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1 Journal of Physiology (1992), 450, pp With 10 figures Printed in Great Britain PHARMACOLOGICAL AND KINETIC PROPERTIES OF m4f82 NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS EXPRESSED IN XENOPUS OOCYTES BY P. CHARNET*, C. LABARCA, B. N. COHEN, N. DAVIDSON, H. A. LESTER AND G. PILARt From the Division of Biology , California Institute of Technology, Pasadena, CA 91125, USA (Received 20 May 1991) SUMMARY 1. Co-injection of RNA synthesized from cloned neuronal acetylcholine receptor (nachr) subunits (a4 and fl2) in Xenopus oocytes produced functional receptors. In macroscopic voltage-clamp experiments, the agonist-induced current exhibited a strong inward rectification. 2. Voltage jumps from + 50 mv to more negative potentials produced relaxations of the agonist-induced current with a single exponential time course. The relaxation rate constant was only weakly voltage dependent. 3. At the single-channel level, three conductances were recorded of 12, 22 and 34 ps. Their burst durations were similar and varied only weakly with voltage (e-fold for 120 to 370 mv), consistent with the poorly voltage-dependent relaxation rate constants. However, the burst durations were less than 10 ms, or less than 1/5 the value expected from voltage-jump relaxations. 4. Hexamethonium (Hex, 0 5 to 8 /lm) inhibited the agonist-induced current and produced voltage-jump relaxations characterized by a rapid conductance increase and a slower conductance decrease. Analysis of these relaxations suggested that the Hex-receptor interaction is open-channel blockade characterized by a forward binding rate of 1 x 107 M-1 s-1 and a dissociation rate constant of about 25 s For the relaxations produced by QX222, the slowest phase was a conductance increase, suggesting that the dissociation rate constant for QX222 is fold greater than for Hex. 6. Hex but not QX222 produced an additional use-dependent blockade that was manifest during repetitive hyperpolarizing pulses. 7. With mouse muscle ACh receptors expressed in oocytes, the blockade by Hex did not depend strongly on voltage. Neither Hex nor QX222 produced appreciable use-dependent block on muscle ACh receptors. 8. Of the four conditions studied (neuronal and muscle receptors, Hex and * Present address: Centre de biochimie macromoleculaire, CNRS, Montpellier, France. t Present address: Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT 06268, USA. MS PHY 450

2 376 P. CHARNET AND OTHERS QX222), only the blockade of the neuronal AChR by Hex is characterized by a residence time longer than the normal open time. 9. It is concluded that the modest differences in primary amino acid sequence between muscle and neuronal receptors lead to profound changes in their interactions with channels blockers. INTRODUCTION The neuronal nicotinic acetylcholine receptor (nachr) is a member of the ligandgated channel superfamily, which includes the nachr from skeletal muscle, GABAA, glycine, kainate, N-methyl-D-aspartate (NMDA), serotonin 5HT3, invertebrate histamine, and perhaps other receptors (Betz, 1990). Recent advances in biochemistry, photoaffinity labelling, electron microscopy, molecular biology and electrophysiology have helped to elucidate the global quaternary structure of the nachr. While the nachr from muscle is a pentamer formed by four different subunits (a62/,y), the receptor expressed in the central and peripheral nervous system seemed to be comprised of only two different subunits; an a-like or agonist binding subunit and a fl-like or structural subunit. Several distinct a and ft subunits have been cloned from cdna libraries from PC12 cells, rat brain, and chicken brain and classified by their homology with the respective ac and f8 subunits from muscle nachr (Boulter, Connolly, Deneris, Goldman, Heinemann & Patrick, 1987; Schoepfer, Whiting, Esch, Blacher, Shimasaki & Lindstrom, 1988; Wada, Ballivet, Boulter, Connolly, Wada, Deneris, Swanson, Heinemann & Patrick, 1988; Deneris, Boulter, Swanson, Patrick & Heinemann, 1989; Papke, Boulter, Patrick & Heinemann, 1989; Couturier, Bertrand, Matter, Hernandez, Bertrand, Millar, Valera, Barkas & Ballivet, 1990). These subunits are localized in various areas of the nervous system to give rise to receptors of probable a2,f3 stoichiometry (Lindstrom, Schoepfer & Whiting, 1987; Cooper, Couturier & Ballivet, 1991), with distinct electrophysiological and pharmacological properties (Luetje & Patrick, 1991). Please note that we use subscripts (i.e. a2) to describe stoichiometry and non-subscripted suffixes (i.e. a2) to describe genetic isoforms of a subunit. Functional expression of receptor hetero-oligomers (cc2, a3, or cc4 with,82 or,f4) or of homo-oligomers (cc7) has been reported after injection of in vitro-synthesized mrna in Xenopus oocytes (Boulter et al. 1987; Ballivet, Nef, Couturier, Rungger & Bader, 1988; Wada et al. 1988; Deneris et al. 1989; Papke et al. 1989; Bertrand, Ballivet & Rungger, 1990; Couturier et al. 1990). Correlation between the type of subunit expressed and the electrophysiological and pharmacological properties of the nachr expressed throughout the nervous system can now be assessed by the use of the oocyte expression system. Papke et al. (1989) have recently shown that a2,f2, cc3fl2, and a4ft2 hybrids possess different elementary conductances and open-time kinetics. Whereas the complete three-dimensional structure at the atomic level must await X-ray analysis, preliminary identifications have already been made for the amino acids that line the pore of the channel and therefore play a role in permeation, gating and block by local anaesthetics (Imoto, Busch, Sakmann, Mishina, Konno, Nakai, Bujo, Mori, Fukuda & Numa, 1988; Leonard, Labarca, Charnet, Davidson & Lester, 1988; Revah, Galzi, Giraudat, Haumont, Lederer & Changeux, 1990; Charnet,

