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J. Biol. Chem., Vol. 278, Issue 45, 44033-44040, November 7, 2003

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The Effects of 3 Subunit Incorporation on the Pharmacology and Single Channel Properties of Oocyte-expressed Human 34 Neuronal Nicotinic Receptors^*

James P. Boorman, Marco Beato, Paul J. Groot-Kormelink, Steven D. Broadbent, and Lucia G. Sivilotti¶

From the Department of Pharmacology, The School of Pharmacy, 29/39 Brunswick Square, London, WC1N 1AX United Kingdom

Received for publication, November 18, 2002 , and in revised form, July 24, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We compared the main properties of human recombinant 343 neuronal nicotinic receptors with those of 34 receptors, expressed in Xenopus oocytes. 3 incorporation decreased the channel mean open time (from 5.61 to 1.14 ms, after approximate correction for missed gaps) and burst length. There was also an increase in single channel slope conductance from 28.8 picosiemens (34) to 46.7 picosiemens (343; in low divalent external solution). On the other hand, the calcium permeability (determined by a reversal potential method in chloride-depleted oocytes) and the pharmacological properties of 3-containing receptors differed little from those of 34. The main pharmacological difference in 343 "triplet" receptors was a 3-fold decrease in the potency of lobeline relative to acetylcholine. Nevertheless, there was no change in the rank order of potency for agonists (epibatidine >> lobeline > cytisine, 1,1-dimethyl-4-phenylpiperazinium iodide, nicotine > acetylcholine > carbachol for both receptors; measured at low agonist concentrations). Sensitivity to the competitive antagonists trimetaphan (0.2–1 µM) and dihydro--erythroidine (30 µM) was similar for the two combinations, with a Schild K_B for trimetaphan of 76 and 66 nM on 34 and 343, respectively. The change in single channel conductance confirms that 3 replaces a 4 subunit in the pentamer. The absence of pronounced differences in the pharmacological profile of the triplet receptor argues against a role for the 3 subunit in the formation of agonist binding sites, whereas the changes in channel kinetics suggest an important effect on receptor gating. The shortening of the burst length of 3-containing receptors implies that any synaptic currents mediated by such channels would have faster decay kinetics.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Among neuronal nicotinic subunits, 3 was long considered an "orphan" as it does not form functional recombinant receptors if expressed as a classical heteromeric combination, i.e. together with either an or a subunit. Because of the sequence similarity between 3 and 5 we tested the hypothesis that, just like 5, 3 would form functional receptors only if co-expressed together with both an and a subunit. By a reporter mutation strategy, we showed that 3 is indeed incorporated into functional recombinant 343 receptors (1). In 343 receptors, 3 is present as a single copy, which replaces one of the subunits (versus two copies each for the 3 and 4 subunits (2)); note that 3 coassembles with other subunits in rat native cerebellar nAChRs¹ (3).

The question now arises of whether the 3 subunit can change the properties of neuronal nicotinic receptors. There are several reasons for investigating this problem. First of all it is important to establish whether any such change introduced by the presence of 3 affects the role that these receptors may have in physiological processes in the central nervous system or in the pharmacology of tobacco addiction. Furthermore, if 3-containing receptors have distinctive biophysical or pharmacological properties, such receptors can in principle be recognized in native tissue by functional assays. Finally, clear changes in the receptor pharmacology and particularly in the binding affinity of competitive antagonists would be a strong indication that the binding sites of the receptor have changed and that 3 directly forms one of the two interface binding sites.

The shorter openings and bursts together with the increased single-channel conductance observed with the co-expression of 3 confirm that 3 is incorporated into the receptor complex. The single-channel conductance change and the stoichiometry of the receptor (2) suggest that 3 takes the place of a classical subunit. The calcium permeability of the receptor and its sensitivity to a range of agonists and antagonists were unchanged apart from a decrease in the relative potency of the agonist lobeline. These results suggest that 3 either does not participate in the formation of the agonist binding site or that the sequences of 3 and 4 in the relevant domains are too similar to allow them to be differentiated.

The extent and nature of changes introduced into the nAChR by the presence of 3 were not sufficient to provide tools for the identification of such receptors in native tissue. Nevertheless, the shorter burst length of these receptors implies a faster decay of any synaptic current that these channels may mediate and suggests a functional role for the 3 subunit in the diverse family of neuronal nicotinic receptor subunits.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Expression of Nicotinic Subunits cRNA in the Xenopus oocyte—cDNAs for the human 3, 3, and 4 (GenBank^TM accession numbers Y08418 [GenBank] , Y08417 [GenBank] , and Y08416 [GenBank] , respectively) containing only coding sequences and an added Kozak consensus sequence (GCCACC) immediately upstream of the start codon (4) were subcloned into the pSP64GL vector, which contains 5'- and 3'-untranslated Xenopus -globin regions (5). All cDNA/pSP64GL plasmids were linearized immediately downstream of the 3'-untranslated -globin sequence, and cRNA was transcribed using the SP6 Mmessage Mmachine Kit (Ambion). The quality and quantity of cRNAs were checked by gel electrophoresis and comparison with RNA concentration and size markers.

