J Biol Chem, Vol. 274, Issue 26, 18335-18340, June 25, 1999

Nicotinic Acetylcholine Receptors Assembled from the 7 and 3 Subunits^*

Eleonora Palma, Laura Maggi, Benedetta Barabino^§, Fabrizio Eusebi^¶, and Marco Ballivet^§

From the  Istituto Pasteur-Fondazione Cenci Bolognetti and Dipartimento di Medicina Sperimentale Universita' di Roma "La Sapienza", ^¶ Laboratorio di Biofisica, Centro Ricerca Sperimentale Istituto Regina Elena, via delle Messi d' Oro 156, 00158 Roma, Italy, and ^§ Département de Biochimie, Université de Genève, 1211 Genève 4, Switzerland

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Intracellular recordings were performed in voltage-clamped Xenopus oocytes upon injection with a mixture of cDNAs encoding the 3 and mutant 7 (^L247T7) neuronal nicotinic acetylcholine receptor (nAChR) subunits. The expressed receptors maintained sensitivity to methyllycaconitine and to -bungarotoxin but exhibited a functional profile strikingly different from that of the homomeric ^L247T7 receptor. The heteromeric ^L247T73 nAChR had a lower apparent affinity and a faster rate of desensitization than ^L247T7 nAChR, exhibited nonlinearity in the I-V relationship, and was inhibited by 5-hydroxytryptamine, much like wild type 7 (^WT7) nAChR. Single channel recordings in cell-attached mode revealed unitary events with a slope conductance of 19 picosiemens and a lifetime of 5 ms, both values being much smaller than those of the homomeric receptor channel. Upon injection with a mixture of ^WT7 and 3 cDNAs, clear evidence was obtained for the plasma membrane assembly of heteromeric nAChRs, although ACh could not activate these receptors. It is concluded that 3, long believed to be an orphan subunit, readily co-assembles with other subunits to form heteromeric receptors, some of which may be negative regulators of cholinergic function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The 11 neuronal nicotinic acetylcholine receptor (nAChR)¹ subunits cloned to date fall into two classes depending on whether an / pair is required for assembly (obligatory heteromers) or whether they can assemble into functional homomeric receptors. Combinations of the 2 and 4 subunits with one or more of the 2 to 6 subunits form functional nAChRs in reconstituted systems (1-6), although the particular combinations of these subunits that co-assemble in vivo are largely unknown. In contrast, the 7 to 9 subunits form homomeric nAChRs in cellular expression systems, even though it is not yet clear whether these subunits can also form heteromeric nAChRs in nerve cells (1, 3, 5, 7-12). 3, the remaining subunit, was long referred to as an orphan because it could not be ascertained whether it assembled into functional nAChRs either in neurons or in cellular reconstitution systems (13-16). Recent biochemical (4) and physiological evidence (17) does strongly suggest, however, that 3 participates in the assembly of nicotinic receptors, together with the 3, 4, 2, and 4 subunits.

When expressed in Xenopus oocytes, the methyllycaconitine (MLA)- and -bungarotoxin (-Bgt)-sensitive homomeric 7 receptor is noncompetitively inhibited by the transmitter serotonin (5-hydroxytryptamine (5HT)) and exhibits fast desensitization, pronounced inward rectification of ACh-evoked current, and low affinity for ACh (7, 18). The L247T mutation of the highly conserved leucine residue in the M2 channel domain of the 7 receptor converts 5HT from antagonist to agonist, abolishes inward rectification, enhances the apparent binding affinity for ACh 100-fold, and considerably decreases the rate of desensitization (18-20).

