©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Alcohols Inhibit a Cloned Potassium Channel at a Discrete Saturable Site
INSIGHTS INTO THE MOLECULAR BASIS OF GENERAL ANESTHESIA (*)

(Received for publication, April 20, 1995; and in revised form, June 12, 1995)

Manuel Covarrubias (1)(§) Tapan B. Vyas (1)(¶) Laura Escobar (1)(**) Aguan Wei (2)

From the  (1)Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107 and the (2)Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The molecular basis of general anesthetic action on membrane proteins that control ion transport is not yet understood. In a previous report (Covarrubias, M., and Rubin, E.(1993) Proc. Natl. Acad. Sci. 90, 6957-6960), we found that low concentrations of ethanol (17-170 mM) selectively inhibited a noninactivating cloned K channel encoded by Drosophila Shaw2. Here, we have conducted equilibrium dose-inhibition experiments, single channel recording, and mutagenesis in vitro to study the mechanism underlying the inhibition of Shaw2 K channels by a homologous series of n-alkanols (ethanol to 1-hexanol). The results showed that: (i) these alcohols inhibited Shaw2 whole-cell currents, the equilibrium dose-inhibition relations were hyperbolic, and competition experiments revealed the presence of a discrete site of action, possibly a hydrophobic pocket; (ii) this pocket may be part of the protein because n-alkanol sensitivity can be transferred to novel hybrid K channels composed of Shaw2 subunits and homologous ethanol-insensitive subunits; (iii) moreover, a hydrophobic point mutation within a cytoplasmic loop of an ethanol-insensitive K channel (human Kv3.4) was sufficient to allow significant inhibition by n-alkanols, with a dose-inhibition relation that closely resembled that of wild-type Shaw2 channels; and (iv) 1-butanol selectively inhibited long duration single channel openings in a manner consistent with a direct effect on channel gating. These results strongly suggest that a discrete site within the ion channel protein is the primary locus of alcohol and general anesthetic action.


INTRODUCTION

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 leq 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

Standard procedures were used to maintain, amplify, and isolate plasmid DNA(26) . For expression in Xenopus oocytes, Drosophila Shaw2 and hKv3.4 were maintained in pBluescript-MXT. This is a hybrid plasmid that includes the 5`- and 3`-untranslated regions of Xenopus beta-globin flanking the multicloning cassette. hKv3.4 was provided by Dr. B. Rudy (New York University Medical Center). To introduce point mutations we used oligonucleotide-directed mutagenesis (Altered Sites (Promega)). In this procedure, a second mutagenic oligonucleotide confers antibiotic resistance, thereby allowing selection of putative mutant clones. Inserts to be mutated were subcloned in pALTER-1 (Promega). Mutant clones were confirmed by sequencing using a radioisotopic dideoxy method driven by Sequenase (U.S. Biochemical Corp.) or automated sequencing from Jefferson Cancer Institute (Thomas Jefferson University). Identified mutant inserts were isolated and ligated into the original plasmid vector. The hKv3.4-Shaw2 tandem dimer subunit was created by engineering a 7-amino acid linker (LDGSFAT) between glycine 560 at the C terminus of hKv3.4 and the first methionine at the N terminus of Shaw2. This causes a deletion of 23 amino acids at the C terminus of hKv3.4.

Synthesis of cRNAs and Microinjection in Xenopus Oocytes

DNA templates linearized at the 3`-end of the insert with an appropriate restriction endonuclease were used for the synthesis of run-off sense cRNA. In vitro transcription and 5`-capping of the cRNA was done using the MEGAscript (Ambion). Mature oocytes (Stage V or VI) were obtained and microinjected according to established procedures(27) . Injected oocytes were kept at 19 °C for a maximum of 7-10 days in ND96 (in mM: 96 NaCl, 2 KCl, 1.8 CaCl(2), 1 MgCl(2), 5 HEPES, pH 7.4, titrated with NaOH) supplemented with penicillin and streptomycin. This solution was changed daily.

