Aliphatic alcohols (n-alkanols) and volatile anesthetics are lipophilic agents that can affect the transport of ions across the cell membrane. Therefore, this is thought to be the basis for the biological actions of ethanol and other general anesthetics in the nervous system. According to the Meyer-Overton rule the ability of these agents to partition in lipid-like environments correlates with their general anesthetic potency in vivo(1, 2) . Thus, the core of the lipid bilayer was an attractive locus of action (``lipid hypothesis''). This hypothesis predicted that membrane proteins, and especially those involved in ion transport, will be indirectly affected by lipophilic anesthetics as the result of a physical perturbation of the lipid bilayer(3, 4, 5) . However, a number of observations have challenged this hypothesis(2, 5) : (i) membrane disordering caused by anesthetic concentrations of n-alkanols can be mimicked by a temperature change of 1 °C(2) ; (ii) certain enzymes are affected by n-alkanols and volatile anesthetics in the absence of phospholipids(6, 7, 8) ; (iii) long chain n-alkanol derivatives exhibit a cut-off effect in their anesthetic potency(2, 7, 9, 10) , and certain volatile anesthetics display stereoselectivity(11) ; and (iv) only a limited number of ion channels with diverse functional properties are affected by anesthetic concentrations of n-alkanols and/or other general anesthetics. Such targets include a subset of neurotransmitter-gated ion channels, namely -aminobutyric acid receptors and N-methyl-D-aspartate receptors(2, 4, 12) , high voltage and low voltage Ca channels(13) , and certain K channels(11, 14, 15) . Although many of these targets are similarly affected by n-alkanols and volatile anesthetics, some show differential sensitivity(16) , thereby suggesting additional specificity. To explain these observations an alternative hypothesis of general anesthesia and alcohol action proposes that lipophilic anesthetics may act directly on membrane proteins (``protein hypothesis'') through an interaction with relatively specific hydrophobic clefts or pockets(2) . Proteins that are sensitive to these agents may have common structural motifs, which are located in or associated with a region of the molecule that is critical to its function. Low affinity bulk sites and high affinity discrete sites may actually coexist in the membrane, but only the latter would be relevant to the anesthetic effect. Low affinity sites could involve the core of the lipid bilayer, the protein-lipid interface, or putative membrane-spanning segments of the polypeptide. In absolute terms, however, the binding affinity of putative general anesthetic and n-alkanol binding sites seems to be in the high micromolar to millimolar range, which suggests weak and unconstrained interactions(4) . Thus, the term high affinity is used here in a relative manner when compared with that of low affinity sites like those mentioned above.

Although valuable studies have been made to demonstrate the protein hypothesis of alcohol and general anesthetic action(2, 10, 17, 18, 19, 20) most of the evidence is still indirect, and the molecular bases remain unknown. In a previous study we found that a cloned K channel encoded by Drosophila Shaw2 is selectively inhibited by physiologically relevant levels of ethanol in a concentration-dependent manner(14) . Such inhibition was found to be rapid, reversible, and voltage-independent. By contrast, 12 K channels homologous to Shaw2 (all members of the Shaker superfamily) required >200 mM to exhibit significant inhibition(14, 21) . Since the same cell type (Xenopus oocytes) was used in these studies and, therefore, the membrane environment was constant, we proposed that the unique sensitivity of the Shaw2 K channel to ethanol was specified by its protein moiety. Compared to other members of the Shaker superfamily, Shaw2 K channels also bear unique biophysical properties(14, 22) , such as lack of inactivation, activation with no apparent delay, low voltage sensitivity, and low open probability. Thus, they are unlikely to control spike repolarization (typically the role of delayed rectifier K channels) but may help to determine the passive electrical properties of the membrane. Native channels with functional properties analogous to those of Shaw2 have been recorded from nerve and muscle cells(23, 24, 25) . To investigate the molecular mechanism underlying inhibition of Shaw2 K channels by ethanol and its relationship to the bases of general anesthetic action, we studied the interactions between wild-type and mutant channels with members of the homologous series of n-alkanols. These experiments demonstrate the presence of a discrete hydrophobic site of action probably located in the Shaw2 polypeptide. In addition, we show that channel inhibition results from a discrete effect on channel gating.

