Potent inhibition of the aortic smooth muscle maxi-K channel by clinical doses of ethanol

F. S. Walters, M. Covarrubias, and J. S. Ellingson

Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Delta V1/2 congruent  17 mV), a slightly steeper relationship (change in slope factor, Delta k, congruent  -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 beta -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 alpha -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 alpha -subunits or channels from brain neuroendocrine tissues where mainly the alpha -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 alpha - and beta -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.


    METHODS
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INTRODUCTION
METHODS
RESULTS
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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.

To examine individual time constants for selected experiments, the total number of open or closed events were normalized and transformed according to the method of Sigworth and Sine (48). To examine the respective kinetic components of the transformed data, a Simplex maximum likelihood fitting routine (pStat) was used.

Analysis of variance (ANOVA) was employed to examine slope conductance, reversal potential, and single-channel parameter values. In addition, significant differences between single-channel parameter means were determined by Fisher's protected least-significant differences test by use of the F-statistic (49). Po values were transformed using arcsine <RAD><RCD>proportion</RCD></RAD> before ANOVA.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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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Fig. 1.   A: the effect of ethanol (EtOH) on the current through a single bovine aortic smooth muscle (BASM) maxi-K channel incorporated into a planar lipid bilayer. Solutions that bathed both sides of the channel contained 150 mM KCl, 2.5 µM CaCl2, and 10 mM HEPES, pH 7.4; membrane potential was held at +40 mV. Solid lines (left) indicate closed current level for each consecutive trace. EtOH was added to the cis (intracellular) side of the chamber. Data were sampled at 5 kHz and filtered at 500 Hz during acquisition; additional filtering at 400 Hz has been imposed for display only. The data obtained in the absence of EtOH are presented as the "control". B: effect of EtOH on closed and open dwell-time distributions of BASM maxi-K channel kinetics. The total number of events (N) were normalized according to the method of Sigworth and Sine (48) to view individual components; bin density = 20 bins per decade. Data were fit using a Simplex maximum likelihood routine. The mean duration of each component (given as time constants or tau  values) are in milliseconds, with the short and long open time constants represented as tau OS and tau OL, and the short and long closed time constants as tau CS and tau CL, respectively. The fractional contribution of each particular component to the composite fit is given in parentheses. Dotted lines show the individual components; solid lines show the composite fit.



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Fig. 2.   A: the effect of EtOH on the current through a single BASM maxi-K channel incorporated into a planar lipid bilayer. B: the effect of EtOH primarily on the closed dwell-time distribution of BASM maxi-K channel kinetics. The conditions and analysis of this channel are the same as those for the channel in Fig. 1 except that the holding potential was +20 mV.


                              
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Table 1.   Effects of ethanol on BASM maxi-K single-channel parameters

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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Table 2.   Effect of EtOH on dwell-time distributions of BASM maxi-K single-channel kinetics

The inhibition of channel activity was the result of two apparent types of kinetic behavior: 1) a large increase in channel closed time coupled with a decrease in channel open time (Fig. 1B and Table 2), or 2) a dramatic increase in channel closed time coupled with a small decrease or nondetectable change in channel open time (Fig. 2B and Table 2).

In a channel that exhibits the first type of response, the longer mean closed time (tau CLong) more than doubled after treatment with 10 mM EtOH, and its proportional contribution also increased nearly twofold (Fig. 1B and Table 2). The long mean open time (tau OLong) decreased by almost two-thirds while maintaining the same dominant proportional contribution to the overall distribution of open time. Other channels that exhibited this apparent type of response varied in the proportional changes of tau CLong or tau OLong (Table 2). In a channel that exhibited the second type of response to 10 mM EtOH, the longer closed state exhibited a dramatic increase of 732% over the control time, and its proportional contribution also increased by 27% (Fig. 2B). tau OLong decreased from 28 to 22 ms after EtOH treatment with its proportional contribution remaining quite high. A substantial increase in tau CLong (>2-fold change) as described in Figs. 1 and 2 was found in 9 out of 10 experiments where channels were inhibited by EtOH (Tables 1 and 2, in part). Short-duration components of the open and closed distributions were relatively unaffected by EtOH; however, parameter estimates of these components were limited due to the data resolution (see METHODS).