Electrophysiology

The two-microelectrode voltage clamp technique was used to record whole-oocyte currents. Microelectrodes are fabricated of borosilicate capillary tubing (Dagan) and have tip resistances on the order of 0.2-0.5 megaohms when filled with 3 M KCl and immersed in ND96. Voltage clamping was done using a TEV-200 amplifier (Dagan) following established procedures (28) . Pulse protocols and data collection were controlled by a 486 computer equipped with Digidata 1200 acquisition hardware (Axon Instruments) and pCLAMP 5.5 or 6.0 (Axon Instruments). Whole-oocyte currents are generally filtered at 1-2 kHz and digitized at 0.125-1 ms/point. Oocytes were regularly perfused with ND96 using a gravity-driven perfusion system at 3-4 ml/min (chamber volume 250 µl). Drugs dissolved in ND96 were applied in the recording chamber at the indicated rate. Standard gigaseal patch clamp technique was used to record single channel currents(29) . An Axopatch 200 (Axon Instruments) interfaced with a 486 computer was used as indicated above. Patch clamp microelectrodes were fabricated from borosilicate glass (7052, AM-Systems) using a three-step horizontal puller (Sutter Instruments). To reduce background noise, tips were coated with Sylgard 184 silicone elastomer (Dow Corning). Tip resistances ranged between 2 and 5 megaohms. Before patch clamp recording, individual oocytes were prepared as described previously (28) and immediately placed in a recording chamber containing 250 µl of a high potassium bath solution (140 mM potassium aspartate, 10 mM KCl, 1.8 mM CaCl(2), 10 mM HEPES, pH 7.2 (titrated with KOH)). This solution clamped the oocyte membrane potential near zero mV, allowing a direct reading of the membrane potential across the patch when recording in the cell-attached configuration. Single channel currents were low pass filtered at 2.5 kHz (8-pole, -3 db cut-off frequency) and digitized at 50 µs/point. Pipette solution for cell-attached patch recording was ND96. Intracellular solution for inside-out patches contained 98 mM KCl, 1 mM MgCl(2), 1 mM CaCl(2), 11 mM EGTA, and 10 mM HEPES, pH 7.2 (titrated with N-methyl-D-glucamine). All experiments were recorded at room temperature (21-23 °C).

Data Analysis

Voltage clamp records were analyzed using commercial software (pCLAMP 6.0, Axon Instruments), and customized macro routines were written in a Quattro Pro environment (Borland). Leak current was subtracted assuming a linear leak. Capacitive currents were subtracted using smooth scaled templates that best described the capacitive transients. Nonlinear curve fitting of dose-response curves was conducted using Sigmaplot (Jandel). Single channel records were idealized in operator-controlled mode using a 50% amplitude criterion (FETCHAN, Axon Instruments) or a slope criterion (TRANSIT, A. Van Dongen, Duke University). The event list generated from idealized records was used to produce amplitude and interval duration histograms. Fitting of smooth functions to histograms was done using a Levenberg-Marquardt algorithm or a maximum likelihood routine (PSTAT, Axon Instruments). All data were expressed as mean ± S.D.