MATERIALS AND METHODS

Molecular Biology

Synthesis of cRNAs and Microinjection in Xenopus Oocytes

Electrophysiology

Data Analysis

RESULTS

Equilibrium Dose-inhibition Experiments Suggest the Presence of a Saturable Site for n-Alkanols

Figure 1: Equilibrium dose-inhibition curves. A, whole-oocytes Shaw2 K currents evoked by a 450-ms step depolarization from -100 to +40 mV. Currents in the absence and presence of 1-butanol are shown superimposed. Top to bottom (in mM), 0, 4.4, 5.5, 11, 22, 33, 44, and 55. B, normalized current amplitude (I/I) as a function of 1-butanol concentration. Symbols represent three separate measurements. Lines represent minimized fits to a Langmuir adsorption isotherm of the following form: I/I = 1/(1 + ([A]/K)), where [A] is the alcohol concentration, K is the apparent equilibrium constant, and n is the Hill coefficient. Solidline, best fit parameters (K = 16 mM, n = 1.1); dottedline, best fit parameters (K = 14 mM, n = 1.2) assuming a background level of 5%. C, logarithmic dose-inhibition curves for the indicated alcohols. Lines were generated as described above. In general, the background level was estimated to be <10%. However, because the absolute saturating level cannot be reached, for consistency no background level was assumed to obtain the best fit. Table 1summarizes the best fit parameters. D, competition dose-inhibition curve. Lineacrosssolidsymbols (1-hexanol alone, control) is a minimized fit obtained as described above (K = 1 mM, n = 0.7). Lineacrosscircles (1-hexanol concentration varied in the presence of ethanol at 170 mM) was generated according to the following equation: I/I = 1/(1 + [C2]/K + [C6]/K), where [C2] and [C6] are the concentrations of ethanol and 1-hexanol, respectively, and K and K are the corresponding equilibrium constants (their values were obtained from Table 1). This equation describes direct competition between two alcohols for a common binding site. Symbols in C and D represent the mean ± S.D. of 3-6 determinations.

The Site of Action Might Be a Hydrophobic Pocket in Shaw2

Figure 2: Inhibition of Shaw2 currents by n-alkanols is proportional to alkyl chain length. A, apparent equilibrium constants as a function of number of methylene groups in the alkyl chain. Line represents a linear regression (r= 0.99). B, standard free energy of binding as a function of the number of methylene groups in the alkyl chain. Here, K is expressed in unitary mole fraction units. Line represents a linear regression (r= 0.99) with a slope of -2.93 kJ/mol. Circles and opentriangles represent measurements for hKv3.4-Shaw2 tandem dimer and hKv3.4/G371I ( Fig. 3and Fig. 9).

Figure 3: Properties of a hybrid human-fly K channel: current kinetics. A, diagram of a human-fly tandem dimer. Linelength and boxwidth are proportional to polypeptide length. Boxes represent putative transmembrane segments (S1-S6); J indicates the junction between hKv3.4 and Shaw2 polypeptides (see ``Materials and Methods''). B, diagram of a putative configuration of a tetrameric channel made of tandem dimers (HFHF). An alternative configuration (HHFF) cannot be ruled out because the entire linker region between the cores of both subunits is sufficiently long(37, 38) . C, D, and E, whole-oocyte outward K currents expressed by Shaw2, hKv3.4, and human-fly tandem dimer, respectively. Currents were evoked by 900-ms step depolarizations from -100 to +50 mV.

Figure 9: Effect of 1-hexanol on wild-type and mutant hKv3.4 K channels. A, whole-oocyte outward K currents evoked by 125-ms step depolarizations from -100 to +40 mV. Currents were recorded before and after exposing oocyte to 8 mM 1-hexanol. Currents in the presence of 1-hexanol were taken after the inhibitory effect had equilibrated (30-60 s). This effect was reversible. B, dose-inhibition relation for wild type and G371I. The line represents a minimized fit obtained as described in the legend to Fig. 1. Best fit parameters were K = 3.3 mM and n = 1. Dashedline represents the control level. Symbols represent the mean of 3 and 2 determinations from wild type and G371I, respectively.

