Introduction

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Agonist-induced activation of both peripheral and neuronal nicotinic acetylcholine receptors (nAChRs) is enhanced by ethanol (EtOH). (Gage, 1965; Bradley et al., 1980; Forman et al., 1989; Nagata et al., 1996). Similar EtOH enhancement is observed in related ligand-gated channels such as -aminobutyric acid-A, 5-hydroxytryptamine-3, and glycine receptors and it is thought that enhancement of the function of these receptors plays a role in EtOH's behavioral effects (Leidenheimer and Harris, 1992; Aguayo and Pancetti, 1994; Machu and Harris, 1994; Deitrich et al., 1997). The kinetic mechanism underlying activation enhancement by ETOH is uncertain.

In peripheral nAChRs from Torpedo electroplaque and muscle, EtOH enhancement of ACh-gated currents is observed with low concentrations of ACh, but currents elicited by saturating ACh concentrations are unaffected by up to 300 mM EtOH (Forman et al., 1989; Wu et al., 1994). Thus, ACh-response relationships are shifted toward lower concentrations, a phenomenon known as leftward (or sinistral) shift.

The leftward shift of ACh responses by EtOH could be achieved by altering several different steps in the gating mechanism of nAChR. The most obvious possibility is that EtOH might enhance the affinity of agonist binding sites. Secondly, EtOH might increase the probability of channel opening (p_open) after agonist binding. Increasing p_open is predicted to result in leftward shift of ACh responses, but because nAChRs with both agonist sites occupied by ACh are estimated to be open more than 97% of the time, increasing p_open will not dramatically increase peak responses at saturating ACh concentrations. Another possible mechanism, reported for benzodiazepine enhancement of -aminobutyric acid-A receptors, is that nAChR single-channel conductance could be increased by EtOH, although this mechanism should also increase peak current responses at high ACh (Eghball et al., 1997). Furthermore, in experiments where desensitization or agonist channel block moderates the overall measured response, leftward shifts in agonist responses could occur if EtOH reduces these actions.

Nicotinic receptors formed from wild-type , , and  subunits and  subunits containing a channel mutation, T263I, are affected by EtOH in a manner that has not been previously reported. ACh-induced currents from T263I receptors are enhanced by EtOH at both low and high ACh concentrations. A detailed examination of EtOH effects on T263I mutant receptors supports a model where enhanced activation in the presence of EtOH is due to an increased opening probability of ACh-bound nAChRs.

	Materials and Methods

Top Summary Introduction Materials & Methods Results Discussion References

Materials. cDNAs encoding wild-type , , , and  subunits and the Y198F mutant in pSP64T vectors were provided by Dr. James McLaughlin (Tufts Medical School, Boston, MA) and T263I mutant cDNA in pGEM2-SP6 was provided by Dr. Cesar Labarca (California Institute of Technology, Pasadena, CA). Acetylcholine chloride (ACh), EtOH, and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

Xenopus Oocyte Expression. Wild-type and mutant nAChRs were expressed on the surface of Xenopus oocytes after injection of oocytes with messenger RNA mixtures encoding the four nAChR subunits. Detailed methods were previously described (Forman et al., 1995). All procedures with frogs were approved by the Massachusetts General Hospital Animal Care Committee. After microinjection, oocytes were incubated for 48 to 96 h, then manually stripped of their vitelline membranes and used for electrophysiology.

Rapid Perfusion Patch-Clamp Electrophysiology. Electrophysiology recordings were made at room temperature (20-22°C). Borosilicate patch pipettes were polished to give open tip resistance of 2-5 M. For rapid perfusion studies, oocyte membrane patches were pulled in the outside-out configuration and held at 50 mV. Inside and outside buffers were symmetrical K-100 (97 KCl mM, 1 MgCl₂ mM, 0.2 EGTA mM, and 5 K-HEPES mM, pH 7.5). Currents through the patch-clamp amplifier (Axopatch-200A; Axon Instruments, Foster City, CA) were filtered (8-pole bessel, 2 kHz) and digitized at 5 to 10 kHz using a 586-class PC, a 12-bit A/D converter (National Instruments, Austin, TX), and custom software.

