THE NEURONAL NICOTINIC acetylcholine receptor
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1 /04/ $03.00/0 ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH Vol. 28, No. 5 May 2004 Single-Channel Analyses of Ethanol Modulation of Neuronal Nicotinic Acetylcholine Receptors Yi Zuo, Keiichi Nagata, Jay Z. Yeh, and Toshio Narahashi Background: We have previously reported that ethanol potentiates the acetylcholine-induced currents of the 4 2 neuronal nicotinic acetylcholine receptors in rat cortical neurons and of those that are stably expressed in human embryonic kidney cells. The potentiation of the maximal currents evoked by high concentrations of acetylcholine suggests that ethanol affects the channel gating. Methods: We performed single-channel patch-clamp experiments to elucidate the detailed mechanism of ethanol modulation of the 4 2 receptor that is stably expressed in human embryonic kidney cells. Results: At least two conductance states, 40.5 ps and 21.9 ps, were activated by acetylcholine. Acetylcholine at 30 nm predominantly induced the high conductance state currents (85% of total). Ethanol did not affect the single-channel conductance but selectively modulated the high-conductance state currents. The high-conductance state currents exhibited two open time constants. Both time constants were increased by 100 mm ethanol, from 1.9 msec to 2.8 msec and from 9.0 msec to 15.5 msec, respectively. Ethanol also prolonged the burst duration and the open time within burst and increased the probability of channel opening. Conclusions: These changes in single-channel parameters indicate that ethanol stabilizes the 4 2 receptor-channel in the opening state, explaining how the maximum acetylcholine-induced whole-cell currents are further potentiated by ethanol. Key Words: Alcohol, Acetylcholine Receptor, Single Channel, 4 2 Receptor, Ethanol. THE NEURONAL NICOTINIC acetylcholine receptor (nachr) is known to be the target site of various chemicals, including nicotine, carbachol, d-tubocurarine, general anesthetics, some insecticides, heavy metals, and several natural toxins (Albuquerque et al., 1989, 1995; Castro and Albuquerque, 1993; Ishihara et al., 1995; Mori et al., 2001; Nagata et al., 1996, 1997, 1998; Swanson and Albuquerque, 1992). Neuronal nachrs play a vital role in synaptic transmission in the central nervous system as they modulate the release of various neurotransmitters (Alkondon et al., 1997, 2000; Barazangi and Role, 2001; Kaiser and Wonnacott, 2000; McGehee and Role, 1995, 1996; Radcliffe and Dani, 1998; Wonnacott, 1997). Ethanol acts on a variety of neuroreceptors and channels, including voltage-gated calcium channels, N-methyl-D-aspartate receptors, GABA A receptors, glycine receptors, and 5-HT 3 receptors (Crews et al., 1996; Lovinger, 1997, 1999; Mihic, From the Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois. Received for publication August 14m 2003; accepted February 3, This work was supported by a grant from the National Institutes of Health AA Reprint requests: Toshio Narahashi, PhD, Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL 60611; Fax: ; narahashi@northwestern.edu. Copyright 2004 by the Research Society on Alcoholism. DOI: /01.ALC A 1999; Mullikin-Kilpatrick and Treistman, 1994; Walter and Messing, 1999; Woodward, 1999). Muscle-type nachrs are also modified by ethanol (Bradley et al., 1980; Forman and Zhou, 1999; Forman et al., 1989; Gage et al., 1975; Linder et al., 1984; Wood et al., 1991; Wu et al., 1994). At the single-channel level, ethanol increases channel open time and decreases channel conductance in muscle nachrs (Aracava et al., 1991; Forman and Zhou, 1999, 2000). We have previously found that the 4 2 neuronal nachrs are sensitive to ethanol modulation, being potentiated over a wide range of ACh concentrations tested (Aistrup et al., 1999; Zuo et al., 2001). Even the maximal currents evoked by high concentrations of ACh are further potentiated by ethanol. However, the underlying mechanism remains to be seen. The potentiation could be due to an increase in channel conductance, an increase in channel open time, or more channels bound by ACh being opened in the presence of ethanol. On the basis of kinetic simulations, we have proposed that ethanol stabilizes channel open state either by an increase in the channel opening rate constant and/or by a decrease in the closing rate constant (Zuo et al., 2001). We now report the results of single-channel patch-clamp experiments that unveiled more detailed mechanisms of ethanol modulation of the 4 2 nachrs that are stably expressed in human embryonic kidney (HEK) cells. At least two conductance state currents were generated by 688 Alcohol Clin Exp Res, Vol 28, No 5, 2004: pp