RESULTS

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

To investigate the presence of a saturable site for aliphatic alcohols, we examined the equilibrium dose-response relation for various members of the homologous series of n-alkanols (C2-C6). Shaw2 K channels were expressed in Xenopus oocytes, and whole-cell currents were recorded under voltage clamp conditions. Drugs were applied externally by bath perfusion, as described before(14) . To ensure equilibrium the bath solution was exchanged with at least 10 volumes (3-4 ml/min), and current was measured as the average amplitude of three traces that did not show further inhibition. All n-alcohols caused inhibition of the whole-cell current (e.g.Fig. 1A). Dose-inhibition relations were hyperbolic, showing clear evidence of saturation especially with longer chain n-alcohols (Fig. 1, B and C). Data were thus analyzed assuming a Langmuir binding isotherm (see legend to Fig. 1). Results were well described by this function with Hill coefficients that ranged between 0.9 and 1.5 (Table 1), implying a one-to-one drug-receptor interaction. To further demonstrate the discrete nature of the binding site we conducted a competition experiment with ethanol and 1-hexanol. These alcohols differ in their apparent equilibrium constants (K) by more than 2 orders of magnitude (Table 1). Oocytes were exposed to a fixed concentration of ethanol (170 mM) and increasing concentrations of 1-hexanol. As expected, in the absence of 1-hexanol 50% of the current is inhibited by ethanol. At low concentrations of 1-hexanol, the action of both alcohols is partially additive, and as the concentration increases, the dose-inhibition relation approaches that of 1-hexanol alone. Thus, the alcohol with higher potency (C6) displaces the one with lower potency (C2). These data are well described by a binding isotherm that incorporates simple competition (see legend to Fig. 1). In fact, all parameters in the equation were constrained to values determined separately for ethanol and 1-hexanol (Table 1). Overall, these results are fully consistent with the presence of a discrete saturable site that mediates inhibition of Shaw2 K channels by 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(0)) 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(0) = 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(0) = 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

The thermodynamic binding parameters of the homologous series of n-alkanols can give information about the nature of the binding site. When K (on a log scale) is plotted as a function of the number of methylene groups in the alkyl chain (Fig. 2A), we find an inverse correlation between these variables (r= 0.99). This is to be expected if binding is a direct reflection of the ability of the anesthetic to partition into organic solvents (i.e. as predicted by the Meyer-Overton rule). The following relation was used to calculate the standard free energy of binding: DeltaG(B) = RTlnK, where R is the gas constant and T is absolute temperature. Since we used unitary mole fraction units for this computation, DeltaG(B) represents the transfer energy from the aqueous solution to the binding site(30) . Accordingly, a plot of DeltaG(B) against the number of methylene groups (Fig. 2B) shows that the change in binding energy per methylene group is -704 cal/mol (-2.9 kJ/mol or 1.3 RT). This value is close to the standard free energy change per methylene group for the transfer of n-alkanols from water into alkanes(31) . Thus, the site of n-alkanol action in Shaw2 might be a hydrophobic pocket and, since no cut-off effect was detected up to 1-hexanol, this pocket can accommodate an alkyl chain of at least six methylene groups. Standard free energy values on the order of -3 kJ/mol have been estimated for the interaction of n-alkanols with voltage-gated Na and K channels from squid axon (32, 33) and muscle nAchR channels(34) . However, compared to Shaw2, n-alkanols act on these channels with lower potency.


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

To test whether Shaw2 subunits carry the sensitivity to n-alkanols, we made a tandem dimer subunit (Fig. 3A) composed of two distinct monomers, one ethanol-sensitive (Shaw2) and the other ethanol-insensitive (hKv3.4). hKv3.4 is a human homologue of Shaw2 (35) with an overall amino acid identity of 50%. (^1)Both are members of the Shaw subfamily; therefore, they can coassemble to form a heteromultimer in Xenopus oocytes(36) . The tandem dimer construct is advantageous because the subunit stoichiometry of the hybrid channel can be constrained(37, 38) . Since K channels are tetrameric proteins(37, 39) , the novel hybrid channel has two subunits of each kind. Fig. 3B represents a possible configuration of a heterotetramer composed of two tandem dimers. Compared with the parent channels, the tandem dimer expressed currents that show intermediate kinetic properties (Fig. 3, C-E). This is probably mediated by a stretch of 28 amino acids at the N terminus of hKv3.4(40) . Moreover, for both ethanol and 1-hexanol, the dose-inhibition relation was also intermediate and uniform (Fig. 4). Thus, the novel phenotype of the heterotetramer is determined by two hKv3.4 subunits conferring intermediate current kinetics and two Shaw2 subunits conferring intermediate n-alkanol sensitivity. As a corollary, we concluded that a Shaw2 subunit itself may carry the n-alkanol binding site.


