Introduction

Top Abstract Introduction Methods Results Discussion References

CAK channel activation increases K⁺ efflux, which repolarizes/hyperpolarizes the membrane potential, decreasing neuronal excitability (Lang and Ritchie, 1987). It has been speculated that drug-induced increases in CAK currents might contribute to the depressant action of EtOH and other sedative/hypnotic drugs on central neurons (Krnjevic, 1972). Several groups have reported increases in CAK responses by EtOH at relevant concentrations. Ca⁺⁺-dependent afterhyperpolarization and Ca⁺⁺-dependent Rb⁸⁶ efflux were used as indexes of CAK responses in these studies (Carlen et al., 1982; Harris and Cadwell, 1985; Madsen and Edeson, 1990), which makes it difficult to address which CAK conductance was involved and the mechanisms underlying the increase in current. We recently found that EtOH, at circulating concentrations found after moderate drinking, reversibly activates BK channels in rat neurohypophysial terminals (Dopico et al., 1996a), which probably contributes to the decreased vasopressin release and plasma levels found after moderate drinking (Dopico et al., 1995a).

BK channel activity is increased with cell depolarization and/or elevated [Ca⁺⁺]_ic (Magleby and Pallota, 1983; Latorre et al., 1989). Thus, it is possible that EtOH activates BK channels by increasing the sensitivity of the channel to voltage and/or [Ca⁺⁺]_ic. In neuroterminals, heterogeneity in the response of native BK channels to changes in [Ca⁺⁺]_ic or transmembrane voltage (Wang et al., 1992; Dopico et al., 1996a) prevented us from testing this hypothesis.

It is postulated that native BK channels from mammalian tissue are heterooligomeric channels that consist of pore-forming () and regulatory () subunits (McManus et al., 1995). BK channel  subunits have been cloned from cDNA obtained from different sources, including Drosophila melanogaster (dslo) and mouse brain (mslo). These cloned channels have been expressed in Xenopus oocytes and characterized electrophysiologically (Atkinson et al., 1991; Butler et al., 1993).

In this work, we used single-channel recordings from excised patches to examine the acute action of EtOH on the activity of mslo BK channels expressed in Xenopus oocytes (where no endogenous  subunits have been reported). Thus, we can address whether the drug interacts (directly or via membrane-bound mediators) with the  subunit, independently of modulatory subunits. A population of cloned channels with nonvariant voltage and/or Ca⁺⁺-sensitivity also allows us to determine the role of changes in the voltage- and/or Ca⁺⁺-sensitivity of the channel in EtOH action. Because the electrophysiological properties examined are associated with functional domains in the  subunit (Wei et al., 1994), EtOH-modified properties also allow preliminary speculation on which regions of this subunit are targeted by EtOH.

Results show that EtOH reversibly activates mslo channels with a potency (EC₅₀ = 24 mM) very close to that reported for native channels and at blood levels considered legal intoxication. EtOH-induced activation results from drug actions on channel gatingin particular, a marked increase in the frequency of channel openings. Channel activation by EtOH is not accompanied by changes in the channel unitary conductance, ion selectivity or voltage dependence of gating. Although EtOH activation of mslo channels is not affected by voltage, it is critically modulated by [Ca⁺⁺] at the intracellular side of the membrane. Preliminary data were presented in abstract form (Dopico et al., 1995b; Dopico et al., 1996b).

