Ethanol sensitivity of BKCa channels from arterial smooth muscle does not require the presence of the beta 1-subunit

Alejandro M. Dopico

Department of Pharmacology and Program in Neuroscience, University of Tennessee Health Science Center, Memphis, Tennessee 38163


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Ethanol inhibition of large-conductance, Ca2+-activated K+ (BKCa) channels in aortic myocytes may contribute to the direct contraction of aortic smooth muscle produced by acute alcohol exposure. In this tissue, BKCa channels consist of pore-forming (bslo) and modulatory (beta ) subunits. Here, modulation of aortic myocyte BKCa channels by acute alcohol was explored by expressing bslo subunits in Xenopus oocytes, in the absence and presence of beta 1-subunits, and studying channel responses to clinically relevant concentrations of ethanol in excised membrane patches. Overall, average values of bslo channel activity (NPo, with N = no. of channels present in the patch; Po = probability of a single channel being open) in response to ethanol (3-200 mM) mildly decrease when compared with pre-ethanol, isosmotic controls. However, channel responses show qualitative heterogeneity at all ethanol concentrations. In the majority of patches (42/71 patches, i.e., 59%), a reversible reduction in NPo is observed. In this subset, the maximal effect is obtained with 100 mM ethanol, at which NPo reaches 46.2 ± 9% of control. The presence of beta 1-subunits, which determines channel sensitivity to dihydrosoyaponin-I and 17beta -estradiol, fails to modify ethanol action on bslo channels. Ethanol inhibition of bslo channels results from a marked increase in the mean closed time. Although the voltage dependence of gating remains unaffected, the apparent effectiveness of Ca2+ to gate the channel is decreased by ethanol. These changes occur without modifications of channel conduction. In conclusion, a new molecular mechanism that may contribute to ethanol-induced aortic smooth muscle contraction has been identified and characterized: a functional interaction between ethanol and the bslo subunit and/or its lipid microenvironment, which leads to a decrease in BKCa channel activity.

maxi-potassium channel; alcohol; aorta; vasoconstriction


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACUTE EXPOSURE TO clinically relevant concentrations of ethanol (EtOH) causes contraction of peripheral, cerebral, coronary, and umbilical arteries in several species, including humans (2-4, 23). There is substantial evidence that EtOH produces vasocontriction by acting on the smooth muscle itself (2, 26, 35, 55). However, the ion channel populations involved in EtOH action are largely unknown.

Large-conductance, Ca2+-activated K+ (BKCa) channel activity controls depolarization and contraction in vascular smooth muscle. Activation of BKCa channels gives rise to positive outward currents, which, in turn, drive the smooth muscle cell membrane potential in a negative direction and, eventually, counteract contraction (5, 8, 30). Therefore, EtOH inhibition of BKCa channels may be the mechanism underlying, or at least contributing to, alcohol-induced contraction of vascular smooth muscle.

BKCa channels consist of pore-forming (alpha , encoded by slo genes) and modulatory (beta ) subunits (33, 51). Heterologous expression of slo channels produces currents that bear most of the features of native BKCa channels: large unitary conductance and selectivity for K+, and Ca2+ and voltage dependence of gating. The presence of beta -subunits shifts the voltage dependence of activation to more negative potentials by increasing the apparent Ca2+ sensitivity of slo channels (1, 9, 41, 42) and modifies channel pharmacology (41, 50).

Acute exposure to clinically relevant EtOH concentration ([EtOH]) reversibly increases BKCa channel activity in neurohypophysial terminals (20), GH3 cells (31), and small neurons in dorsal root ganglia, where alcohol action leads to a reduction in neuronal excitability (27). EtOH activation of BKCa channels is maintained when studied in isolated membrane patches of Xenopus oocytes expressing slo subunits cloned from mouse brain (mslo, mbr5 variant; see Refs. 11 and 18) or in lipid bilayers in which slo subunits from human brain (hslo; see Refs. 1 and 49) were reconstituted (12). These studies suggest that slo subunits and their immediate lipid microenvironment are sufficient targets for EtOH activation of neuronal BKCa channels.

Potentiation of BKCa channels by EtOH, however, is not a universal finding. EtOH (10-100 mM) reversibly increases the activity of BKCa channels in isolated membrane patches from the nerve endings but not from the somata of supraoptic neurons (22). BKCa channel phenotypes in these two areas of the same neuron markedly differ in not only EtOH sensitivity but also gating kinetics, Ca2+ sensitivity, ion conduction, and charybdotoxin sensitivity (22). These differences are likely determined by the presence of different slo isoforms and/or beta -subunits in the two neuronal domains, which could also contribute to differential EtOH sensitivity of the isochannels.

In contrast to its reversible potentiation of several BKCa channels of neuronal origin, EtOH (5-80 mM) significantly inhibits the activity of native bovine aortic smooth muscle BKCa channels after their reconstitution into artificial lipid bilayers (52). It is quite possible that native BKCa channels remain in these bilayers as heterooligomers formed by the tight association of bslo (46) and beta 1-subunits, as found in their native smooth muscle membrane (24). Thus differential responses to EtOH between native bovine aortic smooth muscle BKCa channels and slo channels of neuronal origin when studied in cell-free membranes or bilayers could be attributed to differential alcohol response between bslo and other slo channels and/or control of slo responses to EtOH by the proteolipid microenvironment of the slo subunit, including introduction of EtOH inhibition of bslo channel activity by the presence of beta 1-subunits.

Here, a possible inhibition of bslo channel activity in response to acute exposure to clinically relevant concentrations of EtOH was explored by expressing bslo subunits in Xenopus oocytes and studying their alcohol responses in excised membrane patches under conditions identical to those used to evoke EtOH activation of mslo channels. In addition, a possible modulation of bslo channel responses to EtOH by the presence of beta 1-subunits was determined. Preliminary data were published in abstract form (17).


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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RNA preparation. BKCa channels were expressed in Xenopus oocytes by injection of cRNA transcribed in vitro from cDNA derived from the Slowpoke locus of bovine aortic smooth muscle (bslo; see Ref. 46). Briefly, bslo cDNA inserted in the Kpn I/Not I sites of the pcDNAIII expression vector was linearized with Xba I and transcribed in vitro using T7 polymerase. BKCa channel beta 1-subunit cDNA inserted in the EcoR I/Sal I sites of the pCI-neo expression vector was linearized with Not I and transcribed in vitro using T7 polymerase. Restriction enzymes were obtained from Promega (Madison, WI). In vitro transcriptions were performed using mMessage mMachine transcription kits (Ambion, Austin, TX). Full-length bslo and BKCa channel beta 1-subunit cDNAs were generous gifts from Dr. Edward Moczydlowski (Department of Pharmacology, Yale University) and Dr. María García (Merck Research Laboratories), respectively.