Using the idiosyncratic properties of the ^L247T7 receptor as a tool to investigate whether 3 can participate in functional receptor formation, we injected 3 and ^L247T7 cDNAs into the nuclei of Xenopus oocytes and studied the functional properties of the expressed nAChRs. We report here that coinjection of 3 and ^L247T7 cDNAs causes the formation of an heteromeric nAChR with functional properties clearly different from those of the homomeric ^L247T7 receptor. We also show that 3 co-assembles with wild type 7 (^WT7), but that the resulting heteromeric nAChR is insensitive to ACh.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

cDNAs and Expression Vectors-- The cDNAs encoding chick ^WT₇ and ^L247T₇ neuronal nAChR subunits were cloned into the Flip vector, where transcription is under the control of the SV40 early promoter (7). To maximize expression of the chick 3 subunit, its cDNA was subcloned in the pMT3 expression vector (21) under the control of the efficient adenovirus major late promoter. The same vector was used to express the ^V250T3 and the ^S243C3 mutant subunits.

Site-directed Mutagenesis-- Mutagenesis of 3 was carried out using the QuickChange site-directed mutagenesis system (Stratagene). We induced the point mutations using high performance liquid chromatography-purified mutant oligonucleotides with 15-16 nucleotides flanking the mutated bases. All mutations were confirmed by sequence analysis based on the Sanger dideoxy termination method (manually or with an ABI 377 sequenator). To verify the absence of undesired nucleotide changes, the entire coding region was sequenced.

Methanethiosulfonate (MTS) Derivatives-- Methanethiosulfonate ethylamine hydrobromide (MTSEA), methanethiosulfonate ethyltrimethylammonium bromide (MTSET), and sodium methanethiosulfonate ethylsulfonate (MTSES) freshly dissolved in oocyte Ringer's medium (see voltage-clamp recordings) were added to the oocytes at concentrations of 2.5, 1, and 10 mM, respectively, as detailed elsewhere (22, 23). The 2-min application of MTS derivatives to the oocytes in the presence or absence of 100 µM ACh was preceded and followed by a 5-min perfusion with oocyte Ringer's medium (2, 22).

Oocyte Injections-- Full-length cDNAs encoding the ^WT₇ and ^L247T₇ subunits were expressed as described previously (18). The 7 plasmids were coinjected with ^WT3_, ^V250T3, and ^S243C3 subunit cDNAs at 7/3 ratios ranging between 1:3 and 1:0.5 (0.2-0.8 ng of injected cDNA). Preparation of oocytes and nuclear injection procedures are detailed elsewhere (24, 25). Stage VI oocytes were injected intranuclearly with cDNA clones using a pressure microinjector (Eppendorf) and a Singer micromanipulator.

Voltage-clamp Recordings-- Membrane currents were recorded in the voltage-clamp mode 1-4 days after injection using 2 microelectrodes filled with 3 M KCl. The oocytes were placed in a recording chamber (0.1 ml) perfused continuously with oocyte Ringer's medium (82.5 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl₂, 1 mM MgCl₂, 5 mM Hepes, adjusted to pH 7.4 with NaOH) at controlled room temperature (20-21 °C). During most experiments, the oocytes were held at 60 mV to reduce the possible contribution of endogenous Ca⁺²-activated Cl currents. To construct dose-response relationships, oocytes were held at 60 mV, and the drugs were applied at 3-min intervals. Current-voltage relationships were determined using repetitive exposures to ACh at various potentials, stepping the holding potential from 60 mV to the desired voltage 5 to 10 s before transmitter application. Drugs were dissolved in oocyte Ringer's solution and applied by superfusing the oocyte at a flow rate of 12 ml/min. In particular, MLA was applied for 30 min before testing ACh responses (25). Solution exchange was achieved by using electromagnetic valves (Type III, General Valve). Drugs and chemicals were purchased from Sigma, except MLA (Research Biochemicals International) and the MTS derivatives (Toronto Research Chemicals, Canada).

