Department of Pharmacology and Program in Neuroscience, University of Tennessee Health Science Center, Memphis, Tennessee 38163
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ABSTRACT |
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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 () 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
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
1-subunits, which determines channel sensitivity to dihydrosoyaponin-I and 17
-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
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INTRODUCTION |
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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 (, encoded by
slo genes) and modulatory (
) 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
-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 -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
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
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 1-subunits was determined. Preliminary data
were published in abstract form (17).
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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
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
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 1-subunit
mRNA was coinjected with the
(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
1-subunits. BKCa channel activity after
bslo and
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 M when filled with high-K+ solution (Table 1). All experiments were carried
out at room temperature.
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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).
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RESULTS |
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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.
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EtOH action on bslo channels is unmodified in the presence of
functional 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 +
1 complex, it is conceivable that
the presence of modulatory
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
1-subunits was explored next.
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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+]iEtOH 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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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
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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
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DISCUSSION |
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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
1-subunits, while introducing sensitivity to DHS-I and
17
-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 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
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.
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 CaPathophysiological 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
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ahring, PK,
Strobæk D,
Christophersen P,
Olessen SP,
and
Johansen TE.
Stable expression of the human large-conductance Ca2+-activated K+ channel alpha- and beta-subunits in HEK293 cells.
FEBS Lett
415:
67-70,
1997[ISI][Medline].
2.
Altura, B,
and
Altura B.
Microvascular and vascular smooth muscle actions of ethanol acetaldehyde, and acetate.
Fed Proc
41:
2447-2451,
1982[ISI][Medline].
3.
Altura, B,
and
Altura B.
Peripheral vascular actions of ethanol and its interaction with neurohumoral substances.
Neurobehav Toxicol Teratol
5:
211-220,
1983[ISI][Medline].
4.
Altura, B,
Li Y,
Altura B,
Jelicks L,
Wittenberg B,
and
Gupta R.
Beneficial vs. detrimental actions of ethanol on heart and coronary vascular smooth muscle: roles of Mg2+ and Ca2+.
Alcohol
5:
499-513,
1996.
5.
Asano, M,
Masuzawa-Ito K,
and
Matsuda T.
Charybdotoxin-sensitive K+ channels regulate the myogenic tone in the resting state of arteries from spontaneously hypertensive rats.
Br J Pharmacol
108:
214-222,
1993[Abstract].
6.
Barrett, JN,
Magleby KL,
and
Pallotta BS.
Properties of single calcium-activated potassium channels in cultured rat muscle.
J Physiol
331:
211-230,
1982[ISI][Medline].
7.
Bravo-Zehnder, M,
Orio P,
Norambuena A,
Wallner M,
Meera P,
Toro L,
Latorre R,
and
Gonzalez A.
Apical sorting of a voltage- and Ca2+-activated K+ channel alpha-subunit in Madin-Darby canine kidney cells is independent of N-glycosylation.
Proc Natl Acad Sci USA
97:
13114-13119,
2000
8.
Brayden, J,
and
Nelson M.
Regulation of arterial tone by activation of calcium-dependent potassium channels.
Science
256:
532-535,
1992[ISI][Medline].
9.
Brenner, R,
Pérez GJ,
Bonev AD,
Eckman DM,
Kosek JC,
Wiler SW,
Patterson AJ,
Nelson MT,
and
Aldrich RW.
Vasoregulation by the 1 subunit of the calcium-activated potassium channel.
Nature
407:
870-876,
2000[ISI][Medline].
10.
Brown, DA,
and
London E.
Functions of lipid rafts in biological membranes.
Annu Rev Cell Dev Biol
14:
111-136,
1998[ISI][Medline].
11.
Butler, A,
Tsunoda S,
McCobb D,
Wei A,
and
Salkoff L.
mSlo, a complex mouse gene encoding "Maxi" calcium-activated potassium channels.
Science
261:
221-224,
1993[ISI][Medline].
12.
Crowley, JJ,
Dopico AM,
and
Treistman SN.
Ethanol potentiation of cloned BK channels incorporated into planar lipid bilayers (Abstract).
Soc Neurosci Abstr
26:
1402,
2000.
13.
Crowley, JJ,
Treistman SN,
and
Dopico AM.
Phospholipid and cholesterol modulation of hslo channel activity and ethanol sensitivity in lipid bilayers (Abstract).
Biophys J
82:
586a,
2002.
14.
Cui, J,
Cox DH,
and
Aldrich RW.
Intrinsic voltage dependence and Ca2+ regulation of mslo large conductance Ca-activated K+ channels.
J Gen Physiol
109:
647-673,
1997
15.
Diamond, I.
Cecil Textbook of Medicine. Philadelphia, PA: Saunders, 1992.
16.
DiChiara, TJ,
and
Reinhart PH.
Distinct effects of Ca2+ and voltage on the activation and deactivation of cloned Ca2+-activated K+ channels.
J Physiol
489:
403-418,
1995[Abstract].
17.