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 congruent  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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Fig. 3.   EtOH effect on voltage-dependent gating of the BASM maxi-K channel. In 2 separate experiments (A and B), the channel open probability (Po) is plotted as a function of membrane potential in the presence (open circle ) or absence () of 20 mM EtOH. The lines represent the best fit of the data to a Boltzmann function where Po = Pomax/{1 + exp[(V1/2 - Vm)/k}], Pomax is the maximum Po, Vm is the membrane potential, V1/2 is the midpoint potential for activation, and k is a slope factor. Solutions bathing both sides of the channels contained 150 mM KCl, 2.5 µM CaCl2, and 10 mM HEPES, pH 7.4.

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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Fig. 4.   The effects of the concentration of Ca2+ on the response of the maxi-K channel to EtOH. In the experiment shown, a channel that could be inhibited by EtOH at low-Ca2+ concentrations became very resistant to inhibition after elevating intracellular Ca2+. A: the corresponding currents through the single channel are shown for the concentrations of Ca2+ and EtOH added as noted. Solid lines (left) indicate closed current level. B: the Po is plotted as a function of time, during which additions of Ca2+ and EtOH were made to the intracellular solution as indicated. Solutions that bathed both sides of the channel contained 150 mM KCl, 2.5 µM CaCl2 (initial), and 10 mM HEPES, pH 7.4; membrane potential was held at +20 mV. Channel Po was calculated as described in METHODS, but over consecutive intervals of 10 s.

After the previously inhibited channel (Fig. 4) was restored to a Po of 0.85 by 20 µM Ca2+, the subsequent sequential addition of more EtOH did not cause the strong inhibition observed at lower concentrations of Ca2+ (Fig. 4). This latter property was consistently observed in five other experiments where activation to a high Po by prior elevations of intracellular Ca2+ (20-400 µM) rendered the channels resistant to inhibition by subsequent additions of EtOH ranging from 60 to 200 mM.

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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Fig. 5.   Inhibition of BASM maxi-K activity by low concentrations of EtOH. The Po in the absence and presence of the EtOH concentration indicated was determined, and the percent of the control Po values were calculated. The percent of control Po was then plotted for each experiment at the respective intracellular EtOH concentration. N = 3, 3, 6, or 12 for 5, 7.5, 10, or 20 mM EtOH, respectively. Solutions that bathed both sides of the channels contained 150 mM KCl, 2.5 µM CaCl2, and 10 mM HEPES, pH 7.4.

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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Fig. 6.   Maintenance of BASM maxi-K channel selectivity of K+ over Na+ in the presence of EtOH. The mean amplitudes of single-channel currents are plotted as a function of voltage when the extracellular solution contained 150 mM KCl (black-triangle, triangle ) or 150 mM NaCl (, open circle ). Intracellular solutions contained 150 mM KCl (black-triangle, ) or 150 mM KCl plus EtOH (triangle , open circle ). All solutions contained 2.5 µM CaCl2 and 10 mM HEPES, pH 7.4. Inhibiting concentrations of EtOH used were 10 or 20 mM for the symmetrical KCl or asymmetrical KCl:NaCl conditions, respectively. Each set of data was obtained from a single channel incorporated into the lipid bilayer. The experiment that used symmetrical KCl conditions is the same channel as that shown in Fig. 1.