Experimental Analysis-- The current records were digitized at 50-200 Hz using an analog-to-digital converter (Digidata 1200 Interface, Axon) and stored on a computer for subsequent analysis using pClamp 6.0.2 routines (Axon). For more details, see Ref. 18. To determine the half-dissociation constant (EC₅₀) of ACh, data were fitted using nonlinear fitting routines (included in Sigma Plot, Jandel), to the Hill equation:

(Eq. 1)

where [ACh] is the transmitter concentration, n_H is the Hill coefficient, and I_max is the maximum response. To assess the functional behavior of nAChRs, we defined some parameters as listed below. Receptor sensitivity to the transmitter was estimated by calculating the ratio (in percentage) of current elicited by 0.2 µM ACh (I_0.2) to that elicited at 100 µM ACh (I₁₀₀), which concentrations, respectively, correspond to the EC₅₀ and to the I_max of ^L247T7 (12, 18-20). The time to half-decay (T_0.5) of the inward current activated by ACh (I_ACh), defined as the time taken for the current to decay from peak to half-peak value and the time to 10% decay (T_0.1), were used to estimate the rate of receptor desensitization. The deviation of the I-V curve from linearity was estimated by the ratio of slope conductances at +30 mV and 60 mV. This rectification coefficient (n) (12) ranges between 100% (i.e. linear I-V relationship) and 0% (i.e. full rectification).

Single-channel Recordings-- Single-channel currents were recorded from the animal pole of the oocytes using the patch-clamp technique in the cell-attached mode, as reported (26, 27). Unless otherwise indicated, the ACh concentrations used were in the range 3-5 µM. At these concentrations, the open-channel frequency was variable in the range 2-20 Hz. Recordings were performed using an Axopatch 200B amplifier (Axon). The patch was discarded if no events were detected within 60 s after seal formation at 0 to 40 mV pipette potential or if opening frequency was below 0.5 Hz. Typically, a successful patch was stable for 5-15 min and had >200 opening transitions. No channel openings were observed in uninjected oocytes or in injected oocytes examined with a patch pipette filled with an ACh-free solution. Current recordings were filtered at 2 kHz, sampled at 10 kHz, and analyzed by pClamp 6.0.2 routines (Axon) using a threshold-crossing criterion. Events briefer than 0.2 ms were incompletely resolved and were excluded from the open-time histograms, which thus represent apparent open times. Histograms of amplitudes, apparent open times, and log (shut times) were fitted with a single Gaussian function or with the sum of exponentials as appropriate. Each histogram included from 500 to 2000 events. Burst duration was studied by grouping openings separated by a specific critical time, which was calculated for each patch from the fitted parameters of the shut-time distribution. For each patch, slope conductances were obtained by least squares linear fitting of current-voltage relationships constructed by polarizing the patch potential in the range 80 mV to 100 mV. For more details, see Ref. 27.