3 a4/32 RECEPTOR EXPRESSED IN OOCYTES Labarca, Leonard, Vogelaar, Czyzyk, Gouin, Davidson & Lester, 1990). One use of heterologous expression and site-directed mutagenesis has been the description of the binding sites for open-channel blockers at the muscle receptor (Leonard et al. 1988; Charnet et al. 1990). Several studies have exploited the open-channel blocker, QX222. This study begins to explore the same issues for the neuronal receptor. We considered it most appropriate to employ hexamethonium (Hex), a bis-quaternary ammonium compound that acts as an open-channel blocker on the nachr from different tissues (Ascher, Large & Rang, 1979; Gurney & Rang, 1984; Skok, 1990). The present study, and those of Bertrand et al. (1990), show that this pharmacological property is retained when the neuronal nachr is reconstituted in Xenopus oocytes. The oocyte expression system also enables the comparison of receptors or channels reconstituted from various sources in the same cell. As a result, individual channel properties can be related to the structural characteristics of the proteins, as distinguished from other proteins, lipids, or kinases in the cell of origin. We have therefore also examined the effects of Hex and of QX222 on the nachr from BC3H-1 cells expressed in oocytes. 377 METHODS cdnas used in this study were generously provided by J. Boulter, J. Patrick and S. Heinemann (Salk Institute). cdna transcription, oocyte preparation, mrna injection and electrophysiological recording were performed as previously described (Leonard et al. 1988; Charnet et al. 1990). Briefly mrna was synthesized in vitro using SP6 polymerase, and 50 nl of RNA mixture encoding a4 and fi2 (0-4 ng nl-l in de-ionized, diethyl pyrocarbonate-treated H20) were injected into Xenopus oocytes. Electrophysiological measurements were performed h later. Macroscopic measurements were conducted at 13 C (unless otherwise stated) in a bath solution containing 96 mm-nacl, 2 mm-kcl, 1 mm-mgcl2, and 5 mm-hepes; there were no added Ca2+ salts to avoid activation of the endogenous Cl- channels. Solutions also contained atropine (2 /SM) to avoid activating endogenous muscarinic ACh receptors. We utilized a two-electrode voltageclamp circuit (Axoclamp-2A, Axon Instruments, Foster City, CA, USA) under the control of pclamp programs (Axon Instruments). For voltage-jump relaxations, the membrane potential was stepped from a holding level of -30 mv to + 50 mv for 50 ms, followed by a step to a test potential between -150 and -50 mv. Passive and capacitive currents were eliminated by subtracting records in the absence of ACh. Time constants were fitted with least-square routines in the pclamp series. Single-channel recordings were performed at 13 C on both cell-attached and excised outside-out patches. Both the pipette and the bath contained 100 mm-kcl, 2 mm-mgcl2, 10 mm-hepes, and 10 mm-egta (ph 7-5); thus the oocyte resting potential was near 0 mv. Data were recorded with a Dagan (Minneapolis, MN, USA) 8900 amplifier with a headstage resistor of 10 GQ. Data were sampled continuously at 22 khz by a pulse-code modulator and stored on videotape. Records were played back through an 8-pole Bessel filter, digitized by a Labmaster interface (Scientific Solutions, Solon, OH, USA), and analysed using FETCHAN and pstat in the pclamp series. Single-channel conductances were measured using amplitude histograms obtained at potentials between -190 and mv. Single-channel open times were measured from the time constant of exponential decay fit to distributions of channel openings. Acetylcholine chloride and hexamethonium chloride were obtained from Sigma. QX222 was a gift from Dr B. Takmann (Astra Pharmaceuticals, Worcester, MA, USA). 13-2