Female Xenopus laevis frogs were anesthetized by immersion in neutralized ethyl m-aminobenzoate solution (tricaine, methane sulfonate salt; 0.2% solution weight/volume) and killed by decapitation and destruction of the brain and spinal cord (in accordance to Home Office guidelines) before removal of the ovarian lobes. Clumps of stage V-VI oocytes were dissected in a sterile modified Barth's solution with a composition of 88 mM NaCl, 1 mM KCl, 0.82 mM MgCl₂, 0.77 mM CaCl₂, 2.4 mM NaHCO₃, 15 mM Tris-HCl in high performance liquid chromatography-grade water with 50 units ml^–1 penicillin and 50 µg ml^–1 streptomycin (Invitrogen), pH 7.4, adjusted with NaOH. The dissected oocytes were treated with collagenase (type IA, Sigma; 65 min at 18 °C, 245 collagen digestion units ml^–1 in Barth's solution, 10–12 oocytes/ml), rinsed, stored at 4 °C overnight, and manually defolliculated the following day before cRNA injection (46 nl/oocyte). The oocytes were incubated for 60 h at 18 °C in Barth's solution containing 5% heat-inactivated horse serum (Invitrogen) (6) and then stored at 4 °C. Experiments were carried out at a room temperature of 18–20 °C between 2.5 and 14 days from injection.

cRNA was injected at a ratio of 1:1 in order to express 34 receptors and at a ratio of 1:1:20 (3:4:3) in order to express 343 receptors in conditions that minimized the presence of pair 34 receptors. We have previously found that with this ratio the proportion of current through 34 receptors is too small to be detected by fitting doseresponse curves, since it is 39% when the ratio for 3:4:3 is 1:1:1 (1). The total amount of cRNA to be injected (in 46 nl of RNase-free water) for each combination was determined empirically, with the aim of achieving an optimal signal to noise ratio in the different experiments. Given that most of the experiments described here were carried out at low agonist concentrations, the level of expression we tried to achieve was higher than that needed for full agonist dose-response curves. The average quantity of cRNA injected in each oocyte was 20 and 30 ng for 34 and 343, respectively.

Single Channel Recording—Recordings were obtained in the cell-attached configuration from oocytes that had been stripped of their vitelline membrane after incubation in hyperosmotic solution (10 min with 200 mM sodium methyl sulfate, 20 mM KCl, 1 mM MgCl₂, 10 mM HEPES, pH 7.4, with NaOH). Electrodes were pulled from thick-walled borosilicate glass (GC150F, Warner Instruments) coated with Sylgard® 184 (Dow Corning) and fire-polished to have a final resistance of 12–15 megaohms when filled with low divalent external solution (150 mM NaCl, 2.8 mM KCl, 0.5 mM MgCl₂, 0.5 mM CaCl₂, 10 mM HEPES, 0.5 µM atropine, pH 7.2, with NaOH) which contained 1 µM ACh as agonist. No channel openings other than those attributable to stretch channels were observed in the absence of ACh in the extracellular solution (7). Oocytes were immersed in high potassium solution (100 mM KCl, 10 mM HEPES, 10 mM EGTA, pH 7.2, with KOH) to drive the resting membrane potential of the oocyte to a value consistent and close to 0. Recordings were filtered at 3 kHz (8-pole Bessel filter) and digitized at 30 kHz. The recordings used for amplitude analysis were idealized by time-course fitting; fitted amplitude distributions were fitted with gaussian curves (SCAN and EKDIST, courtesy of D. Colquhoun, UCL; www.ucl.ac.uk/Pharmacology/dcpr95.html) to give a mean value for the amplitudes of the openings at 150-, 125-, 100-, 75-, and 50-mV holding potentials; the mean values from each patch (4 and 3 patches for 34 and 343, respectively) were pooled for the different holding potentials to give the points in Fig. 1C, which were fitted with a straight line to obtain slope conductance values (CVFIT, courtesy of D. Colquhoun, UCL). Errors were calculated from the residuals.

View larger version (28K): [in this window] [in a new window]   FIG. 1. Incorporation of the 3 subunit decreases burst length and increases the conductance of neuronal nicotinic 34 channels. A and B, examples of continuous recordings (cell-attached, transmembrane membrane potential = 100 mV) from oocytes expressing 34 (A) or 343 (B). Underlined sections are expanded in the single sweep to the right, above the open point histograms for these two patches. The open point histograms are scaled to have unitary areas. Fitted amplitudes of single channel currents at different potentials are plotted in C. The lines refer to 343 (3 patches (filled circles)) and 34 (4 patches (filled squares); error bars are S.E.). Note the steeper slope and, therefore, the greater conductance of 343 receptors.