Binding Assay on Oocytes-- Oocytes that had been injected with ^WT7 cDNA or with mixtures of ^WT7 and 3 cDNAs were electrophysiologically tested with 2 mM ACh (28) and assayed for plasma-membrane -Bgt binding. To measure binding, control and injected oocytes were incubated singly for 2 h in 70 µl of oocyte Ringer's medium containing 20 nM [¹²⁵I]-Bgt (Amersham Pharmacia Biotech). After incubation, the labeled medium was removed, and the oocytes were washed five times with Ringer's medium (250 µl each time) and counted individually on a Beckman gamma counter.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Properties of the Homomeric ^L247T7 nAChR-- Oocytes injected with the ^L247T7 subunit cDNA responded to ACh with an inward current whose peak amplitude depended on transmitter concentration. ACh at 100 µM elicited a current (I₁₀₀) with mean peak amplitude of 7.13 µA (18 oocytes, 3 donors [18/3]; range, 4.36 µA to 8.32 µA), whereas at 0.2 µM ACh the current (I_0.2) averaged 4.03 µA ([12/3]; range, 3.1 to 5.87 µA) (Fig. 1, A-B, left). The ratio I_0.2/I₁₀₀, used as a parameter of receptor sensitivity to ACh (27), averaged 61 ± 5% (mean ±S.E.; range, 48-75%), confirming that I_0.2 is close to half the maximum current response obtained at 100 µM ACh (18-20). I₁₀₀ decayed with T_0.5 > 10 s and T_0.1 = 4.6 ± 1.0 s in 13 oocytes (range, 0.9-7 s; 3 donors) and with T_0.1 > 10 s in another 5 cells, indicating a slow rate of desensitization (Fig. 1, B-C, left). The amplitude of I_ACh increased linearly with hyperpolarization and showed a slight rectification at positive potentials. The rectification coefficient (n) ranged from 53.9 to 88.0% (76.6 ± 2.9%; [12/2]), indicating that the I-V relationships in ^L247T7 was nearly linear (Fig. 2A) (18-20). Furthermore, in agreement with our previous report (18), 5HT gave rise to large inward currents (Fig. 1C, left), whose amplitude depended on 5HT concentration. The maximum 5HT-evoked current amplitude (I_5HT = 6.63 µA; range, 3.21 to 8.01 µA; [7/2]) was obtained at 500 µM.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   At a ratio of 1:1, cDNAs encoding the L247T7 and 3 subunits form functional heteromeric nicotinic receptors in Xenopus oocytes. A, examples of inward currents elicited by ACh at the indicated concentrations (in µM). All currents were recorded at 100 mV holding potential, and bars indicate drug application. Note the faster IACh decay in oocytes that express L247T73 compared with L247T7 and the block of I100 after 30 min of incubation with -Bgt (100 nM, superimposed trace). B, examples of inward currents elicited by ACh at the indicated concentrations. Note the reduced ratio I0.2/I100 for L247T73 as compared with L247T 7. C, examples of inward currents elicited by ACh (100 µM) and 5HT (500 µM). Note the weak 5HT agonism in oocytes coinjected with L247T7 and 3.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Oocytes injected with a mixture of L247T7 and 3 cDNAs exhibit fast desensitization and current rectification at positive potentials. A, peak currents evoked by ACh (100 µM) at various holding potentials in an oocyte expressing the L247T 7 receptor. The solid line represents a second order polynomial fit to data. Note the lack of rectification and null potential at 17 mV (data representative of 6 experiments, 3 donors). ACh applied at 3-min intervals, holding potential 50 mV. Inset, superimposed IACh traces. Solid bar, 100 µM ACh application; outward current at +30 mV (top) and inward currents at 30 mV (middle) and 80 mV (bottom) in the same oocyte. B, peak currents as in A (representative of 11 experiments, 3 donors) in an oocyte expressing L247T73 receptor. Null potential, 10 mV. Fitting procedure as in A. Inset, superimposed IACh traces as inset in A.