Single-Channel Studies. Recordings were made at room temperature using excised inside-out patches held at 150 mV. Pipettes were 2 to 5 M resistance and uncoated. Pipette and bath solutions were symmetrical K-100 and ACh in the pipette was 0.2 to 1.0 µM. EtOH, when present, was added to the pipette solution only. Continuous recordings were acquired at 10 kHz digitization with 5 kHz filtering using the FETCHEX program in pClamp6.0 (Axon Instruments).

Data Analysis. In rapid concentration-jump studies, each patch studied under a given set of ACh/EtOH conditions was exposed to these drugs sequentially 8 to 16 times with a recovery period in between each exposure. The ensemble of current traces were averaged. Control ensemble average currents (saturating ACh without EtOH) were assessed before and after experiments where patches were exposed to EtOH. Data was not analyzed if the two control peak currents differed by more than 10%. Experimental peak currents were normalized to the average peak from the two bracketing control measurements in the same patch. For concentration-response studies, normalized data from at least three patches from different oocytes were pooled and averaged for each EtOH concentration studied.

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Kinetic Simulations. MATLAB software (The Mathworks, Natick, MA) was used to both generate simulated nAChR currents by the Q-matrix method and to identify peak current amplitudes in the simulations.

	Results

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EtOH enhances T263I currents at high ACh. Oocyte membrane patches expressing nicotinic receptors containing the T263I mutation produced ACh-induced currents very similar to those of wild-type receptors. Upon exposure to submillisecond ACh concentration jumps, multichannel inward currents rapidly peaked and then desensitized monoexponentially (Fig. 1A, 1 mM ACh trace). As previously reported, T263I mutant nAChRs are characterized by an ACh EC₅₀ that is about 3-fold higher than that for wild type (56 ± 2 µM; Fig. 2), and maximal ACh-induced desensitization proceeds at a rate similar to that for wild-type receptors (Forman, 1997).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   EtOH enhances maximal currents in T263I mutant nAChRs. A, multichannel currents from an outside-out oocyte patch expressing T263I nAChRs were stimulated with 1 mM ACh (18 × EC50). Control currents rise rapidly to a peak and then desensitize monoexponentially with a time constant of 140 ± 13 ms. In the presence of 300 mM EtOH, peak current is enhanced by 29% and the desensitization time constant is reduced to 90 ± 8 ms. B, EtOH-dependent enhancement of T263I currents elicited with 1 mM ACh are shown. Each point represents the average ± S.D. of at least three measurements. The line through the data represents a logistic function: maximal enhancement = 29 ± 3.5%, half-effect EtOH concentration = 80 ± 21 mM, nH = 1.1 ± 0.37.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   EtOH effect on ACh concentration-response relationship for T263I nAChRs. Currents were elicited from outside-out oocyte patches expressing T263I nAChRs. Data points represent average (±S.D.) of at least three peak currents measured from separate patches and normalized to the peak current measured in the same patch at 1 mM ACh in the absence of EtOH. Lines represent logistic equations fitted to data by nonlinear least squares. No EtOH (): Max = 1.0 ± 0.01; EC50 = 56 ± 1.1 µM; nH = 1.6 ± 0.05. 300 mM EtOH (): Max = 1.27 ± 0.037; EC50 = 29 ± 2.5 µM; nH = 1.3 ± 0.15.

EtOH Shifts T263I ACh Response Leftward. The enhancing action of EtOH was quantified by determining the extent of EtOH-induced leftward shift in agonist response curves. In wild-type Torpedo and mouse nAChRs, 300 mM EtOH causes the ACh EC₅₀ to decrease about 2-fold (Forman et al., 1989; Zhou and Forman, submitted for publication.). We therefore measured ACh concentration responses in patches expressing T263I nAChRs both in the absence and presence of 300 mM EtOH.