Recordings at a holding potential of 100 mV were used for obtaining dwell time distributions and idealized by a threshold crossing method with a resolution of 80 µs. Open period and shut time distributions were fitted with a mixture of exponential components (PClamp, Axon Instruments). An approximate correction of the mean open time for missed gaps was carried out as previously detailed (8).

Bursts were defined as groups of openings separated by shut times which were shorter than a particular value, t_crit. We chose the t_crit value by inspecting shut time distributions and the consistency of the time constants of their components across patches, since the time constants of gaps between bursts are expected to vary across patches because they depend on both the agonist concentration and the number of channels in a patch. The t_crit value was on average 168 ms for 34 and 5.6 ms for 343.

Two-electrode Voltage Clamp Recording Conditions—Oocytes, held in a 0.2-ml bath, were perfused at 4.5 ml/min with modified Ringer solution (150 mM NaCl, 2.8 mM KCl, 10 mM HEPES, 2 mM MgCl₂, 0.5 µM atropine sulfate, pH 7.2 adjusted with NaOH) and voltage-clamped at –70 mV using the two-electrode clamp mode of an Axoclamp-2B amplifier (Axon Instruments). Electrodes were pulled from Clark borosilicate glass GC150TF (Warner Instrument Corp.) and filled with 3 M KCl. The electrode resistance was 0.5–1 megaohms on the current-passing side. Experiments were terminated if the total holding current exceeded 2 µA to reduce the effect of series resistance errors. We chose a nominally calcium-free solution to minimize the contribution of calciumgated chloride conductance; this is endogenous to the Xenopus oocyte and may be activated by calcium entry through the neuronal nicotinic channels (9).

Agonist solutions (freshly prepared from frozen stock aliquots) were applied via the bath perfusion for a period sufficient to obtain a stable plateau response (at low concentrations) or the beginning of a sag after a peak (at the higher concentrations); the resulting inward current was recorded on a flat bed chart recorder (Kipp & Zonen) for later analysis. An interval of 5 min was allowed between ACh applications because this was found to be sufficient to ensure reproducible responses. To compensate for possible decreases in agonist sensitivity throughout the experiment, a standard concentration of ACh (approximately EC₂₀ for the particular combination used) was applied every third response. The experiment was started only after checking that this standard concentration gave reproducible responses. The average changes in the response to this ACh standard concentration observed by the end of the experiment for the different combinations ranged from –58 to +60%. All the data are compensated for the response rundown. However, applying this compensation did not affect the conclusions of our work, because the results of analyzing the original data (without compensation) were similar (data not shown). For details specific to each type of experiment, see below.

Agonist Potency Ratios—These were obtained following a previously described protocol (10). The aim of this was to obtain partial (2- or 3-point) dose-response curves at the lowest agonist concentrations that gave an acceptable signal to noise ratio. Care was taken to match the response size for all the agonists tested in a given experiment. These conditions were chosen to reduce the contributions of desensitization and agonist self block and essentially ensured that the slope of the dose-response curves was similar for all the agonists (see the examples in Fig. 3 and 4). Given the large number of agonists tested, only a subset could normally be tested in one cell, but ACh was tested in every experiment and was used as a standard. All curves obtained in each experiment were fitted simultaneously by least squares (CVFIT, courtesy of David Colquhoun, UCL; see www.ucl.ac.uk/Pharmacology/dcpr95.html) with power functions constrained to be parallel (these are the equivalent of fitting with a Hill equation in which the maximum is constrained to be very large with respect to the measured responses). In these simultaneous fits the program estimated the distances between the dose-response curves of the different agonists and that of the standard ACh. Such distances were obtained as concentration ratios with respect to ACh. The potency ratios shown in Table II are the reciprocal of the concentration ratios; errors and confidence intervals for potency ratios were calculated by Fieller's theorem (11). To check that the constraint of parallelism was justified, data were fitted without it to obtain estimates of the Hill slope for each agonist (see Table II). The maximum agonist concentrations used were 0.03 µM for epibatidine, 1.5 µM for lobeline, 5 µM for cytisine, 10 µM for 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), 20 µM for ACh and nicotine, and 200 µM for carbachol. Note that at 20 µM, ACh produces less than 5% of its maximum response for both combinations tested.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Co-expression of the 3 subunit does not affect the potency of most agonists on 34 human neuronal nicotinic receptors expressed in Xenopus oocytes. The traces in A are examples of inward currents recorded from oocytes expressing either 34 (left) or 343 (right) in response to low agonist concentrations. The log-log plots in B are two typical experiments; each set of dose-response curves was obtained from a single oocyte. The curves refer to epibatidine (filled inverted triangles), DMPP (filled diamonds), cytisine (filled triangles), nicotine (filled circles), acetylcholine (filled squares) and carbachol (hollow circles). Note that the potency ranks were the same for both combinations. CCh, carbachol; Nic, nicotine; Cyt, cytisine; Epi, epibatidine.

