Inhibition of the
Po was typically associated with an increase in
the average closed time and a decrease in the average open time (Table
1). Whereas the increase in average closed time was large and
statistically significant, the change in average open time was moderate
and was not found to be statistically significant
[F(1,12) = 2.89, P > 0.10]. The average closed times were lengthened to varying degrees with a range of +82.3 to +960.5% increase. Similar effects were found for those channels that were inhibited by 40 mM or higher
concentrations of EtOH (data not shown). When 20 mM EtOH was added to
the trans (extracellular) face in two experiments, the same
pattern of changes in mean closed and open times was observed as found
for the cis (intracellular) addition. A more detailed
kinetic analysis revealed complex gating that also pointed to a
somewhat weaker effect with regard to the channel open time.
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EtOH alters the gating of the maxi-K channel.
To examine the kinetic basis of the inhibition of
Po, we carried out an analysis of the dwell-time
distributions for 10 single channels across 20-60 mV. Four of
these analyses at 40 mV are summarized in Table
2. In all cases, open and closed time
distributions could be well described by the sum of two exponential
terms indicating the existence of at least two open and two closed
states. This appraisal of the minimal number of kinetic states probably
represents an underestimation of those that actually exist. Other
analyses with a wider recording bandwidth support five or more
exponential components for the closed time distribution and three or
more exponential components for the open time distribution (10,
31). The limited resolution in our data may also be expected to
have overestimated the open times.
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EtOH alters the range of voltage-dependent gating of the maxi-K
channel.
The maxi-K channel is activated by increasingly depolarizing voltages
until it reaches a maximum Po. The inhibition by
EtOH might be the result of a shift in the voltage dependence of the channel. By measuring the Po of the channel at
different voltages in the presence and absence of EtOH, we found that
EtOH caused two changes in the response of the BASM maxi-K channels to
the voltage as interpreted from the description of the
Po-V relation by a Boltzmann
relationship. It decreased the maximum Po and
increased the amount of depolarization required for the
Po to attain a half-maximum Po (V1/2). In two
separate experiments, 20 mM EtOH decreased the maximum
Po from a Po of 0.83 to
0.56 or 0.80 to 0.37 (Fig. 3, A and B, respectively) and shifted the
V1/2 from 17.4 to 43.3 mV or 28.3 to 41.2 mV
(Fig. 3, A and B, respectively). The slope factor, k (mV/e-fold change in
Po; e 2.72), was
also decreased slightly from 8.7 to 7.4 mV or 10.9 to 7.3 mV (Fig. 3,
A and B, respectively).
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The concentration of Ca2+ affects the
response of the maxi-K channel to EtOH.
The maxi-K channel is also activated by increased concentrations of
intracellular Ca2+, and the effects of EtOH on channel
activity may be altered by different concentrations of
Ca2+. The effect of increased concentrations of
Ca2+ on the sensitivity to EtOH was investigated by first
treating a channel with 20 mM EtOH in the presence of 2.5 µM
Ca2+, which potently decreases the
Po (Fig. 4). The
channel inhibited by 20 mM EtOH retained sensitivity to elevated
concentrations of Ca2+ added on the cis
(intracellular) side, and the Po increased as the concentration of Ca2+ was sequentially increased. This
effect was reproduced in a second experiment (data not shown).
Restoration of channel activity by increased intracellular
Ca2+ supports an interpretation that the effects of EtOH on
the BASM maxi-K channel are reversible. In addition, channel
denaturation by EtOH is unlikely to have occurred, because all of the
control single-channel characteristics remain intact with
EtOH-inhibited channels (see Figs. 3 and 6). Reversibility of EtOH
effects on maxi-K channels (13-15) and another K
channel (8, 21) has been documented.
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The effect of different concentrations of EtOH on maxi-K channel
activity.