	Methods

Top Abstract Introduction Methods Results Discussion References

Use of Xenopus oocytes and injection. Xenopus laevis females were purchased from commercial breeders in the United States and were maintained in artificial pond water on a 12-h light/dark cycle. In this habitat, they do not show seasonal breeding behavior, so oocytes are available throughout the year. Stages V and VI oocytes were predominantly used, because they transcribe mRNA into channels efficiently. Oocytes were removed and kept in a Ca⁺⁺-free ND-96 solution (mM): 82.5 NaCl, 2 KCl, 1 MgCl₂, 5 HEPES, pH 7.5, containing 2 mg/ml collagenase (type IV, Sigma, St. Louis, MO) for 2 to 4 h, in order to remove the follicular layer. After defolliculation, oocytes were transferred to a Ca⁺⁺-containing ND-96 saline (mM): 96 NaCl, 2 KCl, 1.8 CaCl₂, 5 HEPES, pH 7.5, supplemented with 2.5 mM sodium pyruvate and 2 mg/ml gentamicin. Injection of mRNA was conducted using a Drummond micropipette modified for microinjection (Drummond Scientific Co., Broomall, PA). Injection volumes of mRNA (0.1-1 µg/µl) ranged from 18.6 to 50.6 nl. The use of varying concentrations and volumes, as well as different intervals between injection and electrophysiological recording (24-72 h), gave us crude control over the amount of BK channel protein expressed in the oocyte's membrane.

RNA preparation and analysis. BK channels were expressed in Xenopus oocytes by injection of mRNA transcribed in vitro from complementary DNA (cDNA) coding for the mbr5 variant of the mslo BK channel, derived from the Slowpoke locus of mouse brain (Butler et al., 1993). Briefly, polyadenylated RNA transcripts were synthesized from Sal 1 linearized mslo templates using T3 RNA polymerase. Full-length cDNA from mslo was a generous gift from Dr. Lawrence Salkoff (Washington University).

Single-channel recordings. Before recordings, oocytes were placed into a dish containing a hypertonic solution (mM): 200 K⁺ aspartate, 20 KCl, 1 MgCl₂, 10 ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid (EGTA), 10 HEPES, pH 7.4, for 10 min. With this treatment the oocytes shrunk, which allowed us to remove the vitelline layer with forceps, exposing the oocyte membrane for subsequent patch recording. Then the oocytes were placed back into ND-96 saline (in this case without gentamicin; for composition, see above) for 10 to 15 min before recording. Single-channel recordings were obtained from excised I/O membrane patches using standard patch-clamp techniques. All solutions were made with ultrapure 18 M and bidistilled water and with high-grade salts. Unless otherwise stated, recordings were obtained in "symmetric conditions"that is, the same solution bathed both the extracellular and the cytosolic sides of the patch membrane (mM): 145 K⁺ gluconate, 5 EGTA, 1 MgCl₂, 15 HEPES, 10 glucose, pH 7.35, containing varying amounts of free Ca⁺⁺. The free Ca⁺⁺ concentration was adjusted to the desired nominal value by adding CaCl₂. The calculation of the final, nominal [Ca⁺⁺]_free in all solutions was done according to Fabiato (1988). In some cases, the extracellular surface of the patch was exposed to a [Ca⁺⁺]_free of 3 to 10 µM. This relatively high concentration of Ca⁺⁺, together with the Mg⁺⁺ present in this solution, helped to increase gigaseal formation. In experiments to study the Ca⁺⁺-sensitivity of BK channels, the intracellular surface of the patch was exposed to (bath) solutions that differed in their final [Ca⁺⁺]_free (0.01-1 µM).

	Results

Top Abstract Introduction Methods Results Discussion References

EtOH-induced increase of mslo channel steady-state activity in excised I/O patches is reversible and concentration-dependent. The acute application of EtOH to the cytosolic surface of I/O patches reversibly increased the steady-state activity (NP_o) of mslo channels expressed in Xenopus oocytes (fig. 1). A flow or osmotic effect was ruled out by exposing the excised patch to a stream of solution with dextrose isosmotically replacing EtOH (control perfusion), which did not change channel activity. Similarly to what we previously described for the activation by EtOH of BK channels in neurohypophysial terminals (Dopico et al., 1996a), whereas EtOH's action was almost immediate in onset (<6 s after switching the perfusion to EtOH-containing solutions), it took approximately 5 to 6 min of drug-free perfusion for channel NP_o to return to pre-EtOH levels in all cases. Because the Ca⁺⁺-sensing sites of the channel seem to be located at the intracellular side of the membrane (McManus, 1991), the fact that EtOH activation was observed in I/O patches more than 15 min after patch excision, with [Ca⁺⁺]_ic highly buffered, indicates that this activation is not mediated by changes in the levels of [Ca⁺⁺]_ic or other freely diffusible cytosolic second messengers. Because EtOH activation was observed during I/O recordings using symmetric [Ca⁺⁺] at positive potentials (i.e., with Ca⁺⁺ flowing outward), EtOH-induced increase in mslo NP_o was not caused by a transient elevation of [Ca⁺⁺] at the intracellular side of the patch in the vicinity of the mslo channel as a consequence of an EtOH-induced transmembrane flux of Ca⁺⁺. Consequently, EtOH's activation of mslo channels expressed in oocytes after injection of the  subunit seems to be due to a direct interaction of EtOH with this protein or some closely associated entity in the patch membrane that, eventually, interacts with the mslo  subunit.