View larger version (18K): [in this window] [in a new window]   FIG. 4. The potency of lobeline relative to ACh is lower in receptors containing the 3 subunit. Traces (A) show the current responses to bath application of low concentrations of ACh or lobeline to 34 (left) or 343 (right) receptors. Concentration-response curves from such traces are plotted in B. The curves refer to lobeline, ACh, and carbachol (left to right). Note that in the experiment on triplet 343 receptors (right) the lobeline curve (filled circles) is closer to that of ACh (filled squares) than it is in oocytes expressing 34 (left). Lob, lobeline.

View this table: [in this window] [in a new window]   TABLE II Potency ratios of a series of nicotinic agonists on human 34 and 343 receptors Potency ratios are expressed relative to acetylcholine (±S.E.), i.e. show how many fold more potent each agonist is than acetylcholine at the foot of the dose-response curve. 95% confidence intervals, calculated using Fieller's theorem, are displayed to ease comparisons.

Antagonist Experiments—The antagonist experiments were carried out to confirm the competitive nature of the antagonist action and to estimate the antagonist dissociation constant by the Schild method. Partial (two-point) concentration-response curves were obtained at low agonist concentrations first in control solution (modified Ringer + 1mM calcium) and then in the presence of an appropriate antagonist concentration; this was present both in the Ringer and in the agonist solutions. Oocytes were incubated with the antagonist for 25 min before responses to ACh were again tested. Only one antagonist concentration was tested in each oocyte in order to minimize the distortion by response rundown (which cannot be quantified in the presence of the antagonist). Only data from oocytes in which the agonist response recovered after antagonist treatment to at least 60% that of control were included in the analysis. In a manner similar to that employed for the agonist potency ratios, the dose ratio r produced by the antagonist was measured for each experiment by fitting to the partial concentration-response curves power functions constrained to be parallel. Unconstrained fits were also performed; there was no significant difference between the slope of the concentration response in control conditions (range 1.59–1.71) and in 0.2–1 µM trimetaphan (range 1.37–1.73, paired t test).

To check that the data were adequately fitted by the Schild equation, data were plotted as a Schild plot and fitted by a power function, r – 1= a[B]ⁿ, where [B] is the concentration of antagonist, and a is a constant, in order to estimate the Schild slope n. Having found n to be close to 1 (see Table III), we refitted the data with the Schild equation, r – 1= [B]/K_B, to obtain K_B, the antagonist dissociation equilibrium constant.

View this table: [in this window] [in a new window]   TABLE III Schild analysis of the antagonist effect of trimetaphan on 34 and 343 receptors n is the total number of oocytes used in the experiments; ranges in square brackets are 2.01-unit maximum likelihood intervals (equivalent to 95% confidence intervals for Gaussian data).

Calcium Permeability—We measured the effect of different calcium concentrations on the reversal potential of the current induced by 2-s voltage ramps from –40 to 40 mV holding potential. Such ramps were applied in the depolarizing direction and in the hyperpolarizing direction both in control conditions (leak) and in the presence of ACh (test) once the agonist current was steady. Current-voltage curves were averaged (n = 4), and leak was subtracted using PClamp (Axon Instruments). Responses were sampled at 2 kHz (Axon Digidata 1320), filtered at 500 Hz (8-pole Bessel filter), and stored on disk for later analysis.

The contribution of the oocyte endogenous calcium-dependent chloride conductance (which would be activated by calcium entry through nicotinic channels) was minimized by preincubating the oocytes in chloride-free low calcium Ringer solution for 24 h (115 mM NaOH, 35 mM sucrose, 2.5 mM potassium gluconate, 10 mM HEPES, 1.8 mM Ca(OH)₂, 0.5 µM atropine bromide, pH 7.2, with methanesulfonic acid). Experiments were also carried out in chloride-free modified Ringer's solution. The composition of the low calcium Ringer was 1.8 mM Ca(OH)₂, 35 mM sucrose, 115 mM NaOH, 2.5 mM potassium gluconate, 10 mM HEPES, 0.5 µM atropine bromide, pH 7.2, with methanesulfonic acid; the high calcium Ringer contained 18 mM Ca(OH)₂ and no sucrose (osmolarity was 263 and 261 mosmol/liter, respectively). All recordings were carried out with electrodes filled with 2.5 M potassium acetate and 10 mM KCl using a 3 M KCl agar bridge as a reference electrode.