The Action of n-Alkanols on Shaw2 Currents Is Probably Mediated by the Channel Polypeptide

()

Figure 4: Properties of a hybrid human-fly K channel: inhibition by n-alkanols. Dose-inhibition curves for ethanol (A) and 1-hexanol (B). Curves were analyzed as described in the legend to Fig. 1. Shaw2 best fit parameters were as follows: K = 168 mM (n = 1) and K = 1 mM (n = 0.7) for ethanol and 1-hexanol, respectively. Tandem dimer best fit parameters were as follows: K = 702 mM (n = 0.8) and K = 5.5 mM (n = 1) for ethanol and 1-hexanol, respectively. Since hKv3.4 exhibits an apparent threshold effect with both ethanol and 1-hexanol, we did not attempt to explain these data quantitatively (see ``Discussion''). Dashedline represents the control level. Symbols represent the mean ± S.D. of two to four determinations.

Selective Inhibition of a Gating Step by n-Alcohols May Explain Inhibition of the Whole-cell Current

Figure 5: Inhibition of Shaw2 single channel currents by 1-butanol. Consecutive single channel currents recorded at 0 mV from an inside out patch under control conditions (left), exposing the cytoplasmic side to 88 mM 1-butanol (center), and after washout (right). Currents shown were low-pass filtered at 2.5 kHz (-3 db, 8-pole) and digitized at 20 kHz. Line across the traces is the zero current level. Analysis of the complete set of records is shown in Fig. 6B and 7.

Figure 6: Effect of 1-butanol on Shaw2 single channel conductance and open time. A, single channel current-voltage relation in the absence () and presence of 44 mM 1-butanol (circo). Mean current amplitudes at each membrane potential were estimated from Gaussian fits to amplitude histograms. The calculated slope conductances (solidlines) were 23 and 25 pS for control and 1-butanol, respectively. B, cumulative histograms of open times. This represents the probability that the open time is equal or shorter than the time on the abscissa.

Figure 7: Effect of 1-butanol on the distributions of closed times and open times. Logarithmic histograms of closed times (A) and open times (B) from single channel records in the absence of alcohol (control), in the presence of 1-butanol, and after washout. The number of binned events in these histograms were (from top to bottom): 1377, 368, and 1738. Solidthicklines represent best fits to a single exponential (closedtime) or a sum of two exponential terms (opentime). Each component of the sum is shown separately by thinsolidlines. The best fit parameters are shown in Table 2(Experiment 9482405/16).

A Single Hydrophobic Mutation in the S4-S5 Loop Confers n-Alkanol Sensitivity in a Human KChannel

Figure 8: The S4-S5 linker of 11 members of the Shaker superfamily of voltage-gated K channels. The letters d, h, m, and r stand for Drosophila, human, mouse, and rat, respectively. Bold and underlinedcharacters represent identity or similarity relative to Shaw2 (assuming S T and K R). Asterisks mark positions that form part of the leucine-heptad repeat(44) . Column on the right indicates whether a channel was significantly inhibited by <200 mM ethanol(14, 21) .

Figure 10: Effect of ethanol on wild-type and mutant Shaw2 K channels. A, whole-oocyte outward K currents evoked by 450-ms step depolarizations from -100 to +40 mV. Currents were recorded before and after exposing oocyte to 170 mM ethanol. Currents in the presence of ethanol were taken after the inhibitory effect had equilibrated (30-60 s). This effect was reversible. B, whole-oocyte Shaw2 K currents evoked by a 9000-ms step depolarization from -100 to +20 and +10 mV (wild type and I319G, respectively). For I319G, current decay at +10 and +60 mV did not seem significantly different.

DISCUSSION

We have studied the interaction of various members of the homologous series of n-alkanols with a cloned K channel encoded by Drosophila Shaw2. These compounds cause depression of the nervous system in vivo and have been commonly used to study the biological bases of general anesthesia and alcohol action(2, 3, 9) . Since Shaw2 membrane currents are selectively inhibited by n-alkanols at low concentrations, we investigated the functional and molecular bases of this action. The results led to the following main conclusions: (i) current inhibition results from a selective reduction in the probability that channels enter a long duration open state; and (ii) the site of n-alkanol action is a discrete hydrophobic cleft or pocket, most likely located in the channel polypeptide.