Use of Xenopus oocytes and RNA injection. Xenopus laevis were purchased from commercial breeders and kept in artificial pond water on a 12:12-h light-dark cycle. Oocytes were removed and defolliculated, as previously described (18). RNA injection was conducted 36-72 h before patch-clamp recordings, using an automated nanoliter injector (Nanojet II; Drummond Scientific, Broomal, PA). The beta 1-subunit mRNA was coinjected with the alpha  (bslo)-subunit mRNA at a concentration of 0.25 µg/µl each for a total volume of 36.8 nl. These subunits were coinjected at a molar ratio >5:1 to ensure that all bslo subunits were interacting with beta 1-subunits. BKCa channel activity after bslo and beta 1 coexpression was recorded and compared with that after injection of 36.8 nl (0.25 µg/µl) of the bslo subunit alone in the same batch of oocytes. Before starting recordings, oocytes were placed in a dish containing a hypertonic solution containing (in mM) 200 K-aspartate, 20 KCl, 1 MgCl2, 10 EGTA, and 10 HEPES, pH 7.4, for 10-15 min. With this treatment, the oocytes shrunk, which facilitates the removal of the vitelline layer with forceps, exposing the cell membrane for patch-clamp recording. Next, the oocytes were placed in ND-96 saline containing (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, and 5 HEPES, pH 7.5, for 15 min before recording.

Data acquisition and analysis. Single channel recordings were obtained from excised inside-out (I/O) or outside-out (O/O) membrane patches using standard patch-clamp techniques. Currents were recorded using a EPC8 patch-clamp amplifier (List Electronics, Darmstadt, Germany), low-pass filtered at either 3 kHz (for dwell time analysis) or 1 kHz [for display and NPo (N = no. of channels present in the patch; Po = probability of a single channel being open) determinations] using an eight-pole Bessel filter (model 900C; Frequency Devices, Haverhill, MA), digitized at a sampling rate of 10 kHz, and stored on an IBM-compatible computer hard drive. Data acquisition and analysis were performed using pClamp software (version 8; Axon Instruments, Union City, CA). An agar bridge containing high-K+ gluconate solution (for composition, see Table 1) was used as a ground electrode. Patch pipettes were pulled from glass microcapillaries (Drummond Scientific). The shank of each patch pipette was coated with Sylgard 184 (Dow Corning, Midland, MI) to reduce capacitance and noise. Immediately before recording, the tip of each electrode was fire-polished on a microforge (MF 200; World Precision Instruments, Sarasota, FL) to give resistances of 5-10 MOmega when filled with high-K+ solution (Table 1). All experiments were carried out at room temperature.

                              
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Table I.   : Components of solutions used to record EtOH action on BKCa single channel function in excised I/O or O/O patches

The product NPo was used as an index of steady-state channel activity. NPo values were calculated from all-points amplitude histograms (18). The slope of the unitary current amplitude (i)-voltage (V) relationship in "symmetric" K+ concentration ([K+]; i.e., the same high-K+ solution bathing the extracellular and the cytosolic sides of the patch membrane) was used as the unitary conductance (gamma ). Values for i were obtained from the Gaussian fit of all-points amplitude histograms by measuring the distance between the modes corresponding to the closed state(s) and the first opening level (20).

In patches when N was low, its value could be determined from the maximal number of unitary current levels under recording conditions that effectively raised Po to ~1. This was achieved by holding I/O patches at positive potentials (+40 to +60 mV) and applying solutions containing 30 µM free Ca2+ concentration to the cytosolic side of the patch. In single-channel patches, open time (to) and closed times (tc) were measured with half-amplitude threshold analysis. A maximum-likelihood minimization routine was used to fit curves to the distribution of to and tc. Determination of the minimum number of terms for adequate fit was established using a standard F statistic table (significance level <0.01). Changes in mean to from multichannel patches of unknown N were calculated as previously described (18). NPo and dwell-time distribution data under a particular experimental condition (drug exposure or isosmotic controls) were calculated from periods of continuous channel recording that typically ranged 20-60 s. For all experiments, voltages given correspond to the potential at the intracellular side of the membrane.

Data are expressed as means ± SE. Statistical analysis was performed with paired or unpaired Student's t-tests, according to experimental design (25). Data plotting and fitting were performed using Origin software (version 6.1; OriginLab, Northampton, MA).

Solutions, chemicals, and construction of concentration-response curves to EtOH. Single-channel recordings were obtained from excised I/O or O/O patches using a variety of solutions (Table 1). The extracellular solutions used in I/O recordings (pipette solution) or O/O recordings (bath solution) contained >= 1 µM free [Ca2+], which helped gigaohm seal formation. Calculation of the nominal free [Ca2+] in these highly buffered solutions was done according to the Max Chelator Sliders software (C. Patton, Stanford University). For solutions with a nominal free [Ca2+] >= 300 nM, the actual free [Ca2+] was checked with a Ca2+-selective and a reference electrode (models 476041 and 476416; Corning; see Table 1).

All solutions were prepared with high-grade salts (Sigma Chemical, St. Louis, MO) and 18 MOmega water (Milli-Q Water System; Millipore, Bedford, MA). Deionized, 100% pure, EtOH (American Bioanalytical, Natick, MA) was freshly diluted in bath solution immediately before recordings. Urea was diluted in bath solution to the desired final concentration from a concentrated stock solution (1 M) of ionic composition identical to the final bath solution. Urea-containing solutions did not usually have any effect on BKCa channel activity when compared with urea-free bath solution, although in a very few cases some inhibition of activity was observed. Dihydrosoyaponin-I (DHS-I; a generous gift from Dr. Owen McManus, Merck Research Laboratories) and 17beta -estradiol (Sigma Chemical) were diluted in bath solution to the desired final concentrations from concentrated stock solutions (1 and 5 mM, respectively) made with 100% DMSO. DMSO-containing solutions (final DMSO concentration <0.001%) were used as controls for 17beta -estradiol- or DHS-I-containing solutions. Handling and application of 17beta -estradiol and corresponding controls were conducted in the dark.

After excision from the oocyte, the intracellular side of the I/O patch was alternatively placed in the mouth of several "sewer" micropipettes (1 mm diameter; WPI), each delivering a solution that contained the desired concentration of EtOH and free [Ca2+]. Bath solution with urea isosmotically replacing EtOH with the corresponding free [Ca2+] was used as a control solution. This gravity-fed delivery system avoids the dilution of agent-containing solutions in the bath solution that continuously perfuses the dish where oocytes/patches are placed. For the construction of concentration-response curves to EtOH action on bslo channels in I/O patches, only "first applications" of EtOH (i.e., acute EtOH exposure to a naive patch) were considered. Thus different EtOH concentrations were applied to different patches.

For the construction of concentration-response curves to EtOH action on bslo vs. bslo + beta 1 channels in O/O patches, both control bath and EtOH-containing solutions were applied to the extracellular surface of the same patch for 20-60 s, using an automated, pressurized, DAD-12 superfusion system (ALA Scientific Instruments, Westbury, NY). NPo in EtOH was compared with its value in control solution applied immediately before EtOH. A similar method of solution application was used for the construction of concentration-response curves to intracellular Ca2+ (Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>) activation of bslo channels in the absence and presence of EtOH, performed in the same I/O patch.