2 ALCOHOL ON SINGLE NICOTINIC RECEPTOR CHANNELS 689 ACh, and ethanol had little or no effect on the channel conductances. Ethanol increased the channel open probability by increasing both the open time within bursts and the burst duration, indicating that ethanol stabilized the open state of the neuronal nachr by a decrease in the closing rate constant. MATERIALS AND METHODS Cell Preparation HEK293 cell line stably expressing the human 4 2 AChR subunits was obtained from SIBIA Neurosciences (La Jolla, CA; now Merck Research Laboratories, San Diego, CA). Cells were cultured in Dulbecco s modified Eagle s medium supplemented with 2 mm L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin (Gibco BRL, Rockville, MD), 6% iron-supplemented calf serum (Sigma, St. Louis, MO), and 100 g/ml G418 (Mediatech, Herndon, VA) at 37 C inanair CO 2 (93 7%, by volume). For patch-clamp experiments, cells were plated on glass coverslips coated with poly-l-lysine and cultured for 1 to 5 days. Solutions for Electrophysiological Recording The external solution contained (in mm) 150 NaCl, 5 KCl, 2.5 CaCl 2, 1 MgCl 2, 10 glucose, 5.5 HEPES acid, 4.5 and Na-HEPES. The ph was adjusted to 7.3 with HCl, and osmolarity was adjusted to 310 mosm with D-glucose. The internal pipette solution contained (in mm) 140 K gluconate, 2 MgCl 2, 1 CaCl 2, 11 EGTA, 10 HEPES acid, 2 Mg 2 ATP, and Na GTP 0.2. The ph was adjusted to 7.3 with KOH, and osmolarity was adjusted to 300 mosm with D-glucose. The junction potential between the internal and external solutions was 12.5 mv, which was corrected before each experiment. Electrophysiological Recording Recording patch pipettes were pulled from 1.5 mm (outer diameter) borosilicate glass capillary tubes with filament (G150F-4; Warner Instrument Corp, Hamden, CT). The electrodes were coated with a semiconductor protective coating material R-6101 (Dow Corning Corporation, Midland, MI) and fire-polished. The resistance of recording electrodes was ~20 M when filled with internal solution. Single-channel currents were recorded using the outside-out configuration of the patch-clamp technique at room temperature (20 25 C). Currents that persisted for 10 to 20 min were used for analysis. The signals were recorded using an Axopatch 200 amplifier (Axon Instruments, Union City, CA) filtered at 5 or 10 khz and stored in a microcomputer via analog to digital converter (Digidata 1200; Axon Instruments). The Fetchex module of the Pclamp6 or Pclamp8 software (Axon Instruments) using gap-free recording modes was used to record the signals. Drug Application Test solutions were applied to the membrane patch by a modified U tube system (Marszalec and Narahashi, 1993) that had a solution exchange rise time (10 90%) of 10 to 15 msec as detected by changes in junction potential. ACh (Sigma) was first dissolved in distilled water to make the stock solution and further diluted with the external solution to make test solutions shortly before experiments. The ethanol used in the present study was absolute ethyl alcohol USP (Pharmco Products, Brookfield, CT). Dihydro- -erythroidine HBr (DH E) was purchased from Research Biochemicals International (Natick, MA). The same patch was tested with ACh and ACh plus ethanol. Data Analysis A cutoff frequency of 2 khz was used for data analyses. Opening and closing of the channels were detected using the 50% threshold criterion (Colquhoun and Sigworth, 1995). Only the events that lasted 400 sec were analyzed. On average, 500 to 1000 events were obtained from a patch. Histograms of the current amplitudes were fitted by a sum of Gaussian functions using the least-square method. The interburst interval was determined by the method of Colquhoun and Sakmann (1985). Histograms of the open times, closed times, and burst durations were fitted by the pstat program of the pclamp software. These results were subsequently compiled for graphical analysis by SigmaPlot 5.0 (SPSS Science, Chicago, IL). Data are expressed as the mean SD or the mean SEM, and n represents the number of patches unless otherwise specified. A Mantel nonparametric test was used to evaluate the effects of 100 mm ethanol on the mean open time and the observed open probability. RESULTS Single-Channel Currents Induced by ACh in 4 2 nachrs Fig. 1A shows a current trace induced by 1 M ACh in an outside-out membrane patch isolated from a HEK cell expressing the 4 2 AChR subunit combination. In