4 378 P. CHARNET AND OTHERS RESULTS Expression of ac4/i2 nachr Figure 1A presents a typical ACh response of an oocyte injected with 50 ng of x4f2 mrna mixture. When the membrane potential was clamped at -80 mv, the application of ACh (1,/SM) induced an inward current that developed over several seconds (corresponding to the time for solution changes in our chamber) and A ACh mv 50 ni 2 20 s --.j b i B +50 mv Fig. 1. Macroscopic responses to ACh. A, current recorded from a representative oocyte injected with 50 ng of a4f2 RNA during application of 1 Sm-ACh at a holding potential of -80 mv (20 C). B, relaxations of the ACh-induced current during a series of voltage jumps from a holding level of + 50 mv to more negative levels (-90, -110, and -150 mv). Top: voltage-step protocol; bottom: current traces (13 C). displayed no desensitization over several minutes. At higher ACh concentration (> 5 #sm) or more prolonged application (> 5 min), desensitization was sometimes observed. This rather limited desensitization seems to be a property of this particular nachr, because more pronounced desensitization was evident with other neuronal nachr hybrids such as a2,b2 (G. Pilar, P. Charnet, C. Labarca & H. A. Lester, unpublished observations). The amplitude of the response to 0-5,sM-ACh at mv (227 ± 27 na, mean + S.E.M., n = 15), although much larger than a similar response recorded with a2fl2 or a3fl2 (Papke et al. 1989; G. Pilar, P. Charnet, C. Labarca & H. A. Lester, unpublished observations) was still lower than that recorded

5 a4fi2 RECEPTOR EXPRESSED IN OOCYTES under identical conditions after injection of equal quantities of muscle nachr mrnas ( na, n = 25). The limited desensitization with the x4fl2 hetero-oligomer allowed us to determine the electrophysiological and pharmacological properties of the channel under quasi- 379 A Voltage (mv) Na+ (100 mm) - - Na+ (50 mm) <i: c cl- 4) a-) B 0 I[ACh] (MM) < C-S O c Voltage (mv) Fig. 2. Current-voltage relations for macroscopic ACh-induced currents. A, tracings of ramp-clamp currents for ACh (1 pm). Membrane potential was ramped from -120 to + 40 mv at 600 mv s-1. B, data for several ACh concentrations from a cell tested with voltage-jump relaxations as in Fig. lb.

6 380 P. CHARNET AND OTHERS equilibrium conditions. In a typical voltage-jump relaxation experiment on the muscle nachr, the membrane potential is held briefly at a positive potential (+ 50 mv in the experiment of Fig. IB), then jumped to a more negative potential (Adams, 1975; Neher & Sakmann, 1975; Sheridan & Lester, 1975). The result is a relaxation to a new, larger population of open channels. For the muscle receptor, this relaxation occurs because the channel lifetime increases at more negative potentials; but the channel opening rate remains nearly constant, so that the equilibrium activation increases at more negative potentials. In the present experiments, when hyperpolarizing voltage steps were generated under continuous perfusion of ACh (1 ftm), the current also relaxed to a new, larger steady-state level with a single exponential time course (Fig. 1B); see also Ifune & Steinbach (1990). These relaxations have been widely used in the past to provide valuable information about the kinetics of the receptor gating and block by non-competitive inhibitors (Adams, 1977; Ascher et al. 1979; reviewed by Adams, 1981). These relaxations were recorded for several different final voltages; and the time course as well as the steady-state current at the end of pulses similar to those shown in Fig. 1B were measured for different ACh concentrations. At all ACh concentrations the equilibrium agonist-induced current displayed inward rectification. In the experiment of Fig. 2A, a voltage ramp was generated at 600 mv s-' from to + 40 mv. The recorded inward current at 100 mm-external NaCl (continuous line) was substantial at potentials between and -60 mv but much smaller between -20 and + 40 mv. Rectification was still present with external K+, Cs+, or Li+ (not shown) and was also observed at all ACh concentrations tested (Fig. 2B). The rectification vitiated accurate measurements of reversal potentials, but there was a clear negative shift of - 20 mv in the position of rectification when the bathing solution was changed to 50 mm-external NaCl (Fig. 2A, dashed line, NaCl replaced by mannitol), with or without external Mg2+ (5 mm). The instantaneous current-voltage relation was measured in two ways. (i) For measurements at negative potentials, the voltage was held at + 50 mv, then jumped to more negative values as in the experiment of Fig. lb (Lester, Changeux & Sheridan, 1975). The instantaneous current-voltage relation, measured just after the jump, rectified much less strongly than the steady-state current-voltage relation (data not shown). (ii) For measurements at positive potentials, the potential was held at mv, then jumped to a positive potential. In this case the instantaneous I-V relation was markedly non-linear with only small outward currents. Similar results have been obtained and analysed more completely for neuronal acetylcholine receptors by Ifune & Steinbach (1990) and by Mathie, Colquhoun & Cull-Candy (1990). The non-linear I-V relation could arise from at least three factors: (i) briefer channel durations at positive potentials; (ii) non-linear single-channel currentvoltage relations; and (iii) an additional, unexplained source of rectification. The hyperpolarizing voltage-jump relaxations were studied in detail. The relaxations can be fitted adequately by a single exponential decay (Fig. 3B). The relation between the rate constant, 1/r, of the relaxation and the value of the final voltage at different ACh concentrations (0'2, 1, 2, and 5 /SM) is illustrated in Fig. 3 C. The rate constant is only slightly affected by voltage, increasing only by 20% over the detectable voltage range from -50 to mv; this would correspond to an e-fold change for mv.