Twenty millimolar EtOH is near the maximum legal intoxication level,
and because 20 and 10 mM EtOH significantly inhibited BASM maxi-K
channel activity, we investigated whether lower EtOH concentrations
could inhibit the channel. In Fig. 5, the
inhibition of the maxi-K channels studied at 5, 7.5, 10, and 20 mM EtOH
is presented. Although the degree of inhibition by a specific
concentration of EtOH varied in the different experiments, the majority
of channels were notably affected. These data also suggest that a
population of channels exist within the BASM preparation that shows
sensitivity to EtOH well below the physiological intoxication range. In
particular, the low concentration of 5 mM EtOH caused a very large
degree of inhibition (70 and 88%) in two experiments (Fig. 5).
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The permeation properties of the BASM maxi-K channel are not
affected by EtOH.
The BASM maxi-K channels in the isolated vesicles used in this study
exhibited the same properties as those reported previously for this
channel reconstituted in planar lipid bilayers (20). The
I-V of the channels incorporated into planar lipid bilayers of POPE/POPC (1:1) showed a slope conductance of 246.5 ± 18 pS (n = 10) in symmetrical 150 mM KCl, a value similar to
249.2 ± 2.5 pS previously reported for the channel incorporated
into a bilayer of neutral lipids composed of POPE/POPC (7:3)
(20), and the reversal potential was near 0 mV. Addition
of 10 mM EtOH to the representative channel analyzed in Fig.
6 resulted in a 52.9% decrease in the
Po but did not affect the slope conductance (248.3 vs. 251.8 pS) or reversal potential (0.8 vs.
1.8 mV) for
control vs. EtOH values, respectively (r2 = 0.999 for both regressions). The lack of an EtOH effect on these
parameters was consistently observed in the BASM maxi-K channels
because the addition of 10 or 20 mM EtOH did not significantly alter
the slope conductance (246.5 ± 18 vs. 248.9 ± 10.9 pS,
P = 0.72) or reversal potential (1.7 ± 2.8 vs.
0.8 ± 2.6 mV, P = 0.49; n = 10);
values are presented without or with EtOH, respectively. In one
experiment, the effect of concentrations of EtOH as high as 80 mM were
examined, and again, these channel properties remained unaltered (data
not shown).
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Variable responses of the BASM maxi-K channel to EtOH. BASM maxi-K channel activity showed a relatively uniform response to EtOH with 90% of channels being inhibited at a concentration of 60 mM or below. As with other channel characteristics, however, there was some variability in this overall robust response. We also observed a few examples in which the response of the BASM maxi-K channel to EtOH was opposite to that observed in the majority of the experiments (see below). Variability of maxi-K channel activity in planar lipid bilayers has been observed by others (20, 25, 38, 42). The data in Table 1 show that in seven different experiments, EtOH decreased the average open time and significantly increased the average closed time. Although in those experiments the average open time always decreased, in an eighth experiment, 20 mM EtOH caused an increase in the average open time of 46.7% (data not shown). This effect, however, was superceded by a large increase in the average closed time (+111.1%) to give the net outcome of a 52.8% reduction in Po (data not shown).
As shown in Fig. 5, the BASM maxi-K channel was inhibited by EtOH concentrations as low as 5.0 mM. We also examined the effects of the lower concentrations of 1.0 and 2.5 mM EtOH on channel activity in three different experiments. In two of the three experiments, the channels had decreased Po at 1.0 mM EtOH by 82% and 22%. By contrast, in the third experiment, the channel activity was activated at 1.0 and 2.5 mM EtOH by 58 and 73%, respectively, before being inhibited by 70% at 5.0 mM. Some variability of channel response toward EtOH was also found in the series of experiments designed to examine the effects of EtOH on voltage dependence of the BASM maxi-K channel. Kinetic analyses of a single channel in one experiment (same channel as in Fig. 3A) revealed a similar inhibitory response as those described in Figs. 1 and 2, with an increase of ![]() |
DISCUSSION |
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EtOH inhibits the BASM maxi-K channel activity and stimulates other
maxi-K channels.