the presence of ACh, multiple openings were initially observed, which were followed by single isolated openings. It was extremely difficult to record the activity from one ion channel by increasing the resistance of patch electrode, probably because of high-density expression of AChRs on the cell membrane. We used lower concentrations of ACh to decrease the occurrence of overlapping single-channel currents and chose the nonoverlapping currents to do singlechannel analysis. When 30 nm ACh was applied to the membrane patch clamped at 70 mv, single-channel currents were observed occurring either as brief isolated openings or as longer openings interrupted by a short closure or gaps (Figs. 1Ba and 3Aa). Two conductance state currents, 1.5 and 2.6 pa, were frequently observed (Figs. 1Ba and 3Aa). A rare lower conductance state current ( 1 pa) appeared when high concentrations of ACh ( 3 M) were applied (data not shown). Because this conductance state current was rarely observed, we analyzed only the two major conductance state currents in the present study. In six patches tested, co-application of 100 nm DH E completely blocked the currents (Fig. 1Bb). Currents recovered within 10 sec after washout with normal external solution (Fig. 1Bc). These results indicate that single-channel currents are mediated by neuronal nachrs. Two Conductance Components Fig. 2A shows typical examples of 30 nm ACh-induced single-channel currents at several holding potentials. Little or no current was induced at positive potentials for most of the patches that we tested (12 of 14 patches). Fig. 2B shows the current voltage (I-V) relationships for two conductance state currents. The slope conductances were calculated from the linear relationship between currents and voltage. The calculated values of 40.5 ps and 21.9 ps represent high and low conductances, respectively.
3 690 ZUO ET AL. Fig. 1. Single-channel currents recorded from an outside-out membrane patch isolated from a HEK cell in which 4 2 human neuronal nicotinic AChRs are expressed. (A) Multiple openings are followed by single isolated openings as induced by a high concentration of ACh (1 M). Part of the current record (from 2700 msec to 3000 msec) is enlarged below. ACh application started at time 0. (B) Current records were taken from one membrane patch. (Ba) Two conductance state currents are observed during application of 30 nm ACh. Arrows indicate high (H) and low (L) conductance openings. C represents the close state. (Bb) Coapplication of 100 nm DH E with 30 nm ACh completely blocks currents. (Bc) Currents recover after 10-sec washout with normal external solution. ACh and DH E were applied through a U-tube system, and the outside-out patch is held at 70 mv. Inward Rectification It has been shown that ACh-induced whole-cell currents in 4 2 AChRs were inwardly rectified (Buisson et al., 1996; Haghighi and Cooper, 1998, 2000). Fig. 2C shows the I-V relationship for 3 M ACh-induced whole-cell current using a ramp protocol. Few or no currents were induced at positive potentials. The lack of single-channel currents at positive potentials (Fig. 2A) provides an explanation for inward rectification of the whole-cell current. Effects of Ethanol on ACh-Induced Single-Channel Currents Conductances. In the presence of ethanol, single-channel currents occurred either as brief isolated openings or as longer openings interrupted by short closures or gaps (Fig. 3Ba) and exhibited two major conductances. The amplitudes of these two conductance state currents were 2.6 and 1.5 pa when clamped at 70 mv in the control (Fig. 3Ab). Coapplication of 100 mm ethanol with 30 nm ACh had little or no effect on these two conductance states. Open Time. The open time distributions for AChinduced high-conductance state currents in the absence and the presence of 100 mm ethanol are shown in Fig. 4. Using one version of displaying data of the Sigworth and Sine method (Sigworth and Sine, 1987), the number of events in each bin is plotted on a linear ordinate and the time axis is drawn on a logarithmic scale so that the effective bin width increases exponentially from left to right. This displays a multiexponential distribution as a series of skewed bellshaped curves whose peaks overlie the time constants of several exponential components (Sigworth and Sine, 1987). The open time distribution for the high-conductance state currents induced by 30 nm ACh and by co-application of 30 nm ACh and 100 mm ethanol clearly indicated multiexponential components (Fig. 4Aa and 4Ba). There were at least two components. The fast component of the open time distribution for 30 nm ACh-induced currents