7 ac4/32 RECEPTOR EXPRESSED IN OOCYTES Voltage-jump relaxations are conveniently analysed in terms of a two-state model for receptor gating: closed = open. (1) Both of the transitions involve several molecular steps, including the binding/dissociation of two agonist molecules and possible conformational changes. The 381 A C 0-1 [ 1000 R 800 C c = hr : :0 ~~ ~~~~i- n [AChl (um) Voltage (mv) D B ms 0.04 V Nmo 1/r [ACh] (,um) 5 6 Fig. 3. Dose-response relations and voltage-jump relaxations. A, currents in a representative cell at the end of a 150 ms pulse to mv, during superfusion of 0-2, 0-5, 2 and 5 /LM-ACh. B, current relaxation recorded during a voltage step from + 50 to mv. A single exponential fit has been superimposed on the record. C, relation between the relaxation rate constant and the final voltage for various ACh concentrations. Note the log scale on the Y-axis. Dashed lines represent linear regression for each concentration and represent e-fold change for 330, 300, 254 and 289 mv for 0-2 #M (seven cells), 1,UM (thirty cells), 2 /tm (eleven cells), and 5 itm (eighteen cells) respectively. D, relaxation rate constant as a function of the ACh concentration for the data in C. sign of the rather weak voltage dependence of IIr in the present experiments is opposite to that for the endplate receptor, recorded both in muscle and in the oocyte expression system (Adams, 1981; Charnet et al. 1990). This observation can be

8 382 P. CHARNET AND OTHERS A B " r I.t C.16. -A-1. A..Ljh 7 "Wlmmr" V., 10.0-o-A 0+16A *4" D ALAJII Current (pa) Lh- i. k. Al 10 A 41 II A,.1 Amup ok-i I IN IL "FlIrrr-lll mr L2 pa 20 ms Current (pa) Fig. 4. Single-channel data. A, traces obtained from an oocyte injected with a4fl2 RNA. Membrane potential, -150 mv. Two different conductance classes can be distinguished. B, all-points amplitude distribution of the trace in A. The largest peak (close to 0 pa) represents the baseline. Amplitudes of the two other classes are -41 and 2 1 pa. C, - same conditions as in A but on a different oocyte. D, amplitude histogram of trace ion C; the two classes had amplitudes of -6 and pa. interpreted by noting that the relaxation rate constant 1/-r is the sum, a + /3'; and the fractional activation is /3'/(x+/)'. The voltage-jump relaxation data therefore suggest that both these quantities increase at more negative potentials, as though

9 ac4/32 RECEPTOR EXPRESSED IN OOCYTES 383 the dominant effect of hyperpolarization is to increase /J'. This conclusion differs from the case for muscle receptors, where the dominant effect of hyperpolarization is to decrease a. Complete dose-response relations were not studied for the a4/32 receptor, but several facts indicate that our measurements were performed in a region where a High Intermediate Low 30 4Ic 1.4 ms 2.6ms 4l1ms c _ 10 -) Fig Time (ms) Open-time distributions of single openings for high (A), intermediate (B) and low (C) conductance openings. [ACh] = 1,UM, -150 mv. substantial fraction of the receptors were activated. (i) The responses increased with increasing [ACh] but less rapidly than with [ACh]2 (Fig. 3A). (ii) As [ACh] was increased, /I increased as well (Fig. 3 C and D). The concentration-dependent part of /I is usually interpreted as /1'; thus /3' varied from about ms-1 at 0-2,UM- ACh to ms-' at 5,tM-ACh. The zero-concentration value for 1/r is usually interpreted as the channel closing rate constant a; values were ms-1. If these - interpretations are correct, the fractional activation /I'/((x +,/') therefore varied from as low as 0-17 to as high as in our experiments. Single-channel recordings The aim of the single-channel measurements was to provide an interpretation of the relaxation measurements. Like previous investigators, we have found that a single hybrid receptor combination gives rise to single-channel recordings with a spectrum of elementary conductances (Papke et al. 1989; Kullberg, Owens, Camacho, Mandel & Brehm, 1990). Cell-attached and outside-out patch clamp recordings were made on oocytes that had been injected with a4fl2. In the outside-out configuration, superfusion of ACh induced channel openings for a few minutes; the openings subsequently disappeared. This behaviour has also been observed for other neuronal nachrs expressed in oocytes (Ballivet et al. 1988). We therefore used the outside-out configuration for conductance determination only, and the more stable cell-attached patches were further analysed for kinetic measurements. Typical cell-attached recordings are displayed in Fig. 4A and C, in the presence of 1 /sm-ach. In the twenty-five patches studied, we observed ACh-activated openings with three distinct conductance levels. When pooled together, these conductances can be classified as low (12 ps), intermediate (22 ps), and high (34 ps). Low and intermediate conductance classes