Although the maxi-K channel displays high permeability to K+, it displays relatively little permeability to the smaller Na+, and this property is known to be maintained in the BASM channel when incorporated into planar lipid bilayers (20). To determine whether EtOH altered the selective permeability for K+ and Na+, we determined the single-channel currents as a function of the membrane potential under bi-ionic conditions with Na+ on the extracellular side of the channel and K+ on the intracellular side. The results in Fig. 6 show that at potentials as negative as -60 mV, no inward current was detected in the presence of 150 mM Na+, showing that the maxi-K channels exhibited the selective permeability for K+ over Na+. The selectivity for K+ was not disrupted in the presence of 20 mM EtOH in the experiment shown in Fig. 6 or in a second experiment (data not shown).

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 tau CLong of 302% over the 20- to 40-mV range and a decrease in tau OLong of 65.3%. In a third experiment of this type, a single channel was not very sensitive to EtOH (requiring 60 mM EtOH to reduce the Po by ~50%). In the presence of 60 mM EtOH, the V1/2 of this channel was again increased (by 13.6 mV), and thus EtOH caused a rightward shift in voltage dependence in all three of these experiments. However, in this third experiment, the k was increased by +4.3 mV by EtOH rather than decreased. Also, whereas the maximum Po value attained in the presence of EtOH was reduced in the first two experiments (Fig. 3, A and B), in the third experiment, the maximum Po values attained with and without EtOH were identical, at about 0.8.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -subunits in the EtOH inhibition of the BASM maxi-K channels, the inhibition of the cloned alpha -subunit of BASM (bSlo) channels expressed in oocytes (14) indicates that the different responses are not strictly dependent on the presence of beta -subunits. It remains possible, however, that beta -subunits may modulate the degree to which a channel is sensitive to EtOH. The beta -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.

Alternatively, the mSlo and bSlo (from BASM) differ by only seven amino acid residues (34), and these structural differences may be responsible for their different responses to EtOH, considering that the alteration of only one amino acid in a voltage-gated channel (KV3.4) confers EtOH sensitivity (9). In addition, 1-alkanols has been found to interact with a specific 13-amino acid region in the Drosophila Shaw2 channel, and mutations of three or more residues resulted in reduced sensitivity (21).

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.

One can interpret the contrasting effects of EtOH and Ca2+ on the resultant Po as reflecting a stabilization or destabilization of the closed state, respectively. Whereas EtOH essentially favors channel closing, as [Ca2+] increases, this response is effectively diminished. As more channel molecules respond to an increase in [Ca2+], they will reside in a closed state that is more prone to open, thereby opposing the EtOH effect on the BASM maxi-K channel. Further single-channel analysis will be required to pinpoint the actual mechanism of interaction.

In a study using chimeras of the tail section (COOH terminus) of maxi-K alpha -subunits from Ca2+-sensitive and insensitive channels, it was proposed that Ca2+ may be involved in interdomain interactions that help to establish the channel activity (44). It was proposed that in the absence of Ca2+, the tail interacts with the core region of the channel and inhibits voltage-gated activation, whereas if Ca2+ is present, it relieves the inhibition by releasing the binding of the tail to the core (44). If this proposal is universally correct for maxi-K channels, it is possible that at low concentrations of Ca2+, EtOH may interfere with the ability of Ca2+ to relieve the inhibition by the tail for the BASM maxi-K channels, thereby promoting the closed state in agreement with the results from our kinetic data. In support of this interpretation, higher concentrations of Ca2+ overcome the EtOH effect and relieve the inhibition of the channel. Once the channel is activated by higher Ca2+ concentrations, presumably by releasing the tail from the core, higher EtOH concentrations of EtOH cannot overcome the Ca2+ activation. An alternative interaction with mSlo or other maxi-K channels that are stimulated by EtOH would have to be invoked assuming that a related mechanism exists. A maxi-K tail sequence that is less inhibitory (shows a left-shifted range of voltage-dependent activation at 0 µM Ca2+) has been discovered (43), therefore, it remains plausible that conformational changes mediated by EtOH could also produce the stimulatory-type response. This would be in accord with the interpretation that EtOH and Ca2+ can behave as partial and full agonists of mSlo, respectively (15).

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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