Materials—The following compounds were purchased from Sigma: acetylcholine chloride, atropine methyl bromide or sulfate, calcium acetate, carbamylcholine chloride (carbachol), (–)-cytisine, dihydro--erythroidine hydrobromide, DMPP, (+)-epibatidine hydrochloride, (–)-lobeline hydrochloride, mecamylamine hydrochloride, (–)-nicotine hydrogen tartrate, potassium acetate, potassium gluconate, and tricaine methanesulfonate. Trimetaphan camsylate was from Cambridge Laboratories Ltd., Wallsend, UK. All other chemicals were from BDH, Analar grade.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Single Channel Properties—As shown by the cell-attached records in Fig. 1, A and B, co-expression of the 3 subunit had a profound effect on the properties of oocyte-expressed 34 neuronal nicotinic receptors. The traces (obtained in the presence of 1 µM ACh at –100 mV) show that the openings of 3-containing receptors were larger and occurred in much shorter bursts (see also the expanded sweeps to the right of the continuous records).

The open point histograms in the same figure (scaled to unitary area) show that a single amplitude class accounted for the openings. Gaussian fits of the open point histogram for these two patches gave amplitude values of 3.13 ± 0.75 and 4.00 ± 0.69 pA for 34 and 343, respectively. Note the width of the open point histogram, which indicates a relatively large open channel noise, a well known phenomenon for neuronal nicotinic receptors be they native or recombinant (12, 13). The slope conductance of these channels (measured by fitting current-voltage plots in Fig. 1C) was 28.8 ± 0.66 picosiemens (4 patches) for the 34 combination and 46.7 ± 1.76 picosiemens for 343 receptors (3 patches).

The most pronounced effect of the presence of the 3 subunit in the receptor complex was on the kinetics of the channel. The continuous recordings of Fig. 1 show clearly that most of the openings of 34 channels occur in very prolonged bursts. These long bursts were completely absent from recordings from 343 channels.

Fig. 2 shows dwell time distributions for representative patches for the two subunit combinations (see Table I). 3 incorporation produced a consistent shift toward shorter apparent open periods (9.4 to 1.8 ms). Thus, the major component in the apparent open period distribution was 3.3 ms for 3-containing receptors (45% of all openings) and 17 ms for 34 (38% of all openings). Shut time distributions were all fitted with a mixture of five exponential components; the shortest components (the first 4 for 34 and the first 2 for 343) were consistent from patch to patch (Table I) and were, therefore, classed as within burst (see "Experimental Procedures"). The faster shut time component is shorter in the 34 patches, and the majority of events in this component will be below experimental resolution; this would result in the apparent lengthening of 34 openings. Given that it is likely that few openings are missed at our experimental resolution of 80 µs, we applied an approximate correction for missed gaps (8); this did not change the effect of 3 incorporation because corrected open times were 5.6 and 1.1 ms in 34 and 343, respectively (Table I).

View larger version (26K): [in this window] [in a new window]   FIG. 2. Dwell time distributions for 34 and 343 channels. Distributions shown are from two representative patches and are fitted by mixtures of exponential components; see Table I. Note the clear shift toward shorter events for apparent open periods of 3-containing receptors.

View this table: [in this window] [in a new window]   TABLE I Analysis of dwell time distributions for cell-attached patches from oocytes expressing 34 or 343 Data from a total of 10 34 patches and 5 343 patches are shown. Note that the areas of the different components were averaged for the total number of patches (i.e. including values of 0 for patches that did not require that particular component in the fitting of the distribution).

Measurements of average burst length confirmed the substantial shortening that is so striking from visual inspection of the traces. Thus mean burst length was 270 and 5.6 ms for 34 and 343 receptors, respectively (Table I). As Fig. 1 shows, a potential complication is that there are signs of heterogeneity in 34 bursts. We therefore considered the possibility that the population of shorter bursts was similar to the 343 bursts and simply became predominant with 3 incorporation. To assess this hypothesis, we measured mean burst length in 34 patches after excluding long bursts (the threshold for exclusion was chosen by inspection and was 274 ms on average in the 10 patches analyzed); the average duration for the "shorter" bursts was 64 ± 14 ms (n = 10) and, therefore, still substantially longer than that of 343 bursts.

Agonist Potency—There were no obvious differences between 34 and 343 receptors in the time course of agonist responses (see Fig. 3A) or the rank order of potency for an extensive series of agonists (epibatidine » lobeline > cytisine, DMPP, nicotine > ACh > carbachol; Table II, Fig. 3B). This rank order of potency is very similar to that found for human or rat 34 receptors by Chavez-Noriega et al. (14) and Meyer et al. (15) by EC₅₀ comparisons in oocytes or HEK293 cells.

Nevertheless, there was a marked decrease in the potency of lobeline with the incorporation of 3, from 23.0 ± 3.70 to 7.14 ± 1.1, relative to ACh for the 34 and 343 receptors, respectively; see the different distances between the partial dose-response curve to lobeline (leftmost in Fig. 4B, filled circles) and that to the standard, ACh (filled squares).

The Hill slopes for each agonist were similar on the two different combinations (see Figs. 3B and 4B, Table II, t test). A fairly wide range of Hill slope values is reported in the literature for the same 34 receptor from values comparable with those observed here (10) to values in excess of 1.9 (14). Note that the latter were observed in the presence of low (0.18 mM) extracellular calcium rather than nominally zero calcium (present study and Ref. 10).