10 384 P. CHARNET AND OTHERS are present in traces displayed in Fig. 4A, and high-conductance and intermediate states are represented in Fig. 4C. Figure 4B and D present the all-points amplitude histogram of the patches for parts A and C. In our recordings, the intermediate conductance state was the most prominent, occurring in 56 % of the patches. Seven of the twenty-five patches displayed more than one type of channel amplitude; five of these displayed the intermediate and low levels. TABLE 1. Single channel properties of neuronal nachr expressed in oocytes High Intermediate Low Conductance (ps) Frequency (%) Mean open time (ms) 1P Voltage dependence (mv for e-fold) Mean burst duration (ms) Voltage dependence (mv for e-fold) Total of twenty-five patches, 28 % with more than one type. All mean open times and mean burst durations are given at mv. Typical open-time distributions for one patch from each conductance class are shown in Fig. 5. We analysed these distributions by fitting a single exponential. The low- and intermediate-conductance classes are characterized by similar mean open times. Burst analysis was conducted by grouping all openings with a gap of < 10 ms (the mean burst duration varied little when the gap duration was varied between 1 and 40 ms). The results for all three types of opening are summarized in Table 1. The burst durations follow the same pattern as the mean open times, i.e. slightly briefer for the high-conductance openings than for the low or intermediate class. On average, bursts consisted of one to two individual openings. Closed-time duration histograms had at least two time constants; one was less than 2 ms and the other greater than 100 ms. The mean open time and the mean burst duration both decreased at more positive potentials for all three classes of opening. This voltage dependence is smaller than usually observed for the muscle receptor, but it has the same direction. On the usual assumption that the rate of channel opening has little or no voltage dependence, the voltage-dependent burst duration predicts the direction of the voltage-jump relaxations in the experiment of Fig. 1B. Because the open time depends less strongly on voltage than for the nicotinic receptor, one expects the voltage-jump relaxations to display an amplitude, relative to the instantaneous component, that is less than the values typically observed for muscle receptors (Sheridan & Lester, 1975, 1977; Adams, 1975, 1977; Neher & Sakmann, 1975). This expectation is borne out; for instance, at mv, the relaxation amplitude is roughly 30 and 60% of the instantaneous ohmic value at -90 and mv, respectively. There is an obvious discrepancy between the results of the voltage-jump relaxation experiments and the single-channel experiments. The former give time constants of the order of 50 ms that decrease at more negative potentials; in the latter, burst durations are < 10 ms at the same temperature and increase at more negative potentials. Further treatment of these points is reserved for the Discussion.

11 ~~~~~~~~~~~~~~~~~~~~Z o a4/32 RECEPTOR EXPRESSED IN OOCYTES 385 Voltage-jump relaxations with hexamethonium When an oocyte injected with a4,82 RNA was superfused with ACh (2 gm) plus Hex (8 fem), the agonist-induced currents decreased relative to the current in the absence of Hex (Fig. 6A). The inhibition was voltage dependent, amounting to 56% at -60 mv and 75 % at -100 mv. A B 200 pa 50 ms Voltage (mv) -150 mv_ -15 mv i ~~~~~~I I J ) [Hex] =8,m -110 mv -500 < ControlC -70 mv _ * Fig. 6. Inhibition of ACh-induced currents by Hex (8 FM). A, voltage-jump relaxations to -150, -110 and -70 mv. After superfusion of hexamethonium the relaxation is smaller and faster. B, current-voltage relationship before and after application of hexamethonium. The voltage was ramped from -120 to + 80 mv at 600 mv s-'. [ACh] = 1 /M. In the presence of Hex and ACh, voltage-jump relaxations were more complex than the simple exponential time course found in the presence of ACh alone (Fig. 6A). There were two distinct exponential components of opposite sign. The faster component was an increase in conductance (rf in Fig. 7); the slower component (with time constant r8) was a much smaller decrease. Relaxations of this sort have been studied previously for acetylcholine receptors in muscle, nerve, and Aplysia neurons (reviewed by Adams, 1981) and are interpreted in terms of open-channel blockade: if G[Hex] closed = open = open (Hex), (2) a F where the rate of blocker-receptor equilibration, O[Hex] +1F, is less than the rate of channel gating, a +f/'. 0 and F are the forward and backward rate constants for channel blocking (Neher & Steinbach, 1978). Figure 7 summarizes the result of analysing such relaxations for [ACh] = 1 /IM (our most reliable data) and with [Hex] ranging from 0'5 to 10 4uM. Data were averaged over voltage jumps to -110, -130 and -150 mv. -Although the data show a substantial amount of scatter, there is a clear increase in l/rt with increasing [Hex]. According to the standard relaxation theory, the sum of the two relaxation rate