In all cases, patches were excised from the cell in "asymmetric" conditions (i.e., high K+ in the electrode and low K+ in the bath solution) at 0 mV. After identifying BKCa channel openings by conductance and Ca2+ sensitivity, patches were exposed to high-K+ solutions delivered through the capillary system. The disappearance of BKCa openings in symmetric K+ concentration ([K+]) was used as a criterion to properly place the patch pipette tip in the mouth of the capillary and determine that the different solutions were effectively reaching the patch. In all experiments, EtOH applications were no longer than 1 min to avoid any possible "desensitization," as observed with protracted exposure in the studies of EtOH action on neurohypophysial BKCa channels (20).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of bslo subunits in Xenopus oocytes renders unitary current events that display all major features of BKCa channels. Single-channel current recordings obtained in I/O patches after expression of bslo subunits in Xenopus oocytes showed all major characteristics of BKCa unitary currents. First, steady-state activity (NPo) reversibly increased with increases in intracellular Ca2+ concentration ([Ca2+]i). At constant voltage (e.g., +40 mV), when the cytosolic side of the same I/O patch was consecutively exposed to solutions with a free [Ca2+] of 0.1, 0.3, and 1 µM, bslo channel NPo reached 0.002, 0.008, and 0.9, respectively (also see Fig. 4). Second, at constant free [Ca2+] at the intracellular side of the patch, NPo increased as the membrane potential was made more positive. From -60 to +40 mV, the voltage dependence of activation could be described by a Boltzmann relationship, such that a plot of the natural log of NPo (or Po) as a function of voltage was linear at low values of Po. From this plot, the reciprocal of the slope is the potential needed to produce an e-fold change in NPo (20, 47). For bslo channels expressed in oocytes, this reciprocal was 12.3 ± 1.6 mV/e-fold increase in NPo (n = 5; a representative patch is shown in Fig. 3). This value is similar to values reported for other slo channels expressed in Xenopus oocytes (11, 16, 18, 28), after reconstitution into artificial lipid bilayers (13), and for arterial smooth muscle BKCa channels studied either in situ (19, 43) or in lipid bilayers (52). From the slope factor, the effective valence (z; see Ref. 48) was obtained: z = 2.6 ± 0.3 (n = 5), which indicates that a minimum of three elementary charges are moved across the electrical field to gate the bslo channel.

Finally, the bslo pore displayed high selectivity and unitary conductance for K+. The i/V relationship obtained from I/O patches in symmetric 145 mM [K+] showed ohmic behavior from -60 to +40 mV, with a slope conductance of 259 ± 20 pS (n = 5). When the bath solution was switched from 145 mM K+ to 100 mM K+/45 mM Na+, the reversal potential shifted from ~0 to 28 ± 4 mV (n = 3), almost identical to the value predicted by the Nernst equation for a channel highly selective for K+ over Na+ (i.e., 30.6 mV). This combination of large conductance and high selectivity for K+ over Na+ is a typical feature of the slo pore (reviewed in Ref. 36).

The majority of bslo channels in excised, I/O patches respond to acute EtOH exposure with a reversible decrease in steady-state activity. EtOH inhibition in this subset of channels is concentration dependent. The acute exposure of the cytosolic surface of I/O patches to 50 mM EtOH usually caused a reversible decrease in bslo channel steady-state activity compared with isoosmotic controls (Fig. 1A), confirming an early finding (21). This [EtOH] was initially chosen because it was reported to cause vasoconstriction in most in vitro artery preparations (see references in 55) and to decrease both amplitude and frequency of spontaneous transient outward currents in aortic smooth muscle cells (56). EtOH action on bslo channel activity was routinely recorded 15 min after patch excision, with both sides of the patch exposed to solutions containing no nucleotides. Thus EtOH-induced channel inhibition is neither secondary to changes in diffusible second messenger levels nor mediated by cellular processes such as GTP-binding protein modulation, NAD(P)H-dependent metabolism/modulation, etc. Furthermore, because bslo channel inhibition by EtOH was observed in I/O patches with both sides of the patch exposed to highly buffered [Ca2+] and at positive potentials, this inhibition is not secondary to a possible decrease in [Ca2+] at the cytosolic side of the patch (which would cause BKCa channel inhibition) resulting from putative EtOH inhibition of Ca2+ transmembrane influx. Thus EtOH inhibition of bslo channels appears to result from a direct interaction of EtOH with these BKCa subunits and/or some closely associated component of the cell membrane.


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Fig. 1.   Ethanol (EtOH) at clinically relevant concentrations usually decreases bslo channel activity after expression of these slo subunits from aortic smooth muscle in Xenopus oocytes. A: EtOH (50 mM) reversibly decreases the steady-state activity of bslo channels in inside-out (I/O) patches. Representative single channel recordings obtained from a multichannel I/O patch before (trace on top), during (trace in middle), and 5 min after (trace on bottom) exposure of the cytosolic side of the patch to EtOH. Channel openings are shown as upward deflections; arrows indicate the baseline level. NPo (N = no. of channels present in the patch; Po = probability of a single channel being open) values were obtained from all-points amplitude histograms (20), constructed from 60-120 s of continuous recording under each condition. The composition of the high-K+ solution facing both intracellular and extracellular sides of the patch is given in Table 1. The membrane potential was set to 40 mV, and nominal free intracellular Ca2+ concentration ([Ca2+]i) was ~300 nM. B: EtOH inhibition of bslo channel activity is observed in the majority (58%) of membrane patches and occurs across a wide concentration range. Scattered points show NPo data obtained from different I/O patches in the presence of EtOH compared with control values. Channel NPo obtained in the presence of a given EtOH concentration ([EtOH]) was normalized to that obtained during the previous wash with control medium (with urea isosmotically replacing EtOH) in the same patch. Overall, reversible inhibition of bslo channel activity by 3-200 mM EtOH was observed in 42 out of 72 patches (58%). An increase in activity was observed in 6 patches (8%), the remaining 24 (34%) being nonresponsive (i.e., NPo = 100 ± 10% of control). Because EtOH modifies slo activity by a constant, multiplicative factor within a wide range of potentials (-60 to 40 mV; Ref. 18 and see also Fig. 3), EtOH-induced changes in NPo obtained at different potentials were pooled. In all cases, the membrane potential was set to -40 to 40 mV, and free [Ca2+]i was buffered at ~300 nM (see Table 1). EtOH-containing or control solutions were applied to the cytosolic side of the patch (see MATERIALS AND METHODS). NPo values were obtained from all-points amplitude histograms (20), constructed from 20 to 60 s of continuous recording. Some graph points correspond to more than one data point (patch). Area between two dotted lines indicate where NPo values fall within 100 ± 10% of control. C: EtOH-induced reduction of bslo channel activity, determined from the subset of sensitive patches, is concentration dependent. Maximal inhibition of bslo activity occurred at 100 mM, at which channel NPo reached 46.2 ± 9% of controls, 200 mM causing no additional inhibition. Inhibition of bslo activity is observed with [EtOH] close to circulating levels of alcohol considered legal intoxication in the United States (~20 mM). Data are expressed as means ± SE (n = 5-8 patches). Data in the presence of a given [EtOH] were compared with those in isosmotic urea before EtOH application from the same patch (control). Statistical significance between means (EtOH vs. control) was determined by paired, 2-tailed, Student's t-test (*P < 0.05 and **P < 0.005). Data were fitted with a sigmoidal function (r2 = 0.97). NPo values were obtained as described in B. For A-C, data were low pass filtered at 1 kHz and sampled at 10 kHz.