View larger version (43K): [in this window] [in a new window]   Fig. 1.   EtOH (50 mM) reversibly increases the activity (NPo) of mslo channels expressed in Xenopus oocytes. Representative single-channel recordings obtained from I/O patches before (upper panels), during (middle panels), and 6.5 min after (lower panels) exposure of the cytosolic side of the patch to EtOH. Channel openings are shown as upward deflections; arrows indicate the baseline level. NPo values were obtained from all-points histograms, constructed from 72 to 126 s of recording under each particular condition. The composition of the solution facing the intracellular and extracellular sides of the patch (symmetric conditions) is described in "Methods." In this case, free [Ca++]ic ~ 32 nM. The membrane potential was set to +20 mV.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   A) The activation of mslo channels by EtOH is concentration-dependent, reaching a "plateau" at 100 to 200 mM EtOH. The EC50, obtained by linear interpolation, is 24.3 ± 4.8 mM. Results are expressed as the ratio of NPo values obtained in the presence and absence of EtOH, determined in the same patch. Each point of the graph is the mean ± S.E. of 4 to 20 determinations; each determination was obtained in a different patch. In all cases, the patch membrane potential was set at 20 to 40 mV, and free [Ca++]ic was 31.6 to 100 nM. B) Logit-log plot of mslo channel activity as a function of [EtOH]. Maximum NPo (maxNPo) was calculated as the mean of NPo values obtained at 100 and 200 mM EtOH, because the EtOH-induced increase in NPo values at those concentrations reached a plateau, as shown in panel A. Data plotted were obtained at 10, 25 and 50 mM EtOH. The Hill coefficient (n, slope of the graph) is 0.87, not significantly different from 1.

EtOH modifies channel gating properties. We have shown above a reversible increase in channel NP_o by EtOH. In the vast majority of cases, we dealt with multichannel patches in which N was unknown. Even in this situation, an increase in NP_o by EtOH may be interpreted as an increase in P_o. Because of the fast and reversible nature of the activation, it is unlikely that EtOH causes an increase in NP_o by introducing more channel-forming mslo  subunits into the patch. Also, in excised (i.e., "cell free") patches, the source of these additional channels would have to be the limited area of membrane under the rim of the pipette, which also makes this possibility unlikely. Nevertheless, we could not rule out a priori the possibility that EtOH might increase channel NP_o by unmasking "silent" channel proteins already present in the patch. However, we were able to observe an increase in channel (N)P_o by EtOH in I/O patches containing only one functional mslo channel protein (as described below for fig. 4). Furthermore, the increase in channel NP_o by 50 mM EtOH (~190% of control) observed in multichannel patches of undetermined N (fig. 3) at 40 mV and 100 nM free [Ca⁺⁺]_ic was similar to the increase in P_o produced by the same concentration of the drug in a single-channel patch (~183% of control; dwell-time distributions for this channel are shown in fig. 4) under identical recording conditions. Thus, the increase in channel P_o produced by EtOH totally accounts for the drug-induced increase in channel NP_o. In conclusion, the reversible increase in channel activity produced by EtOH occurs without any change in N. 