12 386 P. CHARNET AND OTHERS constants is just the sum of all the first-order rate constants; and if l/it is several times greater than 1/1T, one has the approximation: I/Tfz 6x+ +) F+ G[Hex]. (3) For a linear fit of 1/rf with [Hex] according to eqn (3), the slope gives the forward binding rate constant G = 101 x 107 M-1 s-k. Taking the rate constant of the voltage Fa Slowt 150 Control E 100o < [Hex] (OM) Fig. 7. The inverse of the fast relaxation time constant in the presence of Hex is plotted against Hex concentration. Data for 0 5 #M (four cells), 1 LM (four cells), 5/uM (three cells), 6 Mm (three cells), and 10 #M (two cells). The data have been averaged over voltage steps to -150, -130 and -110 mv. Inset: relaxation recorded for a voltage step from + 50 to mv in the presence of ACh (control trace is monoexponential), and during perfusion of Hex (top trace, biexponential, I/Tr and 1/Xr8 are the fast and slow time constants). Total X 107 M-1 S-1. time is 50 ms. [ACh] = 1,M. The superimposed line is drawn according to eqn (4), with a =26s-1, f=3-4 s-, F = 20 s-, G = 1.01 jump relaxations in the absence of Hex (Fig. 3D) at [ACh] = 1,UM, one obtains o + fi' = 26 s-1. Then the intercept at [Hex] = 0 yields F = 9 s-1. The dissociation constant FIG would then be 0 9 #M. The exact relaxation rate constants 1/rf and l/lr are given by quadratic expressions (Adams, 1977) as follows: l/tf,s = [(a+/3' +F+ G[Hex]) ±{(x+fi' +F+ G[Hex])2-4(/3'G[Hex] + fl'f + azf)}i]/2. (4) This prediction for 1/-f (corresponding to the minus sign in eqn (4)) is plotted on the data of Fig. 7; considering the scatter, it accounts for the data about as well as eqn (3). The estimate for F from eqn (4) is about 4 s-1, yielding F/G = 0-4 /,M. The component associated with l/'r was too small for further study. Systematic efforts were not made to analyse the kinetics of Hex blockade over a wide voltage range, but the available kinetic data agree with the equilibrium data in suggesting that the Hex blockade is quite sensitive to membrane potential. We found a 2- to 3-fold increase in G over the range from -110 to mv and a roughly 1-5 to 2-fold decrease in F over the same range. Thus the data suggest that Hex does bind within the membrane field, but the available precision does not allow an estimate of the fraction of the field sensed by the blocker.

13 cc4/,2 RECEPTOR EXPRESSED IN OOCYTES 387 Use-dependent blockade With Hex at the ox4f32 receptor, we found an additional, slow component of block that was manifested most clearly as a cumulative decrease during repetitive voltagejump relaxations to large negative potentials (Fig. 8). Such block may also be termed A 100 na 40 ms B Pulse number Fig. 8. Use-dependent blockade by Hex. A, currents recorded during repetitive voltagejump relaxations from 0 to mv at a frequency of 2 Hz. The 1st, 10th and 25th episodes are displayed. B, time course of the use-dependent block induced by Hex in the experiment of A. [ACh] = 1 /,M, [Hex] = 8 um. 13 'C. 'use-dependent'. In the absence of Hex, there was little decrement (< 5 %) over such a series. We studied use-dependent block as a function of membrane potential with [ACh] = 5 /tm, [Hex] = 8 /tm, but taking the ratio of the 10th to the 1st agonistinduced current. At mv, this ratio was 0-89; at mv, It is thus clear that the use-dependent blockade is voltage dependent, increasing at more negative potentials. Blockade of a4,82 receptor by QX222 We performed voltage-jump relaxation analyses of the blockade of the a4fl2 receptor by QX222 (Fig. 9). Relaxations were prolonged, so that 1/TQ, in the presence of QX222 and ACh, was less than 1/'r in the presence of ACh alone. This result

14 388 P. CHARNET AND OTHERS indicates open-channel blockade with equilibration rates that are faster than the time scale of channel gating. An intuitive explanation for this effect is that the channel burst duration is prolonged as the blockers enter the channel and induce a flickering between the open and blocked states (Leonard et al. 1988; Charnet et al. A [QX2221= 40,uM 300 na 100 ms Control B 1000 j Voltage (mv) Fig. 9. Effects of QX222. A, voltage-jump relaxation from + 50 to -150 mv in the presence of ACh alone (1 #M, Control) and ACh plus QX222 (40 am). Note the slowing of the relaxation. B, voltage dependence of the dissociation constant on semilog scale, calculated from eqn (5). Average of four cells. 1990). These results generally resemble those found for the interaction between QX222 and the mouse muscle AChR. However, it is also clear that QX222 blocks the a4fl2 receptor much more weakly than the mouse muscle receptor. Thus 20 /M, the concentration usually tested with muscle ACh receptors, produced no detectable change in the voltage-jump relaxations. When the waveforms at higher [QX222] were analysed with the relation (Adams, 1977): 1/TQ = (1/r)(1 + [QX]/KQ), (5) we found that KQ = 141,UM at mv, vs. 18 JtM for muscle receptors at the same potential. The weak binding prevented a quantitative study of the voltage dependence, for we felt that our data would be vitiated by the possibility that the channel could close upon the bound QX222 molecule (Neher, 1983). Nevertheless