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

The macroscopic membrane current produced by an ensemble of channels is defined as I = (V - V(r))NPo, where is the unitary conductance, V the membrane potential, V(r) the reversal potential, N the number of active channels, and Po the open probability. Since n-alkanols mainly decrease the whole-cell current (I(M)), inhibition of one or more factors in that equation could explain such an effect. To investigate the mechanism underlying n-alcohol-dependent inhibition of Shaw2 currents, we conducted experiments to study the single channel properties of Shaw2 in the absence and presence of 1-butanol. These experiments were carried out using the cell-attached or inside-out configurations of the patch clamp technique. Drugs were applied either extracellularly (cell-attached) or directly to the cytoplasmic face of the patch (inside out). The method of application did not significantly affect the results (see below), probably because short chain alkanols equilibrate rapidly across the membrane(41) . Since Shaw2 currents do not inactivate (Fig. 3C), even during 10-s step depolarizations, single channel currents can be recorded under steady-state conditions at a constant depolarization (0 mV). Single channel openings are overall very brief and occur mostly in isolation (Fig. 5), indicating a low open probability. 1-Butanol (88 mM) exerted a conspicuous inhibition of the opening frequency and apparently reduced the mean open time. These effects were fully reversible upon washout. Unitary conductance and reversal potential were not significantly affected (Fig. 6A), but in the presence of 1-butanol the cumulative histogram of open times was clearly dominated by very brief open durations (Fig. 6B). Similar but less pronounced results were observed with 85 mM ethanol and 22 and 44 mM 1-butanol (data not shown). Using a nonparametric test (Kolmogorov-Smirnov test) we found that this effect was significant at p leq 0.01(42) . To further investigate the effect of 1-butanol on channel gating, we analyzed logarithmic closed and open time distributions (43) from cell-attached and inside out patches in the absence and presence of 22, 44, and 88 mM 1-butanol. Closed time histograms were well described by a single exponential (Fig. 7A) with a time constant that simply represents the mean interval between individual openings. Consistent with a reduced opening frequency, this time constant was longer in the presence of 1-butanol (Table 2). Control and experimental open time histograms were well described by the sum of two exponential components (Fig. 7B, Table 2), indicating the presence of two open states (brief duration and long duration). 1-Butanol significantly reduced the proportion of long duration openings, with little or no effect on the time constants (Table 2). This explains the apparent reduction in open time and the rapid rise of the cumulative open time histogram. Thus, 1-butanol seems to inhibit selectively a gating step that leads to long duration openings. Inhibition of the whole-cell Shaw2 current by n-alcohols is, therefore, mainly due to a reduction in Po. As a secondary effect, there is a distinct possibility that N is reduced too. However, this seems unlikely, since the height of the brief component of the open time distribution, which is proportional to the contribution of brief duration openings, was almost equal in all conditions. We therefore assumed a homogeneous class of channels with complex gating kinetics involving two open states and at least one closed state (see ``Discussion''). Only the long duration open state is affected by n-alkanols.


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 (bullet) 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