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Changes in mean open time (to, hollow triangles), mean closed time (tc, filled triangles) and steady-state activity (NPo, filled circles) of mslo channels as a function of [EtOH]. Results are expressed as ratios of values obtained in the presence and absence of EtOH. Each point of the graph is the mean ± S.E. of 3 to 9 determinations; each determination was obtained in a different patch. Individual points at a given EtOH concentration for all three variables were simultaneously determined in the same patch. In all patches, the patch membrane potential was set at 40 mV, and free [Ca++]ic was 100 nM. The composition of the solution facing the intracellular and extracellular sides of the patch (symmetric conditions) is described in "Methods." Because data are expressed as ratios, there is a limit for ratios describing decreases induced by the drug (i.e., EtOH-induced decreases in tc ratios are limited to values between 1 and 0), whereas EtOH-induced increases in NPo and to ratios can attain values above 1. C: control values.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Dwell-time distributions of mslo channels in control (panel A, open times; panel B, closed times) and 50 mM EtOH (panel C, open times; panel D, closed times) obtained from a single-channel patch. The membrane potential was set at +40 mV, and the free [Ca++]ic was 100 nM. Exponential curves were fitted using a maximum-likelihood minimization routine. The different components () of each exponential distribution are indicated by dotted lines. In both control and EtOH, both the open-times distribution and the closed-times distributions could be well fitted with two exponentials. A statistical comparison of different fitting models was done using a standard F statistical table (P < .01). Both the actual time value (in milliseconds) and the contribution to the total distribution (as percentages in parentheses) of each component are shown at the right of each panel. These percentages reflect the relative contribution of each component to the exit from the state (open or closed) considered.

EtOH does not affect high permeability and selectivity for K⁺. A remarkable property of all BK channels is the combination of a large conductance and an exquisite selectivity for K⁺: they are practically impermeable to Na⁺ (Latorre, 1994). We previously observed, using native BK channels, that the enhancement of BK currents by EtOH is completely explained by an increase in channel NP_o, with no major changes in single-channel conductance and reversal potential in symmetric conditions (Dopico et al., 1996a). In the present study, we further evaluated BK channel ion conduction properties not only using symmetric [K⁺] but also substituting Na⁺ for K⁺, in order to establish whether EtOH modifies the high selectivity of the channel for K⁺ over Na⁺. Figure 5 shows i/V plots obtained in the absence and presence of 50 mM EtOH, a concentration at which channel NP_o was increased about 160% over control values (fig. 2a). In the absence of EtOH, the single-channel conductance, obtained from the slope of the i/V plot in symmetric 145 mM [K⁺] was 240.1 ± 6.5 pS (r = 0.999), with a reversal potential near 0 mV (0.7 mV). The reversal potential shifted in a positive direction to +28.6 mV (r = 0.998) with 100 mM [Na⁺]/45 mM [K⁺] at the intracellular side, almost identical to the theoretical value of +30.6 mV predicted by the Nernst equation for a channel highly selective for K⁺. Thus, mslo channels exhibit the combination of large unitary conductance and high selectivity for K⁺ over Na⁺ that is characteristic of BK channels. Figure 5 also shows that this characteristic feature was unmodified by EtOH. In the presence of the drug, the single-channel conductance in symmetric 145 mM [K⁺] was 235.7 ± 7.2 pS (r = 0.999), with a reversal potential of 1.3 mV. The shift in the reversal potential to +28.2 mV (r = 0.995) when 100 mM [Na⁺]/45 mM [K⁺] was used at the intracellular side of the patch was almost identical to that observed in controls, following the Nernst prediction. Therefore, EtOH-activated mslo channels retain a typical characteristic of BK channels: the combination of large conductance and high selectivity for K⁺, remaining almost impermeable to Na⁺.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Unitary current-voltage (i/V) relationships of mslo BK channels for both symmetric 145 mM [K+] (circles) and 145 [K+]o/45 mM [K+]i (Na+ substituting for K+) (triangles), in the absence (filled figures) and presence (hollow figures) of 50 mM EtOH, from I/O patches. For composition of the solutions, please see "Methods." In controls, the single-channel conductance (), obtained from the slope of the i/V plot in symmetric [K+], is 240.1 ± 6.5 pS (r = 0.999) with a reversal potential near 0 mV (0.71 mV). The reversal potential shifted in a positive direction to +28.6 mV (r = 0.998) with 45 mM [K+]i, a value very close to the theoretical value of +30.6 mV predicted by the Nernst equation for a channel highly selective for K+. In the presence of EtOH,  in symmetric 145 mM [K+] is 235.7 ± 7.2 pS (r = 0.999), not significantly different from that in controls, and the reversal potential is 1.31 mV. The shift in the reversal potential to +28.2 mV (r = 0.995) with 45 mM [K+]i is almost identical to that observed in controls, which indicates that EtOH does not modify the high selectivity of K+ over Na+ that is characteristic of BK channels. Individual points at each potential were obtained from all-points histograms. Plots for symmetric and asymmetric [K+] have been fitted using first- and second-order regressions, respectively. Each data point is the mean ± S.E. of at least three determinations; each determination was obtained in a different patch.