15 a4c/2 RECEPTOR EXPRESSED IN OOCYTES 389 it was clear that the blockade was highly voltage-dependent, KQ increasing to > 500,M at -110 mv. There were no indications of use-dependent blockade with Q X222 as the blocking drug. Comparison with muscle receptors The nachr from BC3H- 1 cells was expressed in oocytes for the purpose of comparing the blockade by Hex with that by QX222 (Fig. loa and B). Experiments A B [Hex] = 8,u[m22J =20/I Control Control 100 ms C 200 o QX o Hexamethonium Voltage (mv) Fig. 10. Effects of Hex and QX222 on mouse muscle nicotinic AChR expressed in oocytes. Voltage-jump relaxations from +50 to -150 mv; [ACh] = 1 UM. A, Hex (8/,M); B, QX222 (20 /im). Both blockers induced a slowing of the relaxation. Hex induced an additional blockade of the steady-state currents. C, semilog plot of the dissociation constants for Hex and QX222 at different voltages, calculated from eqn (5). Data for Hex (+±S.E.M.) are averaged from three cells; data for QX222 are averaged from fifteen cells, and S.E.M.s are smaller than the size of the symbols. with QX222 have been reported more fully elsewhere (Leonard et al. 1988; Charnet et al. 1990) and are included here for purposes of comparison. Both blockers induced a slowing of the voltage-jump relaxations when voltage steps were applied, indicating a rapidly equilibrating blockade. However, there were important differences in these actions. (i) The slowing by Hex (8,UM) amounted to a decrease of 10 s-' in I1r at all voltages in the range between -70 and mv. This change

16 390 P. CHARNET AND OTHERS in relaxation kinetics was less pronounced than that produced by QX222, despite a much greater steady-state blockade by Hex at the same [ACh]. In the presence of QX222 (20 /LM) at mv, the steady-state blockade was always < 18%, while superfusion of Hex at a lower concentration (8 /tm) induced a steady-state block of % (n = 3) and more than 50% at 20 /tm. (ii) The blockade of equilibrium current by Hex (8 ftm) increased to 62 % at -70 mv; the blockade thus showed the opposite voltage dependence to that for Hex at the a4fl2 receptor or for QX222 at either receptor. (iii) When the apparent dissociation constants for the two drugs were determined according to eqn (5) for voltages from to -90 mv (Fig. 10C), it was found that the KHeX decreases only slightly at more negative potentials, while KQ changes e-fold for 40 mv, respectively). From data obtained with four different - oocytes at -90 mv. KHeX was 30 UM. This is - 4-fold less than the value for QX222 at the same potential (Fig. 10C). These data are considered further in the Discussion. There were no indications of use-dependent blockade with either Hex or QX222 at muscle receptors expressed in oocytes. DISCUSSION Various studies have shown that nachr from ganglionic sources possesses a different pharmacology to that of the muscle receptor. This work extends these observations to the neuronal AChR reconstituted in oocytes. Our results and those of Bertrand et al. (1990) demonstrate that it possesses the typical properties of a neuronal ACh receptor, including inward rectification (Selyanko, Derkach & Skok, 1979; Mathie et al. 1990; Ifune & Steinbach, 1990) and a weak voltage dependence of both open time and burst duration (Mathie et al. 1990; Ifune & Steinbach, 1990). Our measurements showed roughly 4-fold less agonist-induced current from the a4,b2 hybrid receptor than from the mouse muscle receptor after injection of similar quantities of RNA and testing under similar conditions. Many factors could contribute to this smaller response. However, the difference can also simply be accounted for by the known 3-fold differences in burst duration and a roughly 1P5- fold difference between the major conductance class of neuronal receptors (22 ps) and that of muscle receptors under similar circumstances; see for instance Leonard et al. (1988). It remains to be seen whether an equivalent number of receptors appears on the oocyte surface and whether channels open with a similar dependence on agonist concentration. The present partial dose-response relations, the more complete measurements of Bertrand et al. (1990), and the partial measurements of Yoshii, Yu, Mixter-Mayne, Davidson & Lester (1987) suggest that the o4/32 neuronal receptor has a higher sensitivity to ACh than the muscle receptor. Comparison of single-channel and macroscopic kinetics The single-channel recordings suggest burst durations of 6-8 ms, but the voltagejump relaxations at lowest [ACh] yield time constants of 50 ms. Both of these values are usually interpreted as 1/a. This puzzling discrepancy does not occur with mouse muscle receptors expressed in oocytes (P. Charnet, R. J. Leonard and H. A. Lester, unpublished observations). The voltage-jump experiments were performed