EtOH Enhancement Shows No Voltage Dependence. ACh can act as a voltage-sensitive channel blocker (self-inhibition) as well as an agonist. In wild-type nAChRs, channel block is observed at ACh concentrations above 1 mM and at membrane potentials below 50 mV (Sine and Steinbach, 1984). If ACh is a potent blocker of T263I nAChRs, a possible mechanism to explain EtOH enhancement at high ACh is that EtOH weakens channel block by ACh (or other ions). We tested this hypothesis by determining whether EtOH enhancement of both wild-type and T263I mutant channels was dependent on the membrane holding potential.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   The voltage dependence of multichannel nAChR currents in the presence and absence of EtOH. A, multichannel currents elicited with 500 µM ACh () or 500 µM ACh + 300 mM EtOH () from a single oocyte patch expressing wild-type nAChRs. Some inward rectification is apparent in both data sets. Currents are enhanced 8% by EtOH at +100 mV. B, Multichannel currents elicited with 500 µM ACh () or 500 µM ACh + 300 mM EtOH () from a single oocyte patch expressing T263I nAChRs. Pronounced inward rectification is apparent in both data sets. Enhancement at negative holding voltages averages 42 ± 1.5% (n = 4 ± S.E.) and at positive holding voltages averages 40 ± 2.2% (n = 4 ± S.E.). Inset, currents recorded at +100 mV demonstrate 43% enhancement by 300 mM EtOH (solid line).

EtOH Reduces Single-Channel Conductance of T263I nAChRs. We examined whether enhancement of T263I multichannel currents was reflected in single-channel conductances by measuring single-channel conductances in excised inside-out patches using a low concentration of ACh (0.5 µM) in the pipette.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   EtOH reduces the single-channel conductance and increases mean open time of T263I nAChRs. Top panels show current recordings with ACh alone, whereas the lower panels show data recorded in the presence of ACh and 400 mM EtOH in the recording pipette. Left, current recordings from two separate excised inside-out patches with 500 nM ACh (top) or 500 nM ACh plus 400 mM EtOH (bottom). The lower opening frequency in the bottom panel is due to a smaller number of channels. Middle, all-points amplitude histograms of the current traces shown in the left panels. With ACh alone, the histogram peaks are separated by 7.0 pA. Combined data from four patches gave an average (±S.E.) conductance of 47 ± 1.0 pS. In the presence of 400 mM EtOH, the histogram peaks are separated by 5.9 pA (average = 39 ± 1.6 pS, n = 4). Right, open time histograms derived from traces shown in the left panels (10 s total data each). Double exponential fits are overlaid on the histogram data (solid lines). For ACh alone, the data were fit with time constants of 0.15 ± 0.096 ms (79% of openings) and 0.6 ± 0.12 ms (21% of openings). For ACh plus 400 mM EtOH, data were fit with time constants of 0.5 ± 0.34 ms (56% of openings) and 1.8 ± 0.33 ms (44% of openings).

EtOH Increases Apparent Single-Channel Lifetime of T263I nAChRs. In recordings where over 95% of opening events were single openings, open-time duration histograms revealed two distinct T263I channel open lifetimes. Most openings had fitted mean lifetimes below 0.2 ms. (Fig. 4, top right, see legend for details). In the presence of 400 mM EtOH, both short and long opening times increased and there was a shift in the distribution toward more long openings (Fig. 4, bottom right). These EtOH effects on channel lifetimes were consistently observed in a total of eight patches.

	Discussion

Top Summary Introduction Materials & Methods Results Discussion References

The major finding of this study is that EtOH enhances macroscopic currents from T263I mutant nAChRs at both low and high ACh concentrations. This result is in contrast to EtOH actions on wild-type nAChRs, where current enhancement is seen with low ACh, but only weak inhibition is apparent with high ACh concentrations. We investigated the T263I mutant's interactions with EtOH in detail to determine why EtOH affects it differently from wild-type nAChRs. Our analysis demonstrates that EtOH enhancement of nAChR function is due to an increase in the opening probability of agonist-bound receptors.