EtOH differentially modulates the voltage- and Ca⁺⁺-sensitivity of the channel. The study of EtOH's action on cloned channels, compared to natural BK channels, has the advantage of providing a population of channels that show very defined functional characteristicsin particular, voltage/Ca⁺⁺-sensitivity. Dual sensitivities to activating [Ca⁺⁺]_ic and transmembrane depolarizing voltage play a key role in the modulation of BK channel gating, without modifying single-channel conductance or ion selectivity (Magleby and Pallota, 1983; Latorre et al., 1989; McManus, 1991). We have demonstrated above that EtOH increases mslo channel activity by modifying channel gating without altering either single-channel conductance or the selectivity of the channel for K⁺. We next tested whether the drug enhances mslo channel activity by increasing the sensitivity of the channel to voltage and/or [Ca⁺⁺]_ic.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Representative plot of NPo as a function of voltage from mslo channels in the presence (hollow circles) and absence (filled circles) of 50 mM EtOH, obtained in the same I/O patch. Given a fixed intracellular [Ca++] (in this case, [Ca++]free ~100 nM), when the voltage activation is described by a Boltzmann relationship, a plot of the natural log of NPo as a function of voltage should be linear at low values of Po. The reciprocal of the slope, a measure of voltage sensitivity, is the potential needed to produce an e-fold change in NPo: 21.5 mV (r = 0.995) and 22.1 mV (r = 0.993) in the presence and absence of EtOH, respectively. This representative result was confirmed in four other patches from different oocytes, the voltage sensitivity of mslo channels being 17.52 ± 2.18 vs. 18.07 ± 3.61 mV/e-fold change in NPo, in the presence and absence of 50 mM EtOH, respectively (not statistically significant, P > .9). Thus EtOH activates mslo channels without modifying the voltage dependence of activation.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Representative plot of mslo channel NPo as a function of [Ca++]ic, at a fixed potential (+40 mV) in the presence (hollow circles) and absence (filled circles) of 50 mM EtOH, obtained in the same I/O patch. A plot of the natural log of NPo as a function of [Ca++]free should be linear at low values of Po. The slope, a measure of the response in channel activity upon recognition of Ca++-sensing sites to increases in [Ca++]ic, is markedly decreased by EtOH: 1.748 (r = 0.997) vs. 1.044 (r = 0.977) in the presence and absence of the drug, respectively. Data similar to those for this representative case were obtained in six other patches from different oocytes, the slopes being 1.824 ± 0.210 vs. 0.769 ± 0.225 in the absence and presence of 50 mM EtOH, respectively (n = 7; P < .02, paired two-tailed t test).