The inhibition of bslo channel activity in response to 50 mM EtOH shown in Fig. 1A was observed in the majority of patches (6 out of 10) excised from different oocytes. However, no change in activity (3 patches) and even potentiation (1 patch) were also observed. In spite of this variability, bslo channel NPo in the presence of 50 mM EtOH reached an average of 76.4 ± 10.2% of isosmotic control pre-EtOH values (P < 0.01; 2-tailed, paired, Student's t-test; n = 10). To determine whether this response to 50 mM EtOH occurred across a wider concentration range, alcohol action on bslo channel activity was evaluated by applying 3-200 mM EtOH to the cytosolic surface of I/O patches. Concentrations >200 mM usually caused loss of the tight seal.

In the presence of different [EtOH], bslo channel NPo reached the following average values (as %pre-EtOH, isosmotic controls): 3 mM, 95.3 ± 14.7 (not significant, n = 9); 10 mM, 79.5 ± 9.6 (P < 0.05; n = 10); 25 mM, 77.4 ± 15.4 (P < 0.005; n = 12); 50 mM, 76.4 ± 10.2 (P < 0.01, n = 10); 100 mM, 73 ± 10.1 (P < 0.05; n = 14); 200 mM, 71.6 ± 11.6 (P < 0.05, n = 10; 2-tailed, paired, Student's t-test). However, as previously described for data obtained with 50 mM EtOH, both qualitative and quantitative variability among different patches were observed at all concentrations tested. EtOH-dependent inhibition was observed in 5 out of 8 (3 mM), 5 out of 11 (10 mM), 7 out of 12 (25 mM), 6 out of 10 (50 mM), 8 out of 15 (100 mM), and 6 out of 10 (200 mM) membrane patches (Fig. 1B). Overall, EtOH (3-200 mM) reversibly decreased bslo activity in the majority (57%) of patches (37 out of 65 I/O patches), although no response (i.e., NPo = 100 ± 10% of control) was observed in 23 patches (34%), and an increase in activity was observed only in 6 patches (9%). Thus EtOH may reversibly decrease bslo channel activity at concentrations that produce arterial contraction (2, 3, 55), and within legal intoxication levels (~20 mM) and below lethal blood levels (~90 mM) in naive subjects (15).

Differential qualitative responses of bslo channels to a given concentration of EtOH were obtained from membrane patches excised from the same oocyte (n = 6). Thus heterogeneity in the EtOH responses of a cloned ion channel population under identical recording conditions cannot be attributed to interoocyte variability. Because BKCa channel activity was not detected in sham-injected oocytes, it is very unlikely that the qualitative heterogeneity in channel activity responses to EtOH could be explained by alcohol targeting of injected bslo channels in some patches and endogenous slo channels in others. Rather, this heterogeneity is likely explained by heterogeneity in the proteolipid microenvironment of the bslo subunit. However, because bslo subunit-injected oocytes failed to respond to agents that typically evoke modification of channel activity when modulatory beta -subunits are associated with the slo channel (see below), it is unlikely that heterogeneity in bslo responses to EtOH could be explained by the hypothetical presence of endogenous beta -subunits in some patches and not in others.

The overall pattern of bslo responses to acute EtOH exposure markedly differs from the responses to acute EtOH (3-200 mM) of mslo (mbr5 isoform) channels after expression in Xenopus oocytes under recording conditions identical to those used in the present study. Reversible increases in the activity of mslo (mbr5) channels (18) were found in 36 out of 37 I/O patches, the remaining being insensitive. Furthermore, when bslo and mslo (mbr5) were expressed in the same batch of oocytes and their activity was recorded in response to 50 mM EtOH exposure, differential responses were still observed: mslo (mbr5) channel NPo was typically increased to an average of 210 ± 21.2% of controls (P < 0.005; paired, 2-tailed, Student's t-test; n = 6, all patches being EtOH sensitive), whereas bslo activity was decreased to an average of 52.8 ± 4% of controls (P < 0.005; paired, 2-tailed, Student's t-test; n = 5, where 5 patches out of 6 tested were EtOH sensitive). Thus differential responses to acute EtOH exposure between bslo and mslo (mbr5) channels when inserted in a similar proteolipid microenvironment may be linked to the existence of nonconserved regions between these slo channel proteins (see DISCUSSION).

The overall pattern of bslo channel responses to acute EtOH exposure shown above (i.e., 59% of channels are inhibited, 8% are potentiated, and 32% are insensitive) also markedly differs from the responses evoked by similar [EtOH] acutely applied to native aortic smooth muscle channels reconstituted in bilayers of controlled lipid composition, where EtOH shows very high potency and efficacy in >90% of bilayers (50). Next, we decided to determine EtOH potency and efficacy on bslo channels from the selective subset of patches that are inhibited by the drug and compare these parameters of drug action with those obtained with native aortic BKCa channels. EtOH-induced inhibition of bslo channel activity, as determined from the subset of sensitive patches, was concentration dependent from 3 to 100 mM (Fig. 1C). Maximal inhibition of bslo activity was obtained with 100 mM, at which average NPo in the presence of EtOH reached 46.2 ± 9% of controls (P < 0.005; paired, 2-tailed, Student's t-test; n = 4). The highest concentration routinely tested, 200 mM, had no additional effect over that obtained at 100 mM (Fig. 1C). This range of concentrations at which EtOH causes a decrease in bslo channel activity is similar to those reportedly causing vascular smooth muscle contraction (2, 55) and inhibition of native bovine aortic smooth muscle BKCa channels in artificial lipid bilayers (52).

EtOH action on bslo channels is unmodified in the presence of functional beta 1-subunits. Data obtained with native BKCa channels in bilayers, however, differ from current results with bslo channels in two major aspects. First, 90% of native BKCa channels in bilayers (52), but only 59% of bslo channels, are inhibited by EtOH exposure; second, 7.5-10 mM EtOH are enough to totally shut down the activity of native BKCa channels in bilayers, while the maximal alcohol effect (achieved with 100 mM EtOH) on bslo channels, determined from the subset of sensitive patches (Fig. 1C), only decreases activity by ~54%. Thus the "effectiveness" (i.e., no. of patches wherein BKCa channel activity was decreased/no. of patches tested), potency, and efficacy of EtOH action on native bovine aortic smooth muscle BKCa channels reconstituted into artificial lipid bilayers are significantly higher than those from current EtOH data obtained with bslo channels in I/O patches of natural membranes. Because in the bilayer preparation the native BKCa channel most likely exists as a heterooligomeric bslo + beta 1 complex, it is conceivable that the presence of modulatory beta 1-subunits in the bilayer-aortic smooth muscle BKCa channel preparation determines, or at least contributes to, the differences in EtOH effects on native aortic BKCa channels in bilayers vs. bslo channels expressed in oocytes. Thus a possible modification of EtOH action on bslo channels in Xenopus oocytes by the presence of modulatory beta 1-subunits was explored next.