17 a4f2 RECEPTOR EXPRESSED IN OOCYTES with extracellular solutions containing primarily Na+, and the single-channel experiments were performed with symmetrical high-k+ solutions. Another possible source for the difference is that in the single-channel experiments the entire oocyte membrane is held near 0 mv for prolonged periods. Also, in some cases patch formation distorts gating kinetics (Morris & Horn, 1991). Further experiments should be performed to test whether these differences account for the discrepancies. In most experiments on nicotinic receptors, voltage-jump relaxations are produced by the voltage dependence of channel closing and have a rate constant that decreases with hyperpolarization. It should be pointed out that this situation probably does not hold in the present study. Thus, further studies are warranted on the voltage dependence of a4,82 receptors. Comparison with earlier studies In a study on the a4,82 subunits expressed in Xenopus oocytes at room temperature, Papke et al. (1989) reported only two conductance levels of 13 and 7 ps. These conductances are smaller than our measurements at 12 'C. The solutions of Papke et al. (1989) contained Ca2+; ours contained no Ca2+ and 10 mm-egta. Calcium blockade of the single-channel currents might thus explain the lower conductances measured by Papke et al. (1989). Ballivet et al. (1988), in a study on chick a4fl2 receptors expressed in oocytes, found a major conductance of 20 ps in solutions containing 1-8 mm-ca2+ at 'C. Hex blockade of muscle receptors The actions of Hex on muscle receptors are not completely understood (Rang & Rylett, 1984). The blockade by Hex was not strongly voltage dependent at muscle receptors. One interpretation of these data is that Hex is blocking primarily closed states of the muscle receptor. For example, Hex might compete with ACh at the agonist binding site (Rang & Ritter, 1971; Rang & Rylett, 1984). Such a mechanism would account for the decreased inhibition with hyperpolarization, because the effective binding constant of the agonist increases. Furthermore, rapid competitive inhibition would be accompanied by a modest slowing of the voltage-jump relaxations because of a decrease in /3' (Sheridan & Lester, 1977; Krouse, Lester, Wassermann & Erlanger, 1985). An additional possibility is that Hex is a permeant blocker at the muscle AChR. One might therefore expect that high negative potentials would relieve the blockade by increasing the rate of dissociation to the interior of the cell. A final possibility is that Hex can be trapped in the pore and produce a closedblocked state; the premature termination of a burst would limit the lengthening of the relaxation at high negative potentials and therefore give an artifactually high apparent dissociation constant for Hex binding. These hypotheses should be tested with single-channel recording. Contrasts among non-competitive blocker kinetics In the present study, blockade of ACh receptors has been studied with two distinct types of receptor (neuronal and muscle) and with two distinct blockers (Hex and QX222). Of these four conditions, three are characterized primarily by non- 391

18 392 P. CHARNET AND OTHERS competitive blockade (Hex on muscle receptors might act primarily via competitive blockade at the agonist binding site). Three of the four conditions are also characterized by blocker residence times less than the channel burst duration. In only one case - Hex at the neuronal receptor - does the blocker remain bound for a time ( ms) comparable to or longer than the burst duration. This fact is deduced from the distinctive voltage-jump relaxation waveforms observed in this case (Ascher et al. 1979). On the other hand, the forward rate constant for Hex binding to the neuronal receptor is about 107 M-1 s-'. A similar value has been obtained (i) from observations of Hex effects on postsynaptic currents in rat submandibular cells (Rang, 1982), and (ii) for the binding of QX222 to the muscle receptor (Neher & Steinbach, 1978; Leonard et al. 1988; Charnet et al. 1990). The long residence time might also account for the use-dependent blockade which is also unique to the action of Hex on neuronal AChR in our study. Concrete suggestions for the mechanism of use-dependent blockade include the following: (i) the channel can close directly on the bound blocker (reviewed by Lester, 1992); (ii) the receptor can undergo a transition into a desensitized state (Neher, 1983). In either case, there seems to be a transition to a further blocked state (to the right of the open (Hex) state in eqn (2)). The transitions in question are quite rare, as shown by the following calculation. We estimate that the channels are open with an average probability of 0'6 at 5 jtm-ach (see Results). In the experiment of Fig. 8, 160 ms pulses were given at a rate of 1 s-1, so that after 25 s the average channel has been open for a time equal to 0-6 x 0-16 s x 25 = 2-5 s. The use-dependent block eventually closed - 3of the channels during this time; assuming that a channel remains in this further blocked state until the voltage gradient is reversed (Bertrand et al. 1990), the average channel would therefore undergo the transition with a time constant of 2-5 x 2-5 s - 6 s. The average residence time for Hex on the receptor was calculated as 1/F = ms. Therefore it appears that one in every thirty to sixty Hexchannel bindings results in a transition to a further blocked state. Conclusions These experiments thus provide a first description of the kinetic basis of the interaction between Hex and neuronal nicotinic receptors expressed in Xenopus oocytes. Considering the very similar amino acid sequences between neuronal and muscle receptors in the M2 region that is thought to line the ion channel pore, it is surprising that Hex and QX222 display such marked differences in their blocking characteristics for interactions with these two classes of receptors. Future goals should include (i) extending the blockade measurements to the single-channel level and (ii) employing site-directed mutagenesis in an effort to study the particular amino acid residues responsible for the blocker-receptor interaction, as has recently been attempted for the muscle receptor (Leonard et al. 1988; Charnet et al. 1990). We thank J. Boulter, J. Patrick and S. Heinemann for the cdna clones. This work was supported by grants from the National Institutes of Health (NS and NS-10338), by the California Tobacco-Related Diseases Program (RT 365), by the Fondation pour la Recherche Medicale, and by the Muscular Dystrophy Association.

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