Inhibition of a long duration open state by n-alkanols can be interpreted according to Fig. SI, where C represents a single closed state in the activation pathway or a very rapid equilibrium between several closed states that precede channel opening and O and O represent brief duration and long duration open states, respectively. The rates that control channel opening and closing are represented as and , respectively. The following arguments suggest that Fig. SIis a minimal mechanism that qualitatively explains gating of Shaw2 channels. (i) The rising phase of Shaw2 currents shows no delay, even at negative voltages, and gating is weakly voltage-dependent(48) . Closed time histograms suggested only one closed state directly in equilibrium with open channels (Fig. 7A, Table 2). (ii) The presence of two open states (O and O) was suggested from the analysis of open time histograms, which is best described by the sum of two exponential components (Fig. 7B). (iii) Direct transitions O O do not seem likely because 1-butanol selectively affects the frequency of long duration openings with little effect on the time constants of long and brief duration openings (Table 2). Accordingly, parameters extracted from the analysis of open and closed time histograms can be related to rate constants in Fig. SI: long mean open time () = 1/; brief mean open time () = 1/; mean closed time () = 1/( + ); the fractional contribution of long duration openings to the the distribution of open times (f) equals the probability of C O (Table 2). Then, P(C O) = /( + ) and therefore, = f/. Similarly, = f/, where f represents the fractional contribution of brief duration openings to the distribution of open times (Table 2). Our data suggest that mainly is reduced by the action of n-alkanols. However, an accurate estimation of the opening rates is presently limited because we do not know whether the analyzed records represent the activity of a single channel, even in the absence of overlapped openings (owing to a low open probability). Also, is overestimated because of missed brief openings (Fig. 7B and Table 2). Nevertheless, as an approximation, we find from the inside out patch data (Table 2) that is reduced from 8 to 0.5 s (16-fold), whereas is reduced only from 9 to 4 s (2-fold). If the proposed mechanism can account for inhibition of the macroscopic membrane current by n-alkanols, we can predict that at a given concentration f(+)/f(-) should equal the remaining fraction of the membrane current (see below). f(-) and f(+) are the fractional contributions of long duration openings in the absence and presence of 1-butanol, respectively. In addition, since there is little effect on brief openings and the time constants of long and brief open times may differ 5-10-fold (Table 2), the proposed mechanism also predicts that at high concentrations of n-alkanol a small proportion of the membrane current (contributed by brief openings only) should be relatively resistant to inhibition. The dottedline in Fig. 1B describes the data, assuming a simple binding isotherm plus a constant background level representing 5% of total current. Thus, at 88 mM 1-butanol (Fig. 1B and Table 2), (I/I) - 0.05 = 0.1 and f(+)/f(-) = 0.2. The close agreement between these values (within 10% of total current) suggests that inhibition of can explain the effect of n-alkanols on the macroscopic current. As suggested earlier, an additional effect on the number of active channels(N) seems, therefore, unlikely.

Figure SI:

A novel hybrid channel composed of two Shaw2 subunits and two hKv3.4 subunits expressed currents with a sensitivity to n-alkanols greater than that of hKv3.4 alone. We therefore concluded that such a phenotype is determined by the Shaw2 moiety. A similar observation has been made with mutant Shaker channels and tetraethylammonium(37, 38, 49) . Tetraethylammonium is a K channel blocker that is known to interact with specific amino acids near the outer and inner mouths of the pore(50, 51) . Especially interesting in our study was the finding that the apparent threshold effect observed with hKv3.4 (resembling a sigmoidal dose-inhibition relation) is eliminated in the case of hKv3.4-Shaw2 hybrids. In fact, ethanol and 1-hexanol dose-inhibition relations for hybrid channels were hyperbolic with Hill coefficients of about 1.0 (Fig. 4). Thus, it seems that interaction with a high affinity site dominates in the hybrid channel, and such a site might be located in the Shaw2 moiety. In hKv3.4 (and probably in other related channels insensitive to alcohols) there may be multiple low affinity sites, and higher occupancy is required to cause inhibition (resembling positive cooperativity). For instance, multiple low affinity sites may be located in the protein-lipid interface surrounding the channel oligomer. In Shaw2 however, there is a single high affinity site possibly located at the protein-water interface. Consistent with the latter suggestion, we found a standard free energy change per methylene group on the order of -3 kJ/mol (Fig. 2). This value corresponds with the free energy change estimated for the transfer of n-alkanols from water to alkanes(31) . Moreover, we showed that a hydrophobic point mutation in a putative cytoplasmic loop of hKv3.4 (G371I) was sufficient to eliminate apparent sigmoidicity of the dose-inhibition relation and introduce higher affinity (Fig. 9B). The dose-inhibition relation for hKv3.4/G371I resembled that of Shaw2 (albeit with somewhat lower affinity; see below). These data suggest that the effect of G371I in hKv3.4 is unlikely to result from a random structural change. G371I may have created a hydrophobic cleft, and binding of n-alkanols to this site allows inhibition of hKv3.4 with higher affinity. Alternatively, we cannot rule out that G371I causes an allosteric shift between low and high affinity conformations of the channel(2, 7) .