The core of a K channel subunit includes six putative membrane-spanning domains named S1 to S6 (Fig. 3A). Previous studies have suggested that the linker region located between transmembrane segments S4 and S5 plays an important role in controlling the gating of voltage-gated K channels(44, 45) . Since n-alkanols have a selective effect on gating of Shaw2 K channels, it is possible that the S4-S5 loop might contribute to the site of n-alkanol action. According to current molecular models of K channels, the S4-S5 loop is supposed to be cytoplasmic(46) . Sequence alignment of several K channels revealed similarities and a striking difference within that region (Fig. 8). Isoleucine occupies position 319 in Shaw2 (ethanol-sensitive), whereas glycine occupies the equivalent position in all ethanol-insensitive K channels. Compared to glycine, isoleucine is a highly hydrophobic amino acid (47) . Also, relative to Shaw2, other differences within the S4-S5 loop do not involve a hydrophobic substitution that can be related to alcohol sensitivity (Fig. 8). This was an important consideration because earlier results suggested a hydrophobic pocket as the site of n-alcohol action (Fig. 2). To test whether isoleucine affects n-alcohol sensitivity, glycine 371 in hKv3.4 (ethanol-insensitive) was mutated to isoleucine (G371I). This mutation produced two important effects. One effect was a 12-mV depolarizing shift in voltage dependence of prepulse inactivation, with little or no change in voltage sensitivity. The midpoint potentials and slopes for wild type and G371I were as follows: -25 ± 3.5 mV and 4.9 ± 0.2 mV/e-fold (n = 4), and -13 ± 1 mV and 4.4 ± 0.2 mV/e-fold (n = 6), respectively. This was not surprising, because mutations affecting hydrophobic residues within this region are known to affect voltage dependence(44, 45) . The other effect was more striking. G371I was sufficient to significantly alter n-alkanol sensitivity (Fig. 9). Inhibition of peak current by 170 mM ethanol increased 3.8-fold (4 ± 2% and 15 ± 1% in control (n = 5) and G371I (n = 10), respectively). As found with Shaw2(14) , this effect was voltage-independent. Wild-type dose-inhibition relations for ethanol and 1-hexanol ( Fig. 4and Fig. 9) showed evidence of a threshold effect, especially apparent with 1-hexanol. By contrast, G371I dose-inhibition relation with 1-hexanol is hyperbolic and shows evidence of saturation (Fig. 9B). Conversely, we introduced glycine at position 319 in Shaw2 (I319G). This mutation did not affect the sensitivity of Shaw2 channels to ethanol (Fig. 10A) but introduced slow current inactivation (Fig. 10B). This is in contrast to the wild-type Shaw2, which shows no current inactivation during long depolarizations. It seems that a hydrophobic pocket that interacts with n-alkanols can be engineered in hKv3.4 by a point mutation (G371I), but the reverse mutation in Shaw2 (I319G) was not sufficient to eliminate n-alkanol sensitivity. This finding suggests that compensatory structural changes may have retained n-alkanol-dependent inhibition and that I319 may be only one of several determinants of the hydrophobic pocket (see ``Discussion''). Further mutagenesis experiments are necessary to locate other determinants. In any event, these experiments demonstrated that, in agreement with the protein hypothesis, a single amino acid substitution within a putative cytoplasmic loop of a K channel can alter the sensitivity to n-alkanols.


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(B) and O(L) represent brief duration and long duration open states, respectively. The rates that control channel opening and closing are represented as alpha and beta, 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(B) and O(L)) 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(B) O(L) 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/beta(1); brief mean open time () = 1/beta(2); mean closed time ((C)) = 1/(alpha(1) + alpha(2)); the fractional contribution of long duration openings to the the distribution of open times (f(L)) equals the probability of C O(L) (Table 2). Then, P(C O(L)) = alpha(1)/(alpha(1) + alpha(2)) and therefore, alpha(1) = f(L)/(C). Similarly, alpha(2) = f(B)/(C), where f(B) represents the fractional contribution of brief duration openings to the distribution of open times (Table 2). Our data suggest that mainly alpha(1) 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, (C) 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 alpha(1) is reduced from 8 to 0.5 s (16-fold), whereas alpha(2) 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(L)(+)/f(L)(-) should equal the remaining fraction of the membrane current (see below). f(L)(-) and f(L)(+) 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)) - 0.05 = 0.1 and f(L)(+)/f(L)(-) = 0.2. The close agreement between these values (within 10% of total current) suggests that inhibition of alpha(1) 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.

(^1)
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

We thank Charles Choe for skilled technical assistance, Dr. R. Horn for fruitful discussions, Dr. A. Thomas for critical comments on this manuscript, and Dr. E. Rubin for encouragement, support, and valuable suggestions. We are also thankful to Dr. M.-Y. G.-M. Covarrubias for sequencing mutant clones.


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