Antagonist Potency—Trimetaphan acts as a competitive antagonist on native ganglion nAChRs (16), which are likely to contain the 3 and 4 subunits. A measurement (by Schild analysis) of the dissociation constant of trimetaphan would be useful for receptor classification and binding site modeling (very few true equilibrium dissociation constants are available in the neuronal nicotinic literature) and might show whether the antagonist site (which overlaps with the ACh site) is changed by 3 (see "Discussion").

In both receptor combinations trimetaphan (0.2–1 µM) produced a parallel rightward shift in the ACh partial concentration-response curves; its competitive mode of action was confirmed by the slope of the Schild plot (close to 1; see Table III). The incorporation of the 3 subunits had only a slight effect on the K_B for trimetaphan, decreasing it from 75.5 ± 1.8 to 66.0 ± 1.7 nM (Table III).

Although not very potent on 34-type receptors, the alkaloid dihydro--erythroidine is likely to be competitive (17). In agreement with this, we observed a small, parallel shift in the ACh concentration-response curve in the presence of 30 µM dihydro--erythroidine, with similar dose ratios for 34 and 343 (3.3 versus 3.9, n = 2), again suggesting that there is no change in the antagonist binding site.

On ganglion-type nAChRs, mecamylamine is a channel blocker at micromolar concentrations (18) but may act competitively at nanomolar concentrations (16). In our experiments, the mecamylamine concentration needed to produce a significant antagonist action was high (1 µM) and had substantial channel blocking effects (seen as a reduction in the slope of the ACh concentration-response curve); again, the effect was very similar for 34 and 343 receptors (data not shown).

Calcium Permeability—This was similar for the two combinations. The reversal potential shift after a 10-fold increase in extracellular calcium was 7.7 ± 1.5 and 5.2 ± 0.8 mV for 34 and 343, respectively (Table IV; see the value of 6.1 reported for 34 receptors (19).

View this table: [in this window] [in a new window]   TABLE IV Effect of changes in external calcium on the reversal potential of the nicotinic current through 34 and 343 receptors The mean reversal potentials (±S.E.) obtained in 1.8 and 18 mM external calcium and the mean (±S.E.) resulting shift in reversal potential are indicated for the appropriate subunit combinations. n is the total number of oocytes used in the experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Our results show that the 3 subunit consistently incorporates into neuronal nicotinic 34 receptors and profoundly changes their single channel properties, markedly shortening open periods and bursts and increasing single channel conductance. On the other hand, the presence of 3 had only small or negligible effects on the receptor sensitivity to a series of nicotinic agonists or to the competitive antagonists trimetaphan and dihydro--erythroidine and on the channel permeability to calcium. None of these changes is likely to be useful in the identification of native 3-containing receptors; the decrease in lobeline potency is small, and the increase in single channel conductance is not helpful because neuronal nAChRs display a large range of conductances, usually with several overlapping levels for each combination (13).

The main difference observed in the single-channel records of 3-containing receptors was a decrease in the duration of apparent open times and burst length. In an ideal record, i.e. with perfect resolution, the duration of open times reflects only the value of the closing rate constant, , and therefore, the gating properties of the receptor. The open times measured from a real recording are, however, distorted by our finite temporal resolution, as undetected short gaps lengthen apparent openings. In our case, because it is likely that we missed only shuttings, we could apply an approximate correction for missed events to the apparent open times. This did not change much the effect of 3 incorporation.

A potentially important consequence of the decrease in burst length we observed is that synaptic currents mediated by such channels would decay faster than those mediated by 34 channels. A caution is that long bursts may be a feature only of recombinant 34 receptors, particularly in the oocyte expression system (13, 20). On the other hand, channel openings similar to recombinant long bursts have been recorded from the medial habenula, which expresses 3 and 4 subunits (21), so it is possible that incorporation of the 3 subunit does exert this effect in native channels.

Another point of interest concerns the role of the 3 subunit in the receptor structure. Our previous work showed that 343 receptors contain two copies each of 3 and 4 and only one copy of 3 (2). Our single channel data are in good accord with this stoichiometry, as the increase in conductance with 3 incorporation strongly indicates that the 3 subunit takes the place of one of the 4 copies in the 34 pentamer. In nicotinic receptors conductance is mostly determined by the net charge at each of three sets of residues, at positions –4', –1', and 20' of the TM2 (second transmembrane) domain, the cytoplasmic, intermediate, and outer rings of charges (22). At the cytoplasmic and intermediate rings, all the subunits we expressed in this study are similar and contribute a negative charge (aspartate or glutamate). However, at the outer ring, 3 and 3 have a negatively charged glutamate, whereas 4 has a positively charged lysine. Hence, 3 can only affect the rings of charges if it takes the place of a 4 subunit; in this case, the charge on the outer ring would become more negative by two units, explaining the substantial conductance increase we observed. For instance, it has been reported that changing lysine to glutamate in the 20' position of one of the three copies of 2 is sufficient to increase the chord conductance of 42 nAChRs by 35% in divalent-free solution and using potassium as the permeant ion (23).