Dopico, AM.
Ethanol decreases bslo channel activity in excised membrane patches of Xenopus laevis oocytes (Abstract).
Biophys J
80:
A443,
2001.
18.
Dopico, AM,
Anantharam V,
and
Treistman SN.
Ethanol increases the activity of Ca2+-dependent K+ (mslo) channels: functional interaction with cytosolic Ca2+.
J Pharmacol Exp Ther
284:
258-268,
1998
19.
Dopico, AM,
Kirber MT,
Singer JJ,
and
Walsh JV, Jr.
Membrane stretch directly activates large conductance Ca2+-activated K+ channels in mesenteric artery smooth muscle cells.
Am J Hypertens
7:
82-89,
1994[ISI][Medline].
20.
Dopico, AM,
Lemos JR,
and
Treistman SN.
Ethanol activates large-conductance, Ca2+-activated K+ channels in neurohypophysial terminals.
Mol Pharmacol
49:
40-48,
1996[Abstract].
21.
Dopico, AM,
and
Treistman SN.
Ethanol has opposite effects on the activity of two cloned large conductance, Ca2+-activated K+ (bslo and mslo) channels (Abstract).
Soc Neurosci Abstr
22:
351,
1996.
22.
Dopico, AM,
Widmer H,
Wang G,
Lemos JR,
and
Treistman SN.
Rat supraoptic magnocellular neurones show distinct large conductance, Ca2+-activated K+ channel subtypes in cell bodies versus nerve endings.
J Physiol
519:
101-114,
1999
23.
Faragó, M,
Szabó C,
Horváth I,
Dorá E,
and
Kovách G.
Differential vascular actions of ethanol in feline middle cerebral and mesenteric artery.
Acta Physiol Hung
78:
119-125,
1991[ISI][Medline].
24.
Giangiacomo, KM,
Garcia-Calvo M,
Knaus HG,
Mullmann TJ,
Garcia M,
and
McManus O.
Functional reconstitution of the large-conductance, Ca2+-activated potassium channel purified from bovine aortic smooth muscle.
Biochemistry
34:
15849-15862,
1995[ISI][Medline].
25.
Glantz, SA.
Premier in Biostatistics. New York: McGraw-Hill, 2001.
26.
Gordon, E,
Nguyen T,
Ngai A,
and
Winn H.
Differential effects of alcohols on intracerebral arterioles. Ethanol alone causes vasoconstriction.
J Cereb Blood Flow Metab
24:
532-538,
1995.
27.
Gruss, M,
Henrich M,
Konig P,
Hempelmann G,
Vogel W,
and
Scholz A.
Ethanol reduces excitability in a subgroup of primary sensory neurons by activation of BK(Ca) channels.
Eur J Neurosci
14:
1246-1256,
2001[ISI][Medline].
28.
Ha, TS,
Jeong SY,
Cho S-W,
Jeon H-K,
Roh GS,
Choi WS,
and
Park C-S.
Functional characteristics of two BKCa channel variants differentially expressed in rat brain tissues.
Eur J Biochem
267:
910-918,
2000
29.
Hille, B.
Ion Channels of Excitable Membranes. Sunderland, MA: Sinauer, 2001.
30.
Jaggar, J,
Porter V,
Lederer W,
and
Nelson M.
Calcium sparks in smooth muscle.
Am J Physiol Cell Physiol
278:
C235-C256,
2000
31.
Jakab, M,
Weiger TM,
and
Hermann A.
Ethanol activates maxi Ca2+-activated K+ channels of clonal pituitary (GH3) cells.
J Membr Biol
157:
237-245,
1997[ISI][Medline].
32.
Jover, T,
Altura BT,
and
Altura BM.
Effects of protein kinase C inhibitors on ethanol-induced contractions in isolated rat aorta.
Alcohol
18:
17-22,
1999[ISI][Medline].
33.
Kaczorowski, G,
Knaus H-G,
Leonard R,
McManus O,
and
García M.
High-conductance calcium-activated potassium channels: structure, pharmacology and function.
J Bioenerg Biomembr
28:
255-267,
1996[ISI][Medline].
34.
Klatsky, AL.
Alcohol, coronary disease and hypertension.
Annu Rev Med
47:
149-160,
1996[ISI][Medline].
35.
Knych, ET.
Endothelium-dependent transfer of ethanol tolerance in the aorta.
Life Sci
40:
1903-1908,
1987[ISI][Medline].
36.
Latorre, R.
Molecular workings of large conductance (maxi) Ca2+-activated K+ channels.
In: Handbook of Membrane Channels, edited by Petracchia C.. San Diego, CA: Academic, 1994, p. 79-102.
37.
Lodge, NJ.
Direct vasoconstrictor effects of sandimmune (cyclosporine A) are mediated by its vehicle cremophor EL: inhibition by the thromboxane A2/prostaglandin endoperoxide receptor antagonist ifetroban.
J Pharmacol Exp Ther
271:
730-734,
1994[Abstract].