Properties of nAChRs Composed of Both 3 and ^L247T7-- Receptors assembled after injection of a mixture of cDNAs encoding both the 3 and ^L247T7 subunits (ratio 1:1) were activated by ACh and fully blocked by -Bgt (100 nM [5/2]; Fig. 1A, middle and right). They strikingly differed from the ^L247T7 receptor in a number of physiological parameters. Oocytes injected with the cDNA mixture expressed reduced nAChR activity compared with oocytes injected with ^L247T7 cDNA alone, the amplitude of the elicited currents being significantly impaired (I₁₀₀ = 1.62 µA, [24/5], and 7.13 µA, [18/3], respectively). The expressed nAChRs displayed a reduced transmitter sensitivity (I_0.2/I₁₀₀ = 3.2 ± 0.4%; [11/3], Fig. 1B, right) and a faster rate of desensitization (T_0.5 = 1.97 ± 0.21 s; T_0.1 = 0.48 ± 0.05 s; [21/5], Fig. 1, A-C, right) than the homomeric ^L247T7 nAChR. This fast rate of current decay was maintained at 30 mV (T_0.1 range, 0.28-1.3 s, [6/2]), close to the chloride reversal potential, suggesting that this property is not because of a contribution of Ca²⁺-activated Cl currents. 5HT (500 µM) acted as a weak agonist, eliciting an inward I_5HT whose amplitude was only 5% that of the ACh response at 100 µM (I₁₀₀ = 1.72 µA; I_5HT = 88 nA, [17/3], Fig. 1C, right). In addition, when maintained in the bathing fluid for 20-60 s at 500 µM, 5HT reduced the I₁₀₀ response to ACh by 95% (94.6 ± 2.5%, [13/3], data not shown). Furthermore, nAChRs displayed a nonlinear I-V relationship (n=19.8 ± 4.7%; range, 5-25%, [11/3]) (12), as shown in Fig. 2, which compares typical I-V relationships in oocytes injected with mixed cDNAs (Fig. 2B) or with ^L247T7 cDNA alone (Fig. 2A). Finally, the ACh dose-response relationships constructed in 9 oocytes from 2 donors yielded EC₅₀ and n_H of 3.4 µM and 1, respectively, thus showing an affinity for the transmitter decidedly lower than that of the homomeric ^L247T7 receptors (Fig. 3) (18, 20). The EC₅₀ and n_H values were obtained by fitting data to a single Hill plot, with no significant improvement resulting from fitting data with a sum of multiple Hill equations, an indication that the pure homomeric ^L247T7 population was poorly expressed and obscured by the dominant expression of an heteromeric receptor population.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   The ACh dose-response relationship shifts to the right in oocytes expressing L247T73 nAChRs () compared with oocytes expressing L247T7 nAChRs (). The peak currents evoked by ACh at the concentrations indicated on the abscissa were normalized to I100. Ordinate, normalized response to ACh in percentage (9 oocytes, 2 donors). Best-fitting with the Hill equation yielded EC50 = 0.37 µM, nH = 1.5 for L247T7, and EC50 = 3.36 µM, nH = 1 for L247T73. All oocytes were held at 60 mV.

The ACh responses in mixture-injected oocytes were sensitive to MLA, as is the case for oocytes injected with ^L247T7. For instance, the I_0.2 peak amplitude decreased by 34% at 100 pM MLA in ^L247T7 oocytes and by 85% in mixture-injected oocytes. Furthermore, I_ACh peak amplitude in mixture-injected oocytes decreased by 30% at 3 µM ACh and 100 pM MLA, equal to the block on ^L247T7 nAChR when activated near its EC₅₀ by 0.2 µM ACh (Fig. 3). Taken together these data indicate that MLA acts on both putative heteromeric and homomeric nAChRs in a competitive manner.

It is well established that the clamp-holding current is far greater for cells injected with ^L247T7 than with ^WT7. This observation, together with the fact that MLA reduces the holding current, has been explained by the spontaneous, ACh-independent activation of the mutant 7 receptor (29). In our experiments, oocytes injected with the cDNA mixture showed a spontaneous, MLA-sensitive inward current (2.4 ± 1.3% of the I₁₀₀ peak amplitude, [5/2]), quite similar to that estimated in oocytes injected with ^L247T7 (4.5 ± 1.6%, [9/2]; not shown).

To investigate whether the ratio of cDNAs in the mixture could influence the functional properties of the resulting nAChRs, ^L247T7 and 3 cDNAs were injected into oocytes at 7/3 ratios ranging from 1:1 to 1:6. No obvious differences in current decay, ACh sensitivity, or I-V relationships were observed. In contrast, the amplitude of the elicited I_ACh was significantly impaired as the 7/3 ratio was increased (not shown).