Incorporation of the 5 subunit is also known to increase single channel conductance of neuronal nAChR (13, 24, 25), probably by a similar effect on the outer ring (note, however, that 5, but not 3, incorporation should also reduce by one unit the negative charge on the cytoplasmic ring). If 3 replaced an rather than a subunit, the only change in the residues important for conductance would be a conservative serine for threonine swap in position 2', which in muscle nicotinic receptors produces a small increase in conductance, detectable only if potassium is the main permeant ion (22, 26). However, knowing that 3 replaces a 4 subunit still leaves open the question if 3 could participate directly, as the , or complementary (–) subunit, to one of the two agonist binding sites at the subunit interface.

Our antagonist data discount major changes in the binding sites given that the antagonist binding was not affected by 3 (judging from the results of our Schild analysis for trimetaphan and dihydro--erythroidine). Of course minor changes in the agonist binding residues may not be resolvable by competitive antagonists because their binding site overlaps but does not coincide with that of the agonist (note also that the range of competitive antagonists available is relatively small). This may well be the case for a 4 to 3 swap, as the sequences of 4 and 3 in loops D, E, and F are fairly similar; note, however, that the important residues in these domains are not as well characterized as those for loops that form the -side of the interface (27). Additionally, 3 can in principle only change one of the binding sites, not both. The Schild method measures the dissociation constant of the highest affinity site, as antagonist occupancy of one site only is sufficient to block receptor activation (muscle nAChR with one agonist and one antagonist molecule bound have very low P_open; see Sine and Taylor (28)). Thus, any 3-induced decrease in the site affinity for the antagonist would not be detectable.

Our data from the agonist experiments also argue against major changes in binding sites; despite testing a wide range of nicotinic agonists, the only change we observed with 3 incorporation was a decrease in the relative potency of lobeline. In principle, differences in agonist potency can be due to changes in either the binding or gating steps, which cannot be distinguished at macroscopic level (29). The analysis of open periods provides strong evidence of a gating impairment because of the shorter open times observed in 3-containing receptors. A gating change seems, therefore, to be the most likely explanation of the effects of 3 incorporation. Further work is needed to clarify why, as we previously reported, in receptors containing 3 there is no change in the EC₅₀ of ACh or nicotine and in the maximum response (relative to ACh) of the partial agonist nicotine (1). Our data show that the receptor binding sites are not appreciably changed by 3 incorporation. Thus, either 3 plays the role of a "structural" subunit or its contribution to the binding site cannot be detected because the sequences of the relevant domains are too similar to those of 4. This issue may only be decided conclusively by a different strategy, such as expressing 3 after the introduction of mutations aimed at increasing binding site differences for antagonists. Designing such mutants could for instance exploit the evidence on the determinants of binding for dihydro--erythroidine and -conotoxin MII provided by Luetje and co-workers (30–32).

	FOOTNOTES

 
^* This work was supported by Wellcome Trust Project Grants 055524 and 064652, Medical Research Council Cooperative Grant G98194 [GenBank] 00 Ph.D. studentship (to S. D. B.), and a School of Pharmacy Millennium Ph.D. Studentship (to J. P. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: Dept. of Biology, University College London, London WC1E 6BT, UK.

Current address: Dept. of Pharmacology, University College of London, London WC1E 6BT, UK.

^¶ To whom correspondence should be addressed. Tel.: 44-20-7679-3693; Fax: 44-20-7679-7298; E-mail: l.sivilotti{at}ucl.ac.uk.