38.
Magleby, KL,
and
Pallota BS.
Calcium dependence of open and shut interval distributions from calcium-activated potassium channels in cultured rat muscle.
J Physiol
344:
585-604,
1983[Abstract].
39.
Malinowska, B,
Pietraszek M,
Chabielska E,
Pawlak D,
and
Buczko W.
The effect of ethanol and serotonin on blood vessels of the rat.
Pol J Pharmacol
42:
333-342,
1990.
40.
McManus, OB,
Harris GH,
Giangiacomo KM,
Feigenbaum P,
Reuben JP,
Addy ME,
Burka JF,
Kaczorowski GJ,
and
Garcia ML.
An activator of calcium-dependent potassium channels isolated from a medicinal herb.
Biochemistry
32:
6128-6133,
1993[ISI][Medline].
41.
McManus, OB,
Helms LM,
Pallanck L,
Ganetzky B,
Swanson R,
and
Leonard RJ.
Functional role of the beta subunit of high conductance calcium-activated potassium channels.
Neuron
14:
645-650,
1995[ISI][Medline].
42.
Meera, P,
Wallner M,
Jiang Z,
and
Toro L.
A calcium switch for the functional coupling between (hslo) and
subunits (KV. Ca
) of maxi K channels.
FEBS Lett
382:
84-88,
1996[ISI][Medline].
43.
Mistry, DK,
and
Garland CJ.
Characteristics of single, large-conductance calcium-dependent potassium channels (BKCa) from smooth muscle cells isolated from rabbit mesenteric artery.
J Membr Biol
164:
125-138,
1998[ISI][Medline].
44.
Moczydlowski, E,
and
Latorre R.
Gating kinetics of Ca2+-activated K+ channels from rat muscle incorporated into planar lipid bilayers: evidence for two voltage-dependent Ca2+-binding reactions.
J Gen Physiol
82:
511-542,
1983[Abstract].
45.
Mori, T,
Aistrup GL,
Nishikawa K,
Marszalec W,
Yeh JZ,
and
Narahashi T.
Basis of variable sensitivities of GABAA receptors to ethanol.
Alcohol Clin Exp Res
24:
965-971,
2000[ISI][Medline].
46.
Moss, GWJ,
Marshall J,
Morabito M,
Howe JR,
and
Moczydlowski E.
An evolutionarily conserved binding site for serine protease inhibitors in large conductance calcium-activated potassium channels.
Biochemistry
35:
16024-16035,
1996[ISI][Medline].
47.
Singer, JJ,
and
Walsh JV, Jr.
Characterization of calcium-activated potassium channels in single smooth muscle cells using the patch-clamp technique.
Pflügers Arch
408:
98-111,
1987[ISI][Medline].
48.
Toro, L,
Ramos-Franco J,
and
Stefani E.
GTP-dependent regulation of myometrial KCa channels incorporated into lipid bilayers.
J Gen Physiol
96:
373-394,
1990[Abstract].
49.
Tseng-Crank, J,
Foster CD,
Krause JD,
Mertz R,
Godinot N,
DiChiara TJ,
and
Reinhardt PH.
Cloning, expression, and distribution of functionally distinct Ca2+-activated K+ channel isoforms from human brain.
Neuron
13:
1315-1330,
1994[ISI][Medline].
50.
Valverde, MA,
Rojas P,
Amigo J,
Cosmelli D,
Orio P,
Bahamonde MI,
Mann GE,
Vergara C,
and
Latorre R.
Acute activation of maxi-K channels (hSlo) by 17-estradiol binding to the
subunit.
Science
285:
1929-1931,
1999
51.
Wallner, M,
Meera P,
and
Toro L.
Determinant for -subunit regulation in high-conductance voltage-activated and Ca2+-sensitive K+ channels: an additional transmembrane region at the N terminus.
Proc Natl Acad Sci USA
93:
14922-14927,
1996
52.
Walters, F,
Covarrubias M,
Giangiacomo K,
and
Ellingson J.
Potent inhibition of the aortic smooth muscle maxi-K channel by clinical doses of ethanol.
Am J Physiol Cell Physiol
279:
C1107-C1115,
2000
53.
Wei, A,
Solaro C,
Lingle C,
and
Salkoff L.
Calcium sensitivity of BK-type KCa channels determined by a separable domain.
Neuron
13:
671-681,
1994[ISI][Medline].
54.
Welti, R,
and
Glaser M.
Lipid domains in model and biological membranes.
Chem Phys Lipids
73:
121-137,
1994[ISI][Medline].
55.
Werber, A,
Morgan R,
Zhou P,
and
Yang C.
Intracellular mechanisms of constriction of rat aorta by ethanol.
Alcohol
14:
351-360,
1997[ISI][Medline].
56.
Wu, S-N,
and
Chao C-T.
Inhibitory effect of ethanol on voltage-dependent potassium currents in single aortic smooth muscle cells.
Kaohsiung J Med Sci
11:
514-520,
1997.