4 ALCOHOL ON SINGLE NICOTINIC RECEPTOR CHANNELS 691 Fig. 2. The inward rectification of AChinduced currents in 4 2 AChRs. (A) Singlecell currents induced by 30 nm ACh at different holding potentials from one membrane patch. (B) Current voltage relationships for two conductance states. Two conductances were estimated to be 21.9 ps and 40.5 ps. Error bars show the SDs of the Gaussian distribution. Number of patches: 4 for 100 mv, 10 for 70 mv, and 5 for 40 mv. (C) Current voltage relationship for 3 M AChevoked whole-cell currents. had a time constant of 1.9 msec (61.5%), and the slow component had a time constant of 9.0 msec (38.5%; Fig. 4Aa). Ethanol of 100 mm prolonged the fast time constant to 2.8 msec (70.8%) and the slow time constant to 15.5 msec (29.2%) (Fig. 4Ba). The mean open times were significantly prolonged from msec to msec (n 5; p 0.05). The open time distribution for the low-conductance state currents induced by 30 nm ACh and by co-application of 30 nm ACh and 100 mm ethanol also has two components (Fig. 4Ab and 4Bb). The fast component of the open time distribution for 30 nm ACh-induced currents had a time constant of 2.0 msec (76.7%), and the slow component had a time constant of 21.0 msec (23.3%; Fig. 4Ab). Ethanol at 100 mm did not change the open time distribution of the low-conductance state currents: the fast component of the open time distribution had a time constant of 2.1 msec (68.9%), and the slow component had a time constant of 21.0 msec (31.1%; Fig. 4Bb). Closed Time. The distribution of the closed time had at least three components in the presence and absence of 100 mm ethanol (Fig. 5). The fast component of the closed Fig. 3. Single-channel currents induced by application of ACh and co-application of ACh and ethanol in 4 2 AChRs expressed in HEK293 cells. Outside-out patches were held at 70 mv. (A) Current traces (a) and amplitude histogram (b) of single-channel currents induced by 30 nm ACh. Two conductance states are observed with amplitudes of 2.6 pa (85.0% of a total of 855 events, five separate patches combined) and 1.5 pa (15.0%). (B) Current traces (a) and amplitude histogram (b) of single-channel currents observed during co-application of 30 nm ACh and 100 mm ethanol: 2.6 pa (80.4% of a total of 669 events, five separate patches combined) and 1.5 pa (19.6%).
5 692 ZUO ET AL. Fig. 4. Open time distribution for two conductance state currents induced by ACh and co-application of ACh and ethanol. Outside-out patches were held at 70 mv. The distributions are displayed on a logarithmic time axis, and the best fits of exponential functions are shown. (A) Currents induced by 30 nm ACh. (Aa) The time constants for high conductance state currents are estimated to be 1.9 msec (61.5% of a total of 760 events, five separate patches combined) and 9.0 msec (38.5%). (Ab) The time constants for low-conductance state currents are estimated to be 2.0 msec (76.7% of a total of 95 events, five separate patches combined) and 21.0 msec (23.3%). (B) Currents induced by coapplication of 30 nm ACh and 100 mm ethanol. (Ba) The time constants for high-conductance state currents are estimated to be 2.8 msec (70.8% of a total of 532 events, five separate patches combined) and 15.5 msec (29.2%). (Bb) The time constants for low-conductance state currents are estimated to be 2.0 msec (68.9% of a total of 137 events, five separate patches combined) and 21.0 msec (31.1%). time distribution for 30 nm ACh-induced currents had a time constant of 4.3 msec (29.9%), the middle component had a time constant of 88.3 msec (39.4%), and the slow component had a time constant of 690 msec (30.6%; Fig. 5A). In the presence of 100 mm ethanol, the fast time constant of 3.1 msec (29.6%), the middle time constant of 190 msec (34.7%), and the slow time constant of 664 msec (35.7%) were obtained (Fig. 5B). Ethanol at 100 mm shortened the time constant of the fast component of the closed time distribution. Because we do not know how many channels are in the patch, the changes in the middle and slow closed time constants are less revealing. Burst Duration. The distributions of burst duration for currents induced by 30 nm ACh and by co-application of 30 nm ACh and 100 mm ethanol clearly indicated multiexponential components (Fig. 6Aa and 6Ba). When a burst was defined as a sequence of openings separated by closed periods shorter than 15 msec, there were two components of distribution of the burst duration. The fast component of the burst duration for 30 nm ACh-induced currents