Single Channel Recordings-- To investigate if the co-expression of 3 could alter the ^L247T7 channel parameters, we performed single-channel recordings on mixture-injected oocytes (ratio 1:1). Because the coinjection of 3 and ^L247T7 reduces I_ACh, single-channel recordings were only successful in the subset of oocytes that had a high level of expression. A population of single-channel openings was revealed in the cell-attached mode at 3-5 µM ACh in the pipette and 30 mV pipette potential (PP). Its mean open-time (_op) was 4.7 ± 1.0 ms [7/3] and was made up of a single exponential component. Values of _op remained stable with moderate hyperpolarizations. For instance, at 50 mV PP, _op was 4.2 ± 0.5 ms [7/3], statistically equivalent to the value at 30-mV PP. No obvious channel-flickering activity was observed in our recording conditions, indicating that the transmitter is unable to effect an open channel block at the concentrations used. This was evidenced by opening bursts whose mean duration was only slightly longer (6.3 ± 0.8 ms; [7/3]) than _op. Channel activity (16 Hz at 30-mV PP) and amplitude were rather stable over time at a given PP. As the patch was hyperpolarized, the amount of voltage required to change the opening frequency e-fold was 27 mV. Analyses of unitary events revealed a single class of channels with a slope conductance of 18.8 ± 1.4 pS (range, 14-23 pS; [8/3]). Fig. 4 provides an example (representative of seven patches) of ACh-activated unitary events in an oocyte expressing the ^L24773 receptor.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   ACh activates nAChR channels in oocytes injected with a mixture of L247T7 an 3 cDNAs. A, examples of single channel currents at pipette potentials as indicated. Inward currents are represented by upward deflections. B, mean channel current amplitudes plotted versus pipette potential and fitted by linear regression (solid line), yielding the indicated slope conductance. C, histogram of open durations at pipette potential +30 mV, same patch as in A, fitted by a single exponential. op as indicated (n = 1025). D, distribution of single-channel amplitudes at the same pipette potential and in the same patch. Histogram is best fitted by a single Gaussian with a mean of 1.43 ± 0.03 pA. ACh was at 3 µM in the recording pipette.

Properties of nAChRs Obtained upon Injection into Oocytes of 3 and ^WT7 cDNAs-- A number of experiments were performed in oocytes injected with a mixture of 3 and ^WT₇ cDNAs to determine whether these subunits can co-assemble into functional nAChRs. In agreement with the experiments using a mixture of 3 and ^L247T7 cDNAs, the amplitudes of the current responses to ACh were considerably reduced in oocytes injected with 3 and ^WT7 cDNAs, as compared with those injected with ^WT7 cDNA alone. For instance, at 100 µM ACh the average I_ACh recorded in oocytes injected with ^WT7 cDNA was 693.4 nA [24/3], and 45.4 nA [21/3] with a mixture of ^WT7 and 3 at 1:1. Despite this strong inhibition, no differences in the functional properties of the expressed nAChRs were observed. Furthermore, when estimating receptor sensitivity to nicotine by the ratio (in percentage) of the currents elicited by this agent at 10 µM and at 100 µM (which approximate the half-maximum and maximum current amplitudes for ^WT7) (30), we found no significant difference between ^WT7/3 and ^WT7 oocytes [4/1]. Other experiments tested the functional and pharmacological properties of ^WT7/3 nAChRs (Table I). The apparent affinity, desensitization, current rectification, and sensitivities to MLA or 5HT of this receptor species were identical to the corresponding parameters in ^WT7 injected oocytes.

View this table: [in this window] [in a new window]   Table I Functional properties of AChRs expressed in oocytes coinjected with WT 7 and 3 n/n, number of oocytes/number of donors. 5HT (%) and MLA (%) indicates the impairment (in percentage) of the IACh by 5HT (50 µM) or by MLA (25 pM), both acting as nAChR antagonists. n is the rectification coefficient (see "Experimental Procedures"). nH, Hill coefficient. EC50, half-dissociation constant for ACh responses. T0.1, time to 10% decay. T0.5, time to half-decay (see "Experimental Procedures"). Note the overall identity of the receptors resulting from the two injection protocols.

Coinjection of Mutant 3 and ^WT7 cDNAs-- Given that coinjections of 3 and ^WT7 did not provide evidence for the participation