First, pharmacological criteria were used to probe the existence of functional beta 1-subunits associated with the bslo channel before checking the EtOH sensitivity of heterooligomeric channels. Extracellular application of 5 µM 17beta -estradiol increases BKCa currents in isolated membrane patches of Xenopus oocytes that coexpress hslo + beta -subunits but not in those solely expressing hslo subunits (50). Bath application of 17beta -estradiol (5 µM) to O/O patches significantly and reversibly increased the NPo of heterooligomeric bslo + beta 1 channels (Fig. 2A), which reached an average of 184 ± 22% of control (P < 0.01; paired, 1-tailed, Student's t-test). This potentiation was observed in all O/O patches tested (n = 9). In contrast, 5 µM 17beta -estradiol had no effect on the activity of homomeric bslo channels in all O/O patches tested (n = 7; Fig. 2A). Thus introduction of 17beta -estradiol sensitivity by the presence of beta -subunits is not restricted to hslo + beta -subunit complexes but may represent a common phenomenon among slo channels.


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Fig. 2.   The presence of beta 1-subunits, which determines bslo channel sensitivity to 17beta -estradiol and dihydrosoyaponin-I (DHS-I), fails to modify EtOH inhibition of bslo channel activity. A: bars on right show that application of 17beta -estradiol (5 µM) to the extracellular side of outside-out (O/O) patches significantly increased the NPo of heterooligomeric bslo + beta 1 channels, which reached an average of 184 ± 22% of control values. This potentiation was observed in all O/O patches tested (n = 9). On the contrary, in all O/O patches tested (n = 7), 17beta -estradiol (5 µM) had no effect on the activity of homomeric bslo channels, which reached an average of 92 ± 7% of controls. Bars on left show that application of DHS-I (100 nM) to the intracellular side of I/O patches reversibly increased the NPo of heterooligomeric bslo + beta 1 channels, which reached 1,597 ± 576% of controls. Activation was observed in all cases (n = 9). In contrast, DHS-I (100 nM) failed to increase the activity of homomeric bslo channels in all cases (n = 10). Their NPo reached an average of 95 ± 9% of controls. Control solutions for both 17beta -estradiol and DHS-I (final DMSO concentration <0.001%) had no significant effect on channel NPo (n = 35). Data are expressed as means ± SE. Statistical significance between means (drug vs. control) was determined by paired, 1-tailed, Student's t-test (*P < 0.05 and **P < 0.005). B: concentration-response curves to EtOH action on channel NPo of bslo + beta 1 channels (triangle ) vs. bslo channels (down-triangle), obtained from responsive patches, demonstrate that EtOH potency is similar for heterooligomeric and homomeric channels. EtOH effectiveness and efficacy were also identical between heterooligomeric and homomeric complexes (see RESULTS). Thus the presence of functional beta 1-subunits does not modify EtOH action on bslo channel activity.

In addition, the presence of functional beta 1-subunits associated with bslo channels in the oocyte membrane was determined by evaluating NPo changes in response to the application of 100 nM DHS-I to the cytosolic side of I/O patches, including patches excised from the same oocytes wherein 17beta -estradiol and EtOH effects were being tested. As found with the application of 17beta -estradiol to the extracellular side of O/O patches, application of DHS-I (100 nM) to the intracellular side of I/O patches reversibly increased the NPo of heterooligomeric bslo + beta 1 channels in all cases (n = 9) while having no effect on the activity of homomeric bslo channels in all cases (n = 10; Fig. 2A). As previously reported with bovine tracheal smooth muscle BKCa channels incorporated into planar lipid bilayers (40) and mslo + beta  complexes expressed in Xenopus oocytes (41), DHS-I action was characterized by the introduction of intervals of high activity that lasted a few seconds separated by intervals of low activity similar to those in control (data not shown). This resulted in an overall increase of severalfold in steady-state activity when recorded for longer periods of time (typically >60 s), with NPo reaching an average of 1,597 ± 576% of control (P < 0.05; paired, 1-tailed, Student's t-test; n = 9; Fig. 2A, left). Control solutions (final DMSO concentration <0.001%) for both 17beta -estradiol and DHS-I had no effect on channel NPo (n = 35).

Acute exposure of O/O patches expressing homomeric bslo channels to 3-100 mM EtOH resulted in a reversible reduction of steady-state activity in some, but not all, cases (4 out of 7 O/O patches, the other 3 being insensitive). For the construction of these concentration-response curves to EtOH, NPo in the presence of a given [EtOH] was compared with NPo under isosmotic control conditions recorded immediately before alcohol application from the same O/O patch (see MATERIALS AND METHODS). This heterogeneity in EtOH action from patch to patch (i.e., only in 57% of patches was bslo activity reduced by EtOH) was similar to that previously found in a much higher number of I/O patches, where inhibition was observed in 56% of 66 patches (Fig. 1B). A maximal effect was obtained with 100 mM EtOH, at which bslo channel NPo in O/O patches reached 49.2 ± 7% of control (P < 0.005; paired, 2-tailed, Student's t-test). This value does not differ from that obtained with I/O patches (46.2 ± 9%). These similarities in the modulation of bslo channel activity by EtOH when the drug is applied to either the cytosolic surface of I/O patches or the extracellular side of O/O patches would be expected, since EtOH is a small amphiphile thought to readily cross biological membranes to access its site(s) of action.

Acute exposure of O/O patches expressing heterooligomeric bslo + beta 1 channels to 3-100 mM EtOH also resulted in a reversible reduction of NPo in some, but not all, cases (5 out of 9 O/O patches; 55%). From responsive patches, a maximal effect was obtained with 100 mM EtOH, at which NPo reached 53.8 ± 8.9% of control (P < 0.005; paired, 2-tailed, Student's t-test). These findings are almost identical to those found with excised membrane patches expressing bslo channels alone. Furthermore, concentration-response curves from responsive patches to EtOH action on channel NPo of bslo + beta 1 channels vs. bslo channels demonstrate that EtOH potency is similar for heterooligomeric and homomeric channels (Fig. 2B). Overall, this pattern of alcohol action is practically identical to that found in a much larger number of cases with bslo channels from I/O patches (Fig. 1C). Altogether, these data clearly indicate that the presence of functional modulatory beta 1-subunits does not significantly modify EtOH action on bslo channel activity.

EtOH inhibition of bslo channel activity is primarily determined by alcohol-induced reduction in the channel mean tc. The possibility that EtOH causes a decrease in NPo by sequestering channel-forming bslo subunits from the patch membrane is unlikely because of the fast and reversible nature of the inhibition (Fig. 1A). In the vast majority of cases (65 out of 66 I/O patches, all 16 O/O patches), EtOH action on bslo activity was tested in multichannel patches of unknown N. Dilution of cRNA and/or lengthening the interval between cRNA injection and recordings rendered patches containing no channels or the usual multichannel patches. This may be explained by clustering of bslo channels in the oocyte membrane, which is similar to previous findings with mslo channel expression in this system (18). In the single case in which N was certainly equal to one (I/O patch exposed to 50 mM EtOH), the decrease in Po (46% of control) was within the range for the decrease in Po produced by this [EtOH] in multichannel patches of unknown N (Fig. 1B). Thus the decrease in Po produced by EtOH appears to totally account for EtOH-induced reduction in bslo channel NPo.