A number of different EtOH-associated changes in the nAChR gating mechanism could lead to enhancement of multichannel currents under different agonist conditions. A reaction scheme that incorporates the steps leading to channel opening, blocking, and desensitization is shown in Fig. 5. For simplicity, the two ACh binding sites are shown with equal microscopic affinities, because the arguments that follow would be equally valid for a model with distinct binding affinities. In wild-type mouse muscle nAChRs, microscopic rates for ACh binding to agonist sites (k_on) are diffusion limited, near 10⁸ M¹ s¹, whereas the rate for dissociation of ACh (k_off) is estimated to be >6000 s¹ (Lingle et al., 1992; Zhang et al., 1995). After binding of two ligands, opening () occurs at rates up to 60,000 s¹ (Zhang et al., 1995; Maconochie and Steinbach, 1998) and closing () at about 100 to 300 s¹ (Dilger et al., 1991; Zhang et al., 1995). At 21°C and 50 mV, the ACh open-channel blocking rate is about 5 × 10⁶ M¹ s¹ and the unblocking rate is about 3000 s¹ (Sine and Steinbach, 1984). Agonist binding and channel opening and blocking are all very fast compared with desensitization (k_d  10 s¹), so that peak current after submillisecond concentration jumps should reflect only these steps. At saturating (but not blocking) ACh concentrations, all receptors rapidly enter the A₂R state and the resulting maximal macroscopic current will be the unit current, i, times the number of channels times the open probability of doubly ACh-bound receptors p_open = /(+). Channel block by EtOH will also affect maximal currents by reducing the effective channel conductance.

View larger version (7K): [in this window] [in a new window]   Fig. 5.   Kinetic scheme for nicotinic receptor gating, desensitization, and block. Receptor states are depicted as resting (R), ligand bound (A2R), open (A2Ro), and desensitized (A2D). Bimolecular ACh (A) binding rates and unimolecular rate constants for other state transitions are given in the text.

Enhancement of multichannel nAChR currents could be associated with changes in agonist binding, single-channel opening probability, channel conductance, channel block, or desensitization kinetics. Our data directly rule out some of these possibilities. The use of rapid concentration jumps at excised membrane patches enables direct observation of enhancement in multichannel peak currents, which is clearly independent of desensitization (Fig. 1). With longer agonist applications, we can directly observe EtOH effects on the desensitization rate, k_d. Consistent with previous observations in studies of Torpedo nAChR (Forman et al., 1989; Wu and Miller, 1994), we find that EtOH accelerates nAChR desensitization in the presence of both low and high ACh concentrations. In slower current assays, this effect should reduce, not enhance, measured peak currents.

We also show that voltage-dependent agonist blockade of nAChR channels is negligible under the experimental conditions used for this study. I-V relationships for both wild-type and mutant nAChRs show inward rectification due to voltage-dependent channel closing rate (Auerbach et al., 1996), but enhancement of T263I currents by EtOH is equal at both positive and negative holding potentials (Fig. 3). In addition, our single-channel studies demonstrate that EtOH does not enhance single-channel nAChR conductance (Fig. 4), ruling this out as a possible mechanism.

The EtOH-induced leftward shift in agonist concentration responses could be caused by either enhanced agonist binding or by enhanced p_open. Because the p_open of ACh-bound wild-type nAChR is near 1.0, either of these actions would lead to an unchanged maximal current at saturating ACh. Partial agonists such as suberyldicholine, decamethonium, or nicotine bind to the ACh agonist sites on nAChR, but the probability of channel opening when these sites are occupied is low. EtOH and other short-chain alcohols enhance nAChR function at both low and high partial agonist occupancy (Wu and Miller, 1994; Tonner et al., 1992; Liu et al., 1994). These observations suggest that EtOH affects the probability of channel opening after ligands bind, but do not rule out a mechanism where EtOH affects affinity for the agonist site.

A critical problem with the partial agonists is that they are all potent channel blockers, and the overall maximal current observed with these agonists is a function of both channel opening and blockade. Enhancing ligand binding affinity at the agonist sites without affecting p_open or agonist channel blocking affinity of these compounds would increase maximal current (Tonner et al., 1992; Liu et al., 1994). In the absence of agonist channel block, as established for our results using ACh as the agonist, simply enhancing ligand binding affinity would result in a leftward shift in the concentration-response curve without changing maximal current. Because EtOH does not enhance single-channel conductance and the number of receptors in an excised patch is unlikely to change (especially on the rapid time scale of our agonist concentration jumps), the only mechanism that can account for the EtOH-induced increase in maximal T263I multichannel currents is an increase in single-channel p_open.