In this study, the inhibition of BASM maxi-K channels by EtOH is in
sharp contrast to the stimulation of most other maxi-K channels treated
with EtOH (7, 13, 15, 23). Differential activity of EtOH
toward maxi-K channels at two different locations within the supraoptic
neuron (16) has been noted, however, the effects were
classified as stimulatory or neutral, rather than stimulatory vs.
inhibitory. The different responses toward EtOH that we have documented
may be partly due to structural differences. The BASM maxi-K channels
most likely contained associated -subunits (19, 20,
50), whereas the EtOH-activated channels from skeletal muscle
(7) or brain neuroendocrine cells (13, 16,
23) may not have (52). Although this points to a
potential role for
-subunits in the EtOH inhibition of the BASM
maxi-K channels, the inhibition of the cloned
-subunit of BASM
(bSlo) channels expressed in oocytes (14)
indicates that the different responses are not strictly dependent on
the presence of
-subunits. It remains possible, however, that
-subunits may modulate the degree to which a channel is sensitive to
EtOH. The
-subunit is known to affect the activation of maxi-K
channels by two other agents, dehydrosoyasaponin
(30) and estradiol (54). The phosphorylation status affects the response of one maxi-K channel to EtOH
(23), and the different responses to EtOH may arise from
different phosphorylation states of the BASM maxi-K channel as well as
the bSlo expressed in Xenopus oocytes. In
addition, the bSlo and the BASM maxi-K channels may be
transcripts of functionally different splice variants of the
slo gene (2, 24, 51), which may be
differentially affected by EtOH.
EtOH stabilizes the BASM maxi-K channel in the closed state.
In the majority of the bilayers studied here (in the absence or
presence of EtOH), the distributions of closed times and open times
revealed the presence of at least two closed states and two open
states, respectively (Figs. 1 and 2; Table 2). Under our recording
conditions, however (see METHODS), the resolution of brief
closures and openings was limited (the mean duration of both classes of
brief events was ~1 ms). Nevertheless, in the majority of
experiments, it is clear that the channels enter the long-duration open
state for most of the time (>85%). The most consistent and robust
effect of low concentrations of EtOH ( 20 mM) on channel gating at
the single-channel level was to prolong the mean duration of long
closures between 2- and 30-fold in 9/10 single-channel bilayers (Tables
1 and 2, in part). Another less consistent but significant effect
includes an increase in the fraction of long closures (Figs. 1 and 2;
Table 2). These changes suggest stabilization of the closed state(s). A
moderate reduction of the mean duration of long openings (Figs. 1 and
2; Table 2) also suggests a decreased stability of the open state.
Altogether, these effects contributed to a reduced
Po in the majority of the single channels
examined (>80%), and accordingly, a rightward shift of the
Po-V relation accompanied by an
apparent reduction in the maximum Po.
EtOH may alter the Po-V relation by a
combination of effects on the presumed transition rates between
multiple states.
Physiological consequences of the EtOH inhibition. Smooth muscle maxi-K activity appears to be important in the regulation of vascular tone (3, 22, 27, 28, 35, 41). Our results indicate that clinically relevant amounts of 5-20 mM EtOH could inhibit smooth muscle maxi-K channel activity at low concentrations of Ca2+. Although the regulation of vascular tone is complex, an EtOH-induced inhibition of in vivo maxi-K channel activity might be expected to affect the basal tone. Furthermore, maxi-K channels respond directly to a number of vasoconstrictors and vasorelaxants (36, 46), therefore, if EtOH could exert such a marked effect on the in vivo maxi-K component, even normal homeostatic responses to other agents that affect vascular tone might be compromised.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Dr. Kathleen Giangiacomo for generous donation of the bovine aortic smooth muscle preparation and for helpful consultation with the experiments and manuscript preparation. We thank Darren Boehning for technical assistance with the Ca2+ electrode.
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FOOTNOTES |
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This work was supported by National Institute on Alcohol Abuse and Alcoholism Grants AA-07186 (to J. S. Ellingson) and AA-07463 (to F. S. Walters), and in part by AA-10615 (to M. Covarrubias).
Address for reprint requests and other correspondence: J. S. Ellingson, 264 Jefferson Alumni Hall, Dept. of Pathology, Anatomy, and Cell Biology, Thomas Jefferson Univ., 1020 Locust St., Philadelphia, PA 19107 (E-mail: John.Ellingson{at}mail.tju.edu).
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 2 March 2000; accepted in final form 26 April 2000.
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