A decrease in channel Po by EtOH could be determined by a drug-induced decrease in the mean to and/or an increase in the mean tc. Calculated from EtOH-sensitive, multichannel I/O patches ([Ca2+]iapprox 300 nM and V = 40 mV), to was found mildly modified by alcohol exposure, reaching 87% of control values: 2.94 ± 0.46 and 2.55 ± 0.39 ms in the absence and presence of 50 mM EtOH, respectively (P < 0.05; paired, 2-tailed, Student's t-test; n = 4). This [EtOH], however, markedly reduced Po to 50.5% of control values. Thus a major increase in the channel mean tc is the main determinant of EtOH action on Po.

Because multichannel patches of unknown N were found in the vast majority of patches (81 out of 82 I/O or O/O patches), dwell-time distributions were not routinely analyzed. In a single case, the distribution of channel openings could be well fitted with two exponentials, with time constants of 1.5 and 9.7 ms, which contributed 77 and 23% to the total fit area (i.e., to the total time spent in open states). In the presence of 50 mM EtOH, openings could also be well fitted with two exponentials with time constants of 2.9 and 8 ms, which contributed 95 and 5% to the total time spent in open states, respectively. Thus, in the presence of EtOH, the channel population is driven away from longer toward briefer open states. However, the mean duration of brief events is increased, not decreased. Thus, when the duration of the exponential components was weighted, to in the presence of EtOH reaches only 91.2% of control (3.4 and 3.1 ms in the absence and presence of 50 mM EtOH, respectively). This mild decrease in to calculated from a single channel patch is similar to the average change in to obtained from several multichannel patches under identical conditions (see above).

In the absence of EtOH, the distribution of closed times in this single channel patch could be well fitted with two exponentials, with time constants of 1.8 and 35.7 ms and a contribution to the total fit area (i.e., the total time spent in closed states) of 33 and 67%, respectively. Thus the bslo channel exhibits at least two open and two closed states, which is consistent with a previous model proposed for other slo channels (16). In the presence of 50 mM EtOH, time constants were 1.9 (22%) and 65.1 (78%) ms. Thus EtOH slightly shifts the closed channel population toward longer closures and, more important, markedly increases the average duration of these long events. These EtOH-induced changes result in a marked increase in tc from 24.5 to 51.2 ms (209% of control). Therefore, EtOH decreases bslo Po by causing a small decrease in to and a marked increase in tc that is secondary to alcohol-induced stabilization of channel long closed state(s).

EtOH modifies bslo channel gating without markedly affecting the voltage dependence of gating. EtOH targets specific channel dwell states, leading to a reversible reduction in Po (see above). In BKCa channels, whether native or cloned slo subunits, the gating process(es) that determine steady-state Po are controlled by transmembrane voltage (14, 16, 38). Thus we explored whether EtOH inhibition of bslo channel activity occurs by (or with) a drug-induced modification of the voltage dependence of channel gating.

The voltage dependence of channel activity was studied in I/O patches in the presence and absence of 100 mM EtOH, a concentration that caused the maximal effect on bslo channel NPo, as previously shown in Fig. 1C. A logarithmic plot of bslo channel NPo as a function of membrane voltage in the presence of EtOH shows a linear increase as potentials are made more positive at low Po values, as found in controls. Furthermore, the rate-limiting factor was essentially the same in the presence and absence of EtOH: 13.8 (r = 0.97) vs. 12.7 (r = 0.97) mV/e-fold change in NPo when evaluated in the same I/O patch (Fig. 3). Data from this and two other patches rendered an average slope factor of 10.8 ± 1.4 mV/e-fold change in NPo (z = 2.28 ± 0.29) in the presence of 100 mM [EtOH], which is not significantly different (paired, 2-tailed Student's t-test, n = 3) from the average value obtained in the absence of EtOH (see above). Thus EtOH produces a parallel shift in the NPo-voltage relationship toward positive potentials, the number of elementary charges moved across the electrical field to gate the bslo pore being essentially the same in the absence and presence of EtOH.


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Fig. 3.   EtOH modification of bslo channel gating is characterized by a parallel rightward shift in the activity-voltage (V) relationship. Representative plots of NPo as a function of voltage from bslo channels in the presence () and absence (open circle ) of a maximal inhibitory concentration of EtOH (100 mM), obtained in the same I/O patch. Given a fixed intracellular Ca2+ concentration ([Ca2+]; in this case free [Ca2+] approx 300 nM), when the voltage activation is described by a Boltzmann relationship, a plot of the natural log of NPo as a function of voltage is linear at low values of Po. The reciprocal of the slope, a measure of the voltage dependence of gating, is the potential needed to produce an e-fold change in NPo [13.8 mV (r = 0.97) and 12.7 mV (r = 0.97) in the presence and absence of EtOH, respectively]. NPo values were obtained as described for Fig. 1, B and C.

EtOH reduces the Ca2+ dependence of bslo channel gating. A parallel shift in the voltage activation curve of BKCa channels, whether native (6, 44) or cloned from slo genes (14, 16), toward positive potentials is observed when the Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> that is effectively recognized by the "Ca2+ sensor(s)" of the channel is effectively reduced. Thus it is possible that EtOH reduces bslo channel activity by decreasing the channel apparent Ca2+ sensitivity. To explore this possibility, EtOH action on bslo channel activity was evaluated within a wide range of free [Ca2+]i (30 nM-30 µM) at a fixed potential.

For BKCa channels at a fixed voltage, a plot of the natural log of NPo as a function of [Ca2+]i should be linear at low values of activity (6). A representative plot of ln(NPo) as a function of free [Ca2+]i, at -10 mV in the presence and absence of 100 mM EtOH, evaluated in the same I/O patch (Fig. 4), indicates that EtOH decreases the "apparent" Ca2+ sensitivity of bslo channels, since channels in the presence of EtOH require more cytosolic free Ca2+ for any given level of activity. Furthermore, EtOH decreased the slope of the NPo-[Ca2+]i relationship: 1.19 (r = 0.99) vs. 0.84 (r = 0.97) in the absence and presence of 100 mM EtOH. The slope value in the absence of EtOH suggests that bslo channel activation requires either the strong cooperative binding of two Ca2+ ions or the weaker cooperativity of more than two Ca2+ ions. These data are consistent with previous findings obtained in describing the Ca2+ dependence of channel gating after injection of dslo or hslo (16) and mslo subunits (18) in Xenopus oocytes. Similar data to those shown in Fig. 4 were obtained in three other patches from different oocytes, which rendered average slopes of 1.06 ± 0.15 vs. 0.76 ± 0.12 in the absence and presence of 100 mM EtOH (n = 4; P < 0.05, paired, 2-tailed, Student's t-test). This decrease in slope suggests that EtOH alters the capability of the Ca2+-sensing site(s) to respond to increases in [Ca2+]i. These data also indicate that EtOH inhibition of bslo activity is a direct function of [Ca2+]i.


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Fig. 4.   EtOH decreases the Ca2+ dependence of bslo channel gating. Representative plot of bslo channel NPo as a function of [Ca2+]i, at a fixed potential (-10 mV) in the absence (open circle ) and presence () of 100 mM EtOH, obtained in the same I/O patch. A plot of the natural log of NPo as a function of free [Ca2+] should be linear at low values of Po (6). The slope, a measure of the response in channel activity to increases in [Ca2+]i, is markedly decreased by EtOH [1.19 (r = 0.99) vs. 0.84 (r = 0.97) in the presence and absence of alcohol, respectively]. Similar data to this representative patch were obtained in 3 other patches from different oocytes, which rendered average slopes of 1.06 ± 0.15 vs. 0.76 ± 0.12 in the absence and presence of 100 mM EtOH, respectively (n = 4; P < 0.05, paired, 2-tailed, Student's t-test).