If the corresponding site in Shaw2 (I319) were a critical determinant of the native n-alkanol binding site, it seemed that I319G would reduce n-alkanol-dependent inhibition of Shaw2 currents. However, this was not the case, and the main apparent change caused by I319G was the presence of slow current decay (Fig. 10B). We propose the following explanation for this observation; most likely, various residues are important determinants of the binding site. This concept is supported by the fact that the sensitivity of hKv3.4/G371I was intermediate between that of Shaw2 and that of wild-type hKv3.4. If the putative hydrophobic pocket in Shaw2 is structurally critical, it is conceivable that, to preserve a stable conformation, compensatory changes have taken place in Shaw2/I319G. For instance, in I319G another hydrophobic amino acid from a nearby or distant region may play the role of I319. Similar compensatory changes have been proposed to occur in mutants of cystic fibrosis transmembrane conductance regulator(52) . Thus, the determinants of the n-alkanol binding site in Shaw2 might be redundant, and further mutagenesis experiments will be necessary to identify all critical residues.

Additional evidence suggests that the site of n-alkanol action in Shaw2 might be relatively specific. Several functionally important hydrophobic pockets have been located in voltage-gated Na channels and Shaker K channels, mainly in the S4-S5 loop and within S6, where they form part of the inactivation gate receptor and the binding sites of local anesthetics and alkyl quaternary ammonium derivatives(53, 54, 55) . However, n-alkanols and volatile anesthetics affect these channels with very low affinity(14, 21, 32, 33) . Clearly, the presence of functionally important hydrophobic pockets is not necessarily equivalent to general anesthetic action with high affinity. Thus, although the Shaw2 K channel bears significant similarity to those channels, its unique sensitivity to n-alkanols indicates the presence of a relatively specific site of action, which can be partially engineered in a homologous channel by a single amino acid substitution in the S4-S5 loop. It is conceivable that both lipid and protein may form part of the n-alkanol binding site. Recent reports suggest however, that the S4-S5 loop forms part of the inner mouth of the channel(53, 56) , and it is not known how this region may interact with membrane lipids surrounding the protein.

Overall the data provide compelling evidence that favors the presence of a discrete site of n-alkanol action in a cloned K channel and, therefore, support the protein hypothesis of general anesthetic and alcohol action. Evidence of n-alkanol sites has also been reported for ligand-gated ion channels, including N-methyl-D-aspartate receptors, nicotinic acetylcholine receptors and ATP-gated ion channels(5, 10, 19, 20) . However, there is little information available about the nature of the binding sites and their determinants. Nevertheless, n-alkanol binding sites are probably diverse, and future structure-function studies should help to reveal their differences and similarities.

Given the unique biophysical properties of Shaw2, we can relate inhibition of this channel by n-alkanols with a possible anesthetic effect. Shaw2-like K channels have been recorded in nerve and muscle cells(23, 24, 25) , Mainly because of their weak voltage sensitivity and lack of inactivation, they may help to determine the resting membrane potential. Inhibition of Shaw2 by anesthetics would increase membrane resistance. This can cause steady-state depolarization (57) and, in addition, may allow a larger depolarization in response to a given excitatory stimulus. Although these changes may initially cause excitation, their long term effect could lead to inactivation of voltage-gated Na channels and, therefore, inhibition of membrane excitability(58, 59) .

FOOTNOTES

*

This work was supported by United States Public Health Service (USPHS) Program Project Grant AA07186-07 from the NIAAA, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Dept. of Pathology, Anatomy, and Cell Biology, Jefferson Medical College, 1020 Locust St., JAH 245, Philadelphia, PA 19107. Tel.: 215-955-4341; Fax: 215-923-2218.

¶

Supported by USPHS Training Grant AA07463.

**

Supported in part by Hospital de Especialidades, Instituto Mexicano del Seguro Social, Mexico City.

()

Mammalian Shaw homologues share 50% amino acid identity within the core of the polypeptide (S1-S6). The closest homologue (rKv3.3) scores 52%, and hKv3.4 scores 51.5%.

ACKNOWLEDGEMENTS

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