had a time constant of 1.6 msec (45.6%), and the slow component had a time constant of 15.8 msec (54.4%; Fig. 6Aa). Ethanol of 100 mm prolonged the fast time constant to 3.3 msec (68.0%) and the slow time constant to 34.6 msec (32.0%; Fig. 6Ba). Openings Within Burst. We further analyzed the open time distribution within burst (Fig. 6Ab and 6Bb). Two components were used to fit the open time distribution within burst. The fast component of distribution of the open time within burst for the control had a time constant of 2.0 msec (66.0%), and the slow component had a time constant of 9.4 msec (34.0%; Fig. 6Ab). The presence of 100 mm ethanol increased both time constants to 3.2 msec (74.4%) and to 18.9 msec (25.6%), respectively (Fig. 6Bb). Closings Within Burst. Fig. 6Ac and 6Bc shows the closed time distribution within burst in the absence and presence Fig. 5. Closed time distributions for currents induced by ACh and co-application of ACh and ethanol. Outside-out patches were held at 70 mv. The distributions are displayed on a logarithmic time axis, and the best fits of exponential functions are shown. (A) Currents induced by 30 nm ACh. The time constants are estimated to be 4.3 msec (29.9% of a total of 839 events, five separate patches combined), 88.3 msec (39.4%), and 690 msec (30.6%). (B) Currents induced by co-application of 30 nm ACh and 100 mm ethanol. The time constants are estimated to be 3.1 msec (29.6% of a total of 611 events, five separate patches combined), 190 msec (34.7%), and 664 msec (35.7%).
6 ALCOHOL ON SINGLE NICOTINIC RECEPTOR CHANNELS 693 Fig. 6. Burst analysis in the absence and presence of ethanol. Outside-out patches were held at 70 mv. The distributions are displayed on a logarithmic time axis, and the best fits of exponential functions are shown. (A) currents induced by 30 nm ACh. (Aa) Burst duration. The time constants for distribution of burst durations are estimated to be 1.6 msec (45.6% of a total of 562 events, five separate patches combined) and 15.8 msec (54.4%). (Ab) Open time within burst. The time constants for distribution of open time within bursts are estimated to be 2.0 msec (66.0% of a total of 838 events, five separate patches combined) and 9.4 msec (34.0%). (Ac) Closed time within burst. The time constants for distribution of closed time within bursts are estimated to be 0.7 msec (0.8% of a total of 276 events, five separate patches combined) and 6.0 msec (99.2%). (B) Currents induced by co-application of 30 nm ACh and 100 mm ethanol. (Ba) Burst duration. The time constants for distribution of burst durations are estimated to be 3.3 msec (68.0% of a total of 420 events, five separate patches combined) and 34.6 msec (32.0%). (Bb) Open time within burst. The time constants for distribution of open time within bursts are estimated to be 3.2 msec (74.4% of a total of 618 events, five separate patches combined) and 18.9 msec (25.6%). (Bc) Closed time within burst. The time constants for distribution of closed time within bursts are estimated to be 0.7 msec (18.3% of a total of 190 events, five separate patches combined) and 4.6 msec (81.7%). of 100 mm ethanol, respectively. Two components were used to fit the closing time distribution within burst for both the single-channel currents induced by 30 nm ACh alone and those induced by co-application of 30 nm ACh and 100 mm ethanol. The fast component of distribution of the control closed time had a time constant of 0.7 msec (0.8%), and the slow component had a time constant of 6.0 msec (99.2%; Fig. 6Ac). In the presence of 100 mm ethanol, the fast time constant of 0.7 msec (18.3%) and the slow time constant of 4.6 msec (81.7%) were obtained (Fig. 6Bc). Thus, 100 mm ethanol increased the proportion of short close states within the burst without changing the time constants. Open Probability. The mean open probability (P open )of AChRs was increased by a co-application of 30 nm ACh and 100 mm ethanol. P open is defined as the sum of the open times for all channels in a patch divided by the recording time. Figure 7 shows the analyses of the sum of 5 patches. Without ethanol, the P open was (mean SEM, n 637; Fig. 7A). With co-application of Fig. 7. Open probability in the absence and presence of ethanol. Outside-out patches were held at 70 mv. (A) Currents induced by 30 nm ACh. The mean open probability is (mean SEM, a total of 637 events, combined five separate patches). (B) Currents induced by co-application of 30 nm ACh and 100 mm ethanol. The mean open probability is (mean SEM, a total of 692 events, combined five separate patches).