	Discussion

Top Abstract Introduction Methods Results Discussion References

Present results demonstrate that acute exposure to EtOH reversibly increases the activity of cloned BK (mslo) channels expressed in Xenopus oocytes after injection of the channel  subunit. Although the maximal degree of potentiation of mslo channel activity by EtOH is slightly lower than that observed previously in the neurohypophysial BK channel, for which a maximal level of activation (400-450% of controls) occurred at a concentration of 50 mM EtOH, the EC₅₀ found in this study (24 mM) is not only close to legal intoxicating levels in United States but also is almost identical to the EC₅₀ obtained for activation by EtOH of the nerve terminal BK channel (22 mM). In both systems, EtOH-induced BK channel activation occurs immediately after exposure of the cytosolic side of the patch to the drug and decreases to pre-EtOH values after 5 to 6 min of washout with drug-free solution. Furthermore, in both native and cloned channels, the drug modifies similar gating processes: 50 mM EtOH drives the channel away from brief open states toward long open states and destabilizes the long closed state of the channel. In both systems, these modifications, which result in the overall increase in channel activity produced by the drug, occur without altering the channel unitary conductance. These striking qualitative and quantitative similarities support the validity of studying the action of EtOH on cloned channels encoded by slo genes expressed in Xenopus oocytes. Currently, the subunit composition and sequence(s) of the neurohypophysial BK channel are unknown. However, the similarities in the activation by EtOH of two BK channels embedded in two probably different environments (the neurohypophysial and the oocyte membrane) suggest a common site(s) for the action of EtOH on BK channels (probably the subunit itself; see below).

The EtOH-induced increase in mslo channel NP_o is entirely due to actions on channel gating; it occurs without changes in the number of functional channels (in this case,  subunits) present in the patch. Drug actions on gating are a function of EtOH concentration. At [EtOH] = 10 mM, the increase in NP_o is totally explained by a decrease in t_c. On the other hand, at [EtOH]  25 mM, activation results from a combination of concentration-dependent increases in t_o and frequency of channel openings. It is interesting to note that although the overall effect is the opposite (i.e., decrease of channel activity), EtOH's actions on L-type Ca⁺⁺ channel gating in neurohypophysial terminals show a similar dependence on drug concentration: EtOH-induced changes in t_o contribute to the overall decrease in NP_o only at [EtOH]  25 mM. In this system, EtOH's effects on gating are consistent with the interaction of a single drug molecule with a single site (Wang et al., 1994). Although it is possible that the concentration-dependent differential effects of EtOH on mslo channels reflect the interaction of the drug with multiple sites of different affinities, the fact that we found a Hill coefficient not significantly different from 1 for EtOH activation (fig. 2B) indicates that if multiple sites are involved in the functional interaction between EtOH and the channel, this interaction occurs without cooperativity (either positive or negative) among these sites.

The distribution of open and closed times shown in figure 4 indicates that the channel in the absence of the drug exhibits at least two open and two closed states, which is consistent with previous models proposed for BK channels (Barrett et al., 1982; Toro et al., 1990; DiChiara and Reinhardt, 1995). Because of the filtering (1.5 kHz) used to construct idealized records, we worked in the millisecond scale, so the number of open and closed states proposed here may be underestimated. Whether EtOH modifies briefer and/or longer events or switches the channel into a different gating "mode" (a phenomenon that usually occurs in the range of tens of seconds to minutes) remains to be determined. Despite the fact that no additional components in the distribution of either open or closed times were introduced by EtOH, it is possible that a unique EtOH-BK channel dwell time(s) exists but, as a result of fast dissociation, cannot be resolved.