¹ The abbreviations used are: nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine; DMPP 1,1-dimethyl-4-phenylpiperazinium.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Groot-Kormelink, P. J., Luyten, W. H. M. L., Colquhoun, D., and Sivilotti, L. G. (1998) J. Biol. Chem. 273, 15317–15320[Abstract/Free Full Text]
2. Boorman, J. P., Groot-Kormelink, P. J., and Sivilotti, L. G. (2000) J. Physiol. (Lond.) 529, 567–578
3. Forsayeth, J. R., and Kobrin, E. (1997) J. Neurosci. 17, 1531–1538[Abstract/Free Full Text]
4. Groot-Kormelink, P. J., and Luyten, W. H. M. L. (1997) FEBS Lett. 400, 309–314[CrossRef][Medline] [Order article via Infotrieve]
5. Akopian, A. N., Sivilotti, L., and Wood, J. N. (1996) Nature 379, 257–262[CrossRef][Medline] [Order article via Infotrieve]
6. Quick, M. W., and Lester, H. A. (1994) Methods Neurosci. 19, 261–279
7. Methfessel, C., Witzemann, V., Takahashi, T., Mishina, M., Numa, S., and Sakmann, B. (1986) Pfluegers Arch. Eur. J. Physiol. 407, 577–588[CrossRef][Medline] [Order article via Infotrieve]
8. Colquhoun, D., and Sakmann, B. (1985) J. Physiol. (Lond.) 369, 501–557[Abstract/Free Full Text]
9. Sands, S. B., Costa, A. C. S., and Patrick, J. W. (1993) Biophys. J. 65, 2614–2621[Medline] [Order article via Infotrieve]
10. Covernton, P. J. O., Kojima, H., Sivilotti, L. G., Gibb, A. J., and Colquhoun, D. (1994) J. Physiol. (Lond.) 481, 27–34[Abstract/Free Full Text]
11. Colquhoun, D. (1971) Lectures on Biostatistics, pp. 293–297, Clarendon Press, Oxford
12. Mathie, A., Cull-Candy, S. G., and Colquhoun, D. (1991) J. Physiol. (Lond.) 439, 717–750[Abstract/Free Full Text]
13. Sivilotti, L. G., McNeil, D. K., Lewis, T. M., Nassar, M. A., Schoepfer, R., and Colquhoun, D. (1997) J. Physiol. (Lond.) 500, 123–138[Abstract/Free Full Text]
14. Chavez-Noriega, L. E., Crona, J. H., Washburn, M. S., Urrutia, A., Elliott, K. J., and Johnson, E. C. (1997) J. Pharmacol. Exp. Ther. 280, 346–356[Abstract/Free Full Text]
15. Meyer, E. L., Xiao, Y., and Kellar, K. J. (2001) Mol. Pharmacol. 60, 568–576[Abstract/Free Full Text]
16. Ascher, P., Large, W. A., and Rang, H. P. (1979) J. Physiol. (Lond.) 295, 139–170[Abstract/Free Full Text]
17. Xiao, Y., Meyer, E. L., Thompson, J. M., Surin, A., Wroblewski, J., and Kellar, K. J. (1998) Mol. Pharmacol. 54, 322–333[Abstract/Free Full Text]
18. Giniatullin, R. A., Sokolova, E., Di Angelantonio, S., Skorinkin, A., Talantova, M., and Nistri, A. (2000) Mol. Pharmacol. 58, 778–787[Abstract/Free Full Text]
19. Gerzanich, V., Wang, F., Kuryatov, A., and Lindstrom, J. (1998) J. Pharmacol. Exp. Ther. 286, 311–320[Abstract/Free Full Text]
20. Lewis, T. M., Harkness, P. C., Sivilotti, L. G., Colquhoun, D., and Millar, N. (1997) J. Physiol. (Lond.) 505, 299–306[Abstract/Free Full Text]
21. Connolly, J. G., Gibb, A. J., and Colquhoun, D. (1995) J. Physiol. (Lond.) 484, 87–105[Abstract/Free Full Text]
22. Imoto, K., Busch, C., Sakmann, B., Mishina, M., Konno, T., Nakai, J., Bujo, H., Mori, Y., Fukuda, K., and Numa, S. (1988) Nature 335, 645–648[CrossRef][Medline] [Order article via Infotrieve]
23. Cooper, E., Couturier, S., and Ballivet, M. (1991) Nature 350, 235–238[CrossRef][Medline] [Order article via Infotrieve]
24. Ramirez-Latorre, J., Yu, C. R., Qu, X., Perin, F., Karlin, A., and Role, L. (1996) Nature 380, 347–351[CrossRef][Medline] [Order article via Infotrieve]
25. Nelson, M. E., and Lindstrom, J. (1999) J. Physiol. (Lond.) 516, 657–678[Abstract/Free Full Text]
26. Villarroel, A., Herlitze, S., Koenen, M., and Sakmann, B. (1991) Proc. R. Soc. Lond. B Biol. Sci. 243, 69–74[Medline] [Order article via Infotrieve]
27. Brejc, K., van Dijk, W. J., Klaassen, R. V., Schuurmans, M., van der Oost, J., Smit, A. B., and Sixma, T. K. (2001) Nature 411, 269–276[CrossRef][Medline] [Order article via Infotrieve]
28. Sine, S. M., and Taylor, P. (1981) J. Biol. Chem. 256, 6692–6699[Free Full Text]
29. Colquhoun, D. (1998) Br. J. Pharmacol. 125, 923–947[CrossRef]
30. Harvey, S. C., and Luetje, C. W. (1996) J. Neurosci. 16, 3798–3806[Abstract/Free Full Text]
31. Harvey, S. C., Maddox, F. N., and Luetje, C. W. (1996) J. Neurochem. 67, 1953–1959[Medline] [Order article via Infotrieve]
32. Harvey, S. C., McIntosh, J. M., Cartier, G. E., Maddox, F. N., and Luetje, C. W. (1997) Mol. Pharmacol. 51, 336–342[Abstract/Free Full Text]

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