7 694 ZUO ET AL. 100 mm ethanol, the P open was (mean SEM, n 692), representing a 26% increase in comparison with the control (Fig. 7B). Thus, 100 mm ethanol increased the open probability of AChRs. When five patches were individually analyzed, 100 mm ethanol increased P open to (mean SEM) from the control P open of (mean SEM), representing a 20% increase, which is significant at p 0.05 (one-tailed) according to the Mantel nonparametric test. DISCUSSION We have previously characterized the ethanol action on the 4 2 nachrs in detail at the whole-cell level (Aistrup et al., 1999; Zuo et al., 2001). In our simulation, ethanol was postulated to enhance the channel open probability by an increase in the channel opening rate constant and/or by a decrease in the closing rate (Zuo et al., 2001). In the present single-channel study, two levels of single-channel conductance state currents were observed in the 4 2 AChRs that were stably expressed in HEK293 cells. Ethanol was found to change the several single-channel parameters, some of which led to an increase in channel open probability. Ethanol modulation of nachrs is expected to cause profound effects on brain function via various neurotransmitter systems. nachrs are known to be located at presynaptic and preterminal sites as well as postsynaptic membranes in the brain, and those located at presynaptic/ preterminal sites, when activated, cause release of various transmitters, including GABA, dopamine, glutamate, Ach, and norepinephrine (Alkondon et al., 1997, 1999, 2000; Colquhoun and Patrick, 1997; Lindstrom, 1997; Role and Berg, 1996; Wonnacott, 1997). Thus, the potentiation of the AChR activity by ethanol could evoke a cascade of synaptic events involving the GABAergic and dopaminergic systems, which are known to play an important role in ethanol-induced behavioral effects. In the present study, the ethanol concentration of 100 mm was chosen on the basis of our previous whole-cell data. In rat cortical neurons in primary culture, ethanol potentiated 4 2-type currents induced by 3 M ACh (~EC 50 ) in a concentration-dependent manner with 10, 30, 100, and 300 mm ethanol potentiating the currents 14, 27, 56, and 141% of the control, respectively (Aistrup et al., 1999). In HEK cells expressing the 4 2 nachrs, ethanol was less efficacious than in cortical neuron nachrs, with 100 and 300 mm ethanol potentiating the currents induced by 3 mm ACh in 30 and 60% of the control, respectively (Zuo et al., 2001). Thus, ethanol at 100 mm, which produces a moderate degree of current potentiation, was selected for single-channel analyses. Two Conductance Components of 4 2 AChRs The single-channel currents induced by ACh showed at least two amplitude levels in 4 2 AChRs stably expressed in HEK293 cells. The two conductances are estimated to be 40.5 ps and 21.9 ps. Buisson and Bertrand (2001) reported that there were two types of conductance levels in 4 2 AChRs expressed in HEK293 cells with the conductances 38.8 ps and 43.4 ps (high conductance type) and 16.7 ps and 23.5 ps (low conductance type). Several other studies reported multiple conductance levels of currents in neuronal nicotinic AChRs (Bormann and Matthaei, 1983; Weaver and Chiappinelli, 1996). There are two possible explanations for generation of the two conductance state currents. First, the two conductance state currents may arise from two receptor subtypes; different receptor stoichiometries could be expressed when 4 and 2 cdna are co-transfected into the HEK293 cell line. This notion is consistent with the whole-cell ACh concentration-response relationship, which is better fitted by a sum of two Hill equations with high and low receptor affinities for ACh than one Hill equation (Buisson and Bertrand, 2001). A second possible explanation is that two open states with different conductances could arise from a single AChR subtype. It has been known that other nachrs exhibit multiple conductance open states (Hamill and Sakmann, 1981; Morris and Montpetit, 1986; Morris et al., 1983, 1989; Nagata et al., 1998). A single mutation at the partial agonist recognition site in the 7 AChR changed a partial agonist to a full agonist at the whole-cell level (Matsuda et al., 2000, 2001). The partial agonist nitromethylene heterocycle imidacloprid induced subconductance levels of current almost exclusively while the full agonist ACh induced mostly main conductance state currents (Nagata et al., 1996, 1998). Thus, it seems that a single type of AChR can exhibit multiple conductance open states. However, it remains to be seen which explanation will be applicable to the present study. Inward Rectification We observed strong inward