EtOH increased mslo channel activity by shifting the ln(NP_o)-[Ca⁺⁺]_ic relationship to the left while decreasing its slope. The finding that potentiation of mslo activity by EtOH decreased as Ca⁺⁺ levels were increased could be explained by an elevated P_o in the presence of high [Ca⁺⁺]_ic, which would leave little room for further activation by EtOH. However, even at the highest [Ca⁺⁺]_ic used, NP_o values were low, as confirmed by the linearity of the plot of ln(NP_o) vs. [Ca⁺⁺]_ic (fig. 7). The decrease in slope indicates an altered Ca⁺⁺ dependence of channel gating in the presence of EtOH. In the absence of the drug, an approximate two power relationship (limiting slope = 1.82) was observed between free [Ca⁺⁺]_ic and channel NP_o, which suggests that channel activation requires either the strong cooperative binding of two Ca⁺⁺ ions or the weaker cooperativity of more than two Ca⁺⁺ ions. These data are consistent with previous findings obtained in describing the Ca⁺⁺ dependence of channel gating after injection of dslo or hslo  subunits into Xenopus oocytes (DiChiara and Reinhardt, 1995). Mechanistically, these results can be interpreted as meaning that the binding of two Ca⁺⁺ ions is required to open the mslo channel or that, alternatively, only one Ca⁺⁺ is required to open the channel, which, once opened, dwells in open states longer as [Ca⁺⁺]_ic is increased. On the other hand, in the presence of 50 mM EtOH, the limiting slope in the free [Ca⁺⁺]_ic-NP_o relationship was well below 1 (0.77). Because EtOH does not change the number of  subunits (see "Results"), this finding indicates that the drug introduces apparent negative cooperativity in the interaction between Ca⁺⁺ ions in the cytosolic side of the patch and Ca⁺⁺-sensing site(s).

One possibility to explain the decreasing augmentation of channel NP_o by EtOH with increasing [Ca⁺⁺]_ic is that EtOH behaves functionally as a "partial" agonist of the mslo channel, for which [Ca⁺⁺]_ic is a "full" agonist. Two observations are consistent with this hypothesis: 1) EtOH "antagonism" of Ca⁺⁺ on mslo channels (i.e., the decrease in the slope of the NP_o-[Ca⁺⁺]_ic relationship produced by the drug) and EtOH "agonism" (i.e., mslo channel activation) occur at the same EtOH concentration (50 mM), a requirement for partial agonists (Kenakin, 1987); 2) Under conditions in which the basal activation of the system is kept low, the putative partial agonist should function as a competitive antagonist (Kenakin, 1987). To test this proposition, we evaluated the action of 50 mM EtOH on mslo channel NP_o in I/O patches exposed to a high nominal free [Ca⁺⁺]_ic while keeping P_o values low by recording at negative potentials (80 mV). Under these recordings conditions, 50 mM EtOH failed to increase channel activity: NP_o in the presence of the drug was 103.4 ± 14.4% of control (pre-EtOH, obtained in the same patch) values (n = 4). Whether this postulated partial agonism is a consequence of the interaction of EtOH with a shared site at the receptor or reflects interactions with two (or more) discrete sites (one or more that explain activation and another or others that explain the decreased response in channel activity to increases in [Ca⁺⁺]_ic), cannot be determined from functional studies such as those described here. In the absence of binding studies, a variety of possibilities (not mutually exclusive) may be considered in an effort to explain this action of EtOH: an actual decrease in the number of Ca⁺⁺ binding sites, introduction of negative cooperativity in the actual binding of Ca⁺⁺ ions to a conserved number of Ca⁺⁺ recognition sites, and alteration in the Ca⁺⁺ dependence of channel gating per se (that is, EtOH modifies steps subsequent to Ca⁺⁺ interaction with recognition sites).

Although a competitive interaction between EtOH and Ca⁺⁺ has been demonstrated to underlie EtOH's inhibition of Ca⁺⁺-dependent ATPase activity in erythrocytes (IC₅₀ ~ 3 M) (Lopez and Kosk-Kosicka, 1995), our results are the first to show a functional antagonism between Ca⁺⁺ at physiological intracellular levels (about 10-316 nM) and EtOH at pharmacologically relevant concentrations. EtOH activation of BK channels would be most evident in a resting neuron and/or when [Ca⁺⁺]_ic is limited by intracellular storage mechanisms. EtOH is known to inhibit Ca⁺⁺ entry by blocking voltage-dependent Ca⁺⁺ channels (Wang et al., 1994; Mullikin-Kilpatrick et al., 1995). EtOH-blockade of Ca⁺⁺ channels will maintain [Ca⁺⁺]_ic near resting levels, maximizing the activation of BK channels by the drug. Furthermore, BK channels exposed to EtOH retain their high permeability and selectivity for K⁺ over Na⁺ (fig. 5), and these properties allow BK channel activity to contribute effectively to membrane repolarization or hyperpolarization. Thus, EtOH activation of BK channels and blockade of voltage-dependent Ca⁺⁺ channels would act synergistically to result in repolarization or hyperpolarization of the cellular membrane and, eventually, decreased excitability.