EtOH modifies bslo channel steady-state activity without affecting basic ion conduction properties of the bslo pore. The association of large unitary conductance and high selectivity for K+ over Na+ is a typical feature of the slo pore (36), which allows BKCa channels to contribute effectively to membrane repolarization in vascular smooth muscle. The possibility that EtOH-induced reduction in bslo NPo was accompanied by drug-induced modifications in channel ion conduction properties was evaluated next.

All-points amplitude histograms of data collected at -10 mV in the absence and presence of a maximally inhibitory concentration of EtOH show that alcohol, while decreasing channel NPo from 0.071 to 0.030, did not change the unitary current amplitude of the channel [2.66 ± 0.43 vs. 2.64 ± 0.57 pA, in the absence and presence of 100 mM EtOH (Fig. 5A)]. From these histograms, unitary current values at different potentials were obtained for constructing i/V plots. The i/V relationship of averaged data obtained from I/O patches in symmetric 145 mM [K+] in the presence and absence of 100 mM EtOH indicates that alcohol neither modified the reversal potential (near 0 mV) nor introduced any rectification or shift from -60 to +40 mV (Fig. 5B). The slope conductance, obtained by linear regression of data, was 255 ± 22 pS (n = 3), a value similar to that found in controls (see above).


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Fig. 5.   EtOH inhibition of bslo currents occurs without changes in single channel conductance. A: representative all-points amplitude histograms of bslo channel currents in the absence (top) and presence (bottom) of 100 mM EtOH show that alcohol exposure does not modify unitary current amplitude. C and O indicate the mode of the Gaussian components corresponding to the closed state(s) and the open state(s), respectively. Data were fitted using a Simplex least-squares routine to a Gaussian function of two terms (solid lines). Unitary current amplitudes obtained from the fit are 2.66 ± 0.43 and 2.64 ± 0.57 pA (mean ± SD) in the absence and presence of EtOH, respectively. Data were obtained from the same I/O patch in symmetric 145 mM K+ with the potential set at -10 mV. B: unitary current amplitude (i)-voltage (V) plots in the presence () and absence (open circle ; open circles are covered by the filled circles because of the almost complete overlapping of experimental data points) of 100 mM EtOH were constructed from I/O patches in symmetric 145 mM K+. Data were fitted with linear regression, giving similar slope conductances of 277 and 273 pS in the presence (dotted line; r = 0.99) and absence (dashed line; r = 0.99) of EtOH. Individual amplitude current points at each potential were obtained from all-points amplitude histograms like those in A. Data are expressed as means ± SE (n = 4 patches).

In addition, when 100 mM [Na+]/45 mM [K+] was used in the bath with I/O patches, the shift in the reversal potential for EtOH-exposed bslo channels (from near 0 to 29 ± 5 mV; n = 3) was almost identical to that observed in controls (see above). Therefore, bslo channels, when maximally inhibited by exposure to EtOH, retained their characteristic large unitary conductance and high selectivity for K+ over Na+.

All-points amplitude histograms also helped to address the possibility that part of the effect of EtOH on bslo Po could have involved a fast channel blockade by the drug. A fast channel blockade is often evidenced by an apparent reduction in the unitary current amplitude ("subconductance") in the presence of the channel blocker, because fast open-blocked transitions are not resolved by bandwidth limitations (29). Were this the case, all-points amplitude histograms would have shown that, in the presence of EtOH, the increase in the area under the curve (AUC) of the channel closed state(s) would have been accompanied by the appearance of a new AUC component(s) in the Gaussian fit of the histogram, which has a mode lower than that corresponding to the channel "main conductance" (i.e., open state in absence of the blocker). Representative all-points amplitude histograms obtained from data low-pass filtered at 1 kHz demonstrate that EtOH increases the AUC of the closed state(s) (and decreases the AUC of the open channel) without introducing into the fit new modes of lower current amplitude (Fig. 5A).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

An EtOH-bslo subunit functional interaction leads to alcohol inhibition of aortic smooth muscle BKCa channels. Data presented here demonstrate that EtOH inhibits BKCa channel activity in the majority of membrane patches after expression of bslo subunits in Xenopus oocytes. EtOH action on channel function was studied in excised membrane (I/O and O/O) patches in solutions containing no nucleotides 10-15 min after patch excision from the cell. Thus EtOH inhibition of bslo channels is not mediated by cell metabolism or freely diffusible cytosolic messengers. Rather, it results from a direct interaction between EtOH and bslo subunits and/or their immediate proteolipid environment. Furthermore, the presence of modulatory beta 1-subunits, while introducing sensitivity to DHS-I and 17beta -estradiol, failed to affect the EtOH sensitivity of the bslo channel, stressing the fact that bslo subunits seem sufficient for alcohol action.

Differential EtOH sensitivity of bslo channels expressed in oocyte membranes vs. native aortic smooth muscle BKCa channels reconstituted into artificial lipid bilayers. EtOH (5-60 mM) inhibition of native aortic BKCa channels incorporated into artificial bilayers (52) is characterized by higher effectiveness, potency, and efficacy compared with data presented here with bslo channels in excised patches of oocyte membranes. These differences may be potentially attributed to the presence of modulatory beta 1-subunits in the bilayer preparation, different recording conditions (in particular, [Ca2+]i), the presence of modulatory lipid species in the oocyte membrane that are lost in the artificial bilayer, and/or the existence of polymorphism in the slo subunit of aortic BKCa channels. The lack of effect of beta 1-subunit expression on EtOH action on bslo channel activity (Fig. 2B) suggests that these modulatory subunits unlikely contribute to the differential EtOH sensitivity between native bovine aortic BKCa and bslo channels.

Within a wide range of [Ca2+]i (30 nM to 30 µM), EtOH inhibition of bslo channel activity was demonstrated to increase with increases in Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> (Fig. 4). Because EtOH sensitivity of native BKCa channels in artificial bilayers was explored at [Ca2+]i approx  2.5 µM (52) and the bulk of our results from I/O patches were obtained at [Ca2+]i approx  300 nM (Fig. 1B), it is possible that the different levels of Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> used in the two studies may contribute to the differential sensitivity between BKCa channels in bilayers and bslo channels expressed in oocytes. However, it should be noted that, whereas EtOH inhibition of bslo channels increases up to a free[Ca2+]i of 30 µM, EtOH inhibition of BKCa channels in bilayers is decreased when Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> is raised from 2.5 to 20 µM (52). In addition, although bslo channel activity in the presence of 100 mM EtOH and 3 µM [Ca2+]i only reaches an average of 50% of control, the activity of aortic BKCa channels in the bilayer at 2.5 µM [Ca2+]i may be totally shut down with EtOH concentrations well below 100 mM. Thus differences in [Ca2+]i between these systems are unlikely the sole mechanism underlying the differential EtOH sensitivity of the two aortic BKCa channel preparations. Thus, by a process of exclusion, the bases for the differential EtOH sensitivity of native aortic BKCa channels in bilayers vs. bslo + beta 1-subunits in isolated oocyte membrane patches should be focused on the lipid microenvironment of the slo subunit and/or the putative existence of polymorphism in slo subunits of aortic BKCa channels.