rectification in the whole-cell current and no measurable outward single-channel currents at depolarized potentials for most of the outside-out patches of 4 2 AChRs expressed in HEK cells. This is in contrast with earlier reports using either Xenopus oocytes or HEK cells (Buisson et al., 1996; Haghighi and Cooper, 1998, 2000). Haghighi and Cooper (1998, 2000) suggested that spermine and ubiquitous intracellular polyamine interacted with the negatively charged glutamic acid residues at the intermediate ring of AChRs and affected both the rectification and Ca 2 permeability. It is possible that the outside-out configuration of membrane patch resulted in a change in the degree of rectification as a result of modification of intracellular components and/or channel protein itself. Ethanol Stabilizes the Open State of the Receptor Relationship With Whole-Cell Data. Maximum AChinduced currents, even when evoked by high concentrations
8 ALCOHOL ON SINGLE NICOTINIC RECEPTOR CHANNELS 695 of ACh, were augmented by ethanol in both 4 2-type AChRs in rat cortical neurons and the human 4 2 AChRs stably expressed in HEK cells (Aistrup et al., 1999; Zuo et al., 2001). Thus, a question arises as to how ethanol enhances the maximal responses evoked by ACh. This enhancement could not be due to a simple increase in ACh affinity for its binding site, because such an increase in the ACh affinity would not increase the maximal response. It has to be due either to an increase in the single-channel conductance or to more receptors staying in the open state, and the latter could arise from an increase in channel opening rate constant and/or a decrease in the closing rate (Zuo et al., 2001). These hypotheses were tested at the single-channel level in the present study. No change in single-channel conductance was observed by 100 mm ethanol (Fig. 3). However, P open was significantly increased by ethanol. The average P open from the sum of five patches was increased from to (Fig. 7). This increase in P open could be caused by an increase in the mean open time and/or by a decrease in the mean closed time. There was indeed an increase in the mean open time at the high conductance level. The two components of mean open time were increased from 1.9 msec and 9.0 msec to 2.8 msec and 15.5 msec, respectively (Fig. 5Aa and 5Ba). Little or no change in the open time distribution was observed for the low-conductance openings (Fig. 4Ab and 4Bb). It should be noted that the highconductance channels amounted to 80 to 85% of the total population (Fig. 3Ab and 3Bb). In addition, ethanol almost doubled both the burst duration (1.6 msec and 15.8 msec in control; 3.3 msec and 34.6 msec with ethanol) and the mean opening time within burst (2.0 msec and 9.4 msec in control; 3.2 msec and 18.9 msec with ethanol; Fig. 6Aa, 6Ab, 6Ba, and 6Bb). In contrast, there was little or no change in the mean closed time within bursts (Fig. 6Ac and 6Bc). This suggested that ethanol increased the mean opening time for each single opening without affecting the number of openings within the burst. Thus, we concluded that ethanol stabilizes the opening state of the receptor by decreasing the closing rate. Comparison With Muscle Data Numerous works on alcohol have been performed on muscle-type nachrs. Ethanol has been demonstrated to potentiate ACh response (Bradley et al., 1980; Forman and Zhou, 1999; Forman et al., 1989, Gage et al., 1975; Linder et al., 1984; Wood et al., 1991; Wu et al., 1994). Wu et al. (1994) suggested that the major effect of ethanol was to increase the open/closed equilibrium of receptors without changing the receptor affinity for agonist in Torpedo nachrs. Ethanol increased ACh-induced macroscopic currents at both low and high ACh concentrations in a mutant T263I nachrs, which was explained by an increase in P open (Forman and Zhou, 1999). At the single-channel level, 50 to 300 mm ethanol was found to increase the mean open time (Forman and Zhou, 1999). These studies generally agree with the present study in that ethanol increases P open, likely as a result of the stabilization of the opening state of receptor. ACKNOWLEDGMENTS HEK cell lines stably expressing the 4 2 AChR subunits were provided by SIBIA Neurosciences, (La Jolla, CA; now Merck Research Laboratories, San Diego, CA). The authors thank Brian O Neil Claeps of Merck Research Laboratories San Diego for technical assistance with 4 2 cell line culture and Julia Irizarry for secretarial assistance. REFERENCES Aistrup GL, Marszalec W, Narahashi T (1999) Ethanol modulation of nicotinic acetylcholine receptor currents in cultured cortical neurons. 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