EtOH-induced activation of mslo channels occurs in I/O patches when highly buffered Ca⁺⁺ solutions are used at both sides of the patch, in the absence of cyclic nucleotides or other "regenerating" systems. This result indicates that EtOH-induced mslo channel activation is not secondary to EtOH's modulation of freely diffusible second messengers (including cytosolic Ca⁺⁺) but, rather, probably results from a functional interaction between EtOH and the channel or a closely associated entity in the membrane patch. It is currently postulated that native BK channels from mammalian tissues consist of two structurally distinct subunits, termed  and  (McManus et al., 1995). It is unlikely that the  subunit is the subunit mainly responsible in making the BK channel activatable by EtOH. First, EtOH activation was observed when BK channel activity was evoked by injection of the channel  subunit into Xenopus oocytes, an expression system where no endogenous BK  subunits have to our knowledge been observed. Second, preliminary data obtained in I/O patches from oocytes injected with the  subunit of the dslo BK channel also show a reversible, equipotent increase in NP_o by EtOH (Dopico et al., 1996b), and BK  subunits do not seem to regulate dslo channel activity (Meera et al., 1996). Third, bslo (Moss et al., 1996) and mslo  subunits are about 98% homologous, yet only mslo is potentiated by EtOH when they are expressed in the oocyte. Therefore, it is unlikely that an endogenous  subunit is important for EtOH's action on BK channels (Dopico and Treistman, 1996).

The use of mslo  allows us to speculate on which specific regions in this subunit EtOH functionally interacts with. Mslo electrophysiological properties have been related to functional domains (termed "core" and "tail") in the channel protein: 1) both the core (S1-S8) and the tail (S9-S10) are required for function; 2) the core determines ion selectivity and permeability (properties linked to the pore-lining sequence, a region between S5 and S6 termed H5), and voltage dependence of gating (a region defined by S4); 3) the tail contributes to determining apparent Ca⁺⁺-sensitivity (Wei et al., 1994). The lack of change in the voltage dependence of mslo channel gating in the presence of acute EtOH shown here indicates that EtOH does not functionally interact with the voltage sensor. In addition, the unmodified K⁺ permeability and selectivity of mslo channels in the presence of EtOH indicate that the drug does not alter two key properties associated with the H5 region. Together, these data indicate that acute EtOH does not modify several of the major electrophysiological characteristics of mslo channels determined by the core. However, an interaction between EtOH and the core cannot be totally ruled out because the drug increased the channel t_o, which is at least partially determined by this domain (Wei et al., 1994). On the other hand, the EtOH-induced alteration in the response of the Ca⁺⁺-sensing site(s) to increases in [Ca⁺⁺]_ic shown here with mslo channels suggests a functional interaction of the drug with Ca⁺⁺ sensing sites of the channel  subunit, possibly with the S9 to S10 region (tail domain).

Although the simplest explanation for our data is a direct interaction of EtOH with selective regions of the  subunit, we cannot ignore the possibility that EtOH is affecting membrane-bound modulators of BK channel activity, such as G proteins (Scornick et al., 1993; Mullikin-Kilpatrick et al., 1995), protein kinase or phosphatase activities (Reinhardt et al., 1991), redox status (Loguercio et al., 1993; Lee et al., 1994), lipids (Bolotina et al., 1989; Kirber et al., 1992; Abadji et al., 1994), or water-lipid or lipid-protein interfaces (Barry and Gawrich, 1994). The recording of mslo channel activity in I/O patches long after patch excision (>15 min) in the absence of nucleotides in the solutions facing both sides of the patch makes some of these elements unlikely mediators of EtOH action. However, it is intriguing that washout of EtOH's effects in both native and cloned channels was significantly slower than the onset of these effects, requiring 5 min, a result that suggests the need for further experiments to probe the basis for this observation.