Interestingly, we have recently demonstrated that EtOH potentiation of hslo (hbr1 variant; see Ref. 49) channels incorporated in planar lipid bilayers is modulated by different membrane lipid species. Increases in bilayer cholesterol content or decreases in bilayer phosphatidylserine, in particular, blunt EtOH action on hslo activity (13). Thus the presence of modulatory lipid species in the oocyte membrane, but absence in an artificial bilayer made of phosphatidylethanolamine and phosphatidylcholine (52), might contribute to the diminished alcohol action found with bslo channels in the oocyte membrane.

Natural membranes contain lipid microdomains with a lipid composition and physical properties different from the bulk membrane (10, 54), where slo channels have been reported to cluster (7). The association of bslo channels with distinct lipid species in the oocyte membrane may also help to explain the probable clustering of functional slo channels in the oocyte membrane noted here with bslo and previously (18) with mslo channels. Heterogeneity among these lipid microdomains in the oocyte membrane might also contribute to the intraoocyte variability in the EtOH responses of a homogenous population of cloned channel proteins (Fig. 1B).

A major role for the proteolipid microenvironment of an ion channel protein as probable modulator of EtOH action on channel function as postulated here may not be restricted to BKCa but extended to other ion channels. For example, EtOH potentiation of GABAA-mediated currents was observed with alpha 1beta 2gamma 2S-subunits when expressed in Xenopus oocytes but not when the same subunit combination was expressed in HEK cells (reviewed in Ref. 45).

Differential and common responses to EtOH between bslo and other slo channels. One striking finding of the present work is the differential responses of bslo vs. mslo (mbr5) channels to the same range of [EtOH] when studied in the excised oocyte membrane under identical recording conditions. These differences are maintained even when bslo and mslo channel responses are evaluated in the same batch of oocytes. Overall, alcohol activation of mslo channels was found in 42 out of 43 patches (98%), the remaining patch being nonresponsive, while activation of bslo channels was observed only in 6 out of 71 patches (8%), the remaining being either inhibited (58%) or nonresponsive (34%). It is possible that nonconserved regions between bslo and mslo (mbr5) contribute to their differential responses to EtOH, whether by directly interacting with this compound, by "sensing" EtOH action at lipid-protein interfaces, and/or by being differentially targeted after posttranslational processing that modifies EtOH action. Bslo and mslo (mbr5) are 98% identical proteins (11, 46), the least conserved area being the COOH-terminal region. This region is represented by a sequence of 60 amino acids, the first 8 of which are substituted, and the remaining absent in bslo, resulting in a shorter COOH terminus. Thus the longer COOH-terminal end in the mslo protein might be responsible for EtOH potentiation of BKCa channel activity. Alternatively, differential EtOH sensitivity between bslo and mslo (mbr5) could be linked to other nonconserved regions, such as three residues in the linker between S8 and S9.

Bslo and other slo channel responses to EtOH, however, share strong similarities. Despite causing qualitatively opposite effects (i.e., inhibition and activation), in all cases EtOH acts at similar concentrations and modulates NPo primarily by targeting channel long closed states. Furthermore, EtOH-induced changes in NPo occur without major modification of the voltage dependence of gating and the characteristic high-K+ permeability and selectivity over Na+. These data indicate that EtOH does not modify functional properties associated with the slo channel "core" domain [S1-S8 (53) or S0-S8 (51)], a region highly conserved between mslo (mbr5) and bslo subunits.

Finally, for both mslo and bslo channels, EtOH modulates activity with a reduction in the Ca2+ dependence of channel gating. For both channels, the slope of the ln(NPo-[Ca2+]i) relationship is shifted from control values greater than one to values below one in the presence of EtOH. Because EtOH is unlikely to change the number of slo subunits (see RESULTS and also Ref. 18), this reduction in slope suggests that alcohol introduces apparent negative cooperativity in the interaction between Ca2+ in the cytosolic side of the patch and Ca2+-sensing site(s) near or in the slo subunit. Thus the EtOH-induced decrease in the Ca2+ dependence of slo channel gating may be determined by a reduced availability of Ca2+-binding sites without a decrease in the number of channel proteins, introduction of negative cooperativity in the actual binding of Ca2+ to a conserved number of Ca2+ recognition sites, and/or alteration in the Ca2+ dependence of channel gating per se (i.e., EtOH modifies steps subsequent to Ca2+ binding).

EtOH action on mslo (mbr5) channel activity and its Ca2+ dependence is best described by a model in which EtOH behaves as a partial agonist of the mslo channel, for which Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> is the full agonist (18). One possibility is that EtOH "partial agonism" on mslo channels is determined by alcohol interactions with two (or more) discrete sites: one (or more) that determines EtOH activation, and another (or others) that determines EtOH-Ca2+ antagonism. If so, it is conceivable that the site(s) responsible for EtOH activation of mslo channels is related to protein regions not conserved between mslo and bslo (see above), whereas the site(s) responsible for the functional antagonism between EtOH and Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> (found with both mslo and bslo channels) resides in conserved Ca2+ sensors of these two proteins or is linked to the existence of some particular component of the proteolipid microenvironment of the slo subunit in the oocyte membrane.

Pathophysiological implications of the present results. There is wide evidence that changes in vascular tone in response to acute alcohol exposure are primarily the result of EtOH action on the vascular smooth muscle itself (2, 26, 34, 35, 55). At this level, EtOH-induced relaxation and contraction have both been reported, depending upon [EtOH] and, primarily, vessel type (2, 3, 34). In vitro exposure to clinically relevant [EtOH] produces contraction of aortic smooth muscle (32, 37, 39, 55), this effect being partially mediated by protein kinase C (32, 55) and a caffeine-sensitive pool of Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> (55). The ion channel populations involved in EtOH-induced vasoconstriction, however, are largely unknown. Data presented here demonstrate that an interaction between EtOH and the bslo subunit and/or its immediate lipid environment leads to a reduction in BKCa channel activity. Because BKCa channel activity in vascular smooth muscle generates positive outward currents that drive the cell membrane potential in a negative direction and, eventually, counteract contraction (5, 8, 30), EtOH-induced inhibition of bslo channels may be a mechanism underlying or, at least, contributing to, alcohol-induced contraction of aortic smooth muscle.


    ACKNOWLEDGEMENTS

I gratefully acknowledge Dr. Joshua J. Singer for critical reading of the manuscript, Dr. Weihua Huang for advice and help with cDNA cloning and linearization, Dr. Suleiman Bahouth for helpful discussion, and Maria Asuncion-Chin for excellent technical assistance.


    FOOTNOTES

This work was supported by National Institute on Alcohol and Alcohol-Related Diseases Grant AA-11560.

Address for reprint requests and other correspondence: A. Dopico, Dept. Pharmacology, Univ. Tennessee Hlth. Sci. Ctr., 874 Union Ave., Memphis, TN 38163 (E-mail: adopico{at}utmem.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.

First published February 5, 2003;10.1152/ajpcell.00421.2002

Received 13 September 2002; accepted in final form 30 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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