To test whether we could quantitatively account for both the increased maximal current and the decreased EC₅₀ observed in the presence of 300 mM EtOH, we simulated T263I nAChR currents based on a modified scheme (Fig. 5) without agonist block (Fig. 6). Kinetic parameters for the simulation were those given above for wild-type receptors, except that we set the channel closing rate,  = 8000 s¹ based on our open lifetime estimates (Fig. 4). We varied  and found that at  = 20,000 s¹ (/ = 2.5; Fig. 6 ), that the EC₅₀ of the simulated concentration-response data was 53 µM, close to the actual value for T263I receptors (Fig. 2). The maximal p_open in this simulation was 0.71. To simulate the effect of EtOH, we increased the / ratio by either increasing  or decreasing  (with similar results). At / = 10 (Fig. 6 ), the EC₅₀ of simulated data dropped to 25 µM and the maximal p_open increased to 0.91. Thus, a 4-fold increase in / caused a 2-fold decrease in EC₅₀ and a 28% increase in maximal p_open. The remarkable correlation between the simulated data and our measurements in the absence and presence of EtOH is demonstrated in Fig. 6 (right panel), where simulated concentration-response curves are plotted with renormalized electrophysiologic data from Fig. 2.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Simulation of nAChR currents shows that increasing / ratio reproduces EtOH actions. Left, the kinetic parameters shown in Fig. 5  and  were varied in Fig. 6 (modified to remove agonist block), which was used to generate simulated nAChR currents over a range of ACh concentrations. Maximal popen values (open symbols) were calculated as /(+) and EC50s (filled symbols) were derived from logistic fits (eq. 2) to the simulated peak current versus [ACh] relationship at each / ratio. The curves drawn in the right panel were used to generate the parameters plotted for / = 2.5 () and / = 10 (). Parameters from simulations of wild-type receptor kinetics (see text) are represented by squares (/ = 40 at 0 EtOH) and diamonds (/ = 160 at 300 mM EtOH). Right, peak currents from two simulations are drawn as concentration-response curves. The solid line represents a simulation of T263I receptor kinetics and was generated using kon = 108 M1 s1, koff = 6000 s1,  = 20,000 s1,  = 8000 s1, kd = 10 s1, and kd' = 1 s1, and was fitted with EC50 = 53 µM, popen(max) = 0.71, and nH = 1.4. The dashed line used the same parameters, except  = 2000 s1 and was fit with EC50 = 25 µM, popen(max) = 0.91, and nH = 1.5. Overlaid on the simulated curves are electrophysiological data from Fig. 2 that were renormalized by multiplying by 0.71, the T263I maximum popen derived from simulation.  represent normalized T263I peak currents measured in the absence of EtOH;  represent currents measured in the presence of 300 mM EtOH.

Our results confirm prior studies suggesting that EtOH acts on ligand-gated ion channels by stabilizing the open-channel state relative to the closed agonist-bound state (Aracava et al., 1991; Bradley et al., 1994; Wu and Miller, 1994; Zhou and Lovinger, 1998). Furthermore, our simulation of nAChR kinetics suggests that 300 mM EtOH increases / by about 4-fold. Indeed, the linear log-log relationship between predicted EC₅₀ and / seen in Fig. 6 (left panel) has a slope near 0.5, indicating that EC₅₀ depends on (/)^1/2. This result is also predicted by an approximate numeric solution for EC₅₀ based on Fig. 5 (see Appendix).

A direct implication of our observation that EtOH increases maximal currents in T263I receptors is that the microscopic p_open of ACh-bound T263I receptors must be significantly less than 1.0. The simulation shown in Fig. 6 suggests that p_open is near 0.7, but we can also estimate a value directly from our measurements. About 28% enhancement of maximal multichannel currents (at ACh 1 mM) in patches expressing T263I receptors was observed at the highest EtOH concentrations we studied. Thus, assuming p_open for ACh-bound T263I receptors in the presence of 300 to 700 mM EtOH is near 1.0, p_open in the absence of EtOH can be no more than 0.78 (1/1.28). Furthermore, the enhancing actions of EtOH overcome a modest EtOH-dependent reduction in T263I single-channel conductance (Fig. 4). If we correct for the 19% inhibition of single-channel conductance at 400 mM EtOH, p_open in the absence of EtOH is estimated to be at most 0.63 (0.81/1.28).

In effect, ACh is a partial agonist at T263I nAChRs, and our single-channel kinetic data suggest that the T263I mutation destabilizes the open-channel state relative to that of the wild-type receptor. Single-channel T263I currents recorded at low ACh show kinetic behavior consistent with this low opening probability estimate. Channel lifetimes for T263I receptors are at least 20-fold shorter than wild-type channels, demonstrating that the closing rate of T263I receptors is much higher than that of wild-type nAChRs. Although our concentration jumps are not fast enough to directly estimate opening rates, currents from rapidly perfused patches expressing T263I nAChRs rise in under 1 ms (10-90% rise times are 0.5 ms in Fig. 1A), demonstrating that ACh binding and channel-opening rates are not dramatically slower than those in wild-type nAChRs. Of note, a homologous  subunit mutation, S252I, does not confer a T263I phenotype to nAChRs. Both the ACh EC₅₀ and average channel lifetime for S252I nAChRs are near those of wild type, and EtOH does not increase maximal currents in patches expressing S252I receptors, indicating that p_open is near 1.0 (Forman, 1997; Zhou and Forman, submitted for publication).

Finally, at least part of EtOH's effect on p_open is due to a decrease in channel closing rate (), because apparent channel lifetimes were significantly longer in the presence of EtOH. This conclusion agrees with previous reports of single-channel kinetic analysis of EtOH effects in wild-type nAChRs (Aracava et al., 1991; Bradley et al., 1994).

We can closely simulate EtOH's effects on T263I receptors by increasing / in as shown in Fig. 6, and this mechanism can also account for EtOH effects in wild-type nAChRs. Let us assume that the effects of EtOH on the T263I activation mechanism are the same as those in wild-type receptors but adjust our model to incorporate the higher baseline p_open characterizing these channels. This situation is approximately represented by the simulated results in Fig. 6 (left panel) at a / ratio of 40 (, derived from a simulation with  = 20,000 s¹ and  = 500 s¹), giving p_open = /(+) = 0.976 and a fitted EC₅₀ = 12 µM. Assuming 300 mM EtOH causes a 4-fold increase in / to 160 ( and , derived from simulation with  = 20,000 s¹ and  = 125 s¹), Fig. 6 predicts that wild-type EC₅₀ will drop about 2-fold to 6.4 µM while p_open rises to 0.994. Again, the model closely approximates experimental observations (Forman et al., 1989; Wu et al., 1994). To generalize, our model predicts that EtOH will have an equivalent effect on the EC₅₀ derived from macroscopic current in these receptors, but EtOH's effect on currents at maximal agonist occupancy will depend on the p_open for the specific agonist/receptor pair as well as the degree of EtOH channel inhibition.

We confirmed this generalization in another mutant nAChR with a low p_open, Y198F. This mutation is in the agonist binding domain of nAChR (Tomaselli et al., 1991) and, like T263I, is characterized by a low p_open and high ACh EC₅₀. Indeed, in the presence of 300 mM EtOH, ACh concentration responses from patches expressing Y198F are shifted leftward (2-fold reduction in EC₅₀) and maximum currents are enhanced by about 35% (data not shown). As seen with both wild-type and T263I nAChRs, Y198F single-channel conductance is also inhibited by 19 ± 2.7% in the presence of 400 mM EtOH.

We conclude that the effects of EtOH on the gating kinetics of T263I and Y198F receptors are the same as those in wild-type receptors. EtOH shifts ACh-response curves leftward by the same degree (about 2-fold at 300 mM EtOH) in wild-type and mutant nAChRs. The 2-fold leftward shift can be quantitatively accounted for by a 4-fold increase in /, but our data do not rule out a small additional direct EtOH enhancement of agonist binding. EtOH's differential effects on mutant and wild-type receptor currents at saturating ACh concentrations are explained by the different microscopic opening probabilities in the absence of EtOH. The increased p_open is associated with slowed channel closing rates, indicating a stabilized open state, and others have suggested that EtOH may also increase opening rates (Bradley et al., 1994). High-resolution single-channel burst analysis may define the relative contributions of opening and closing rate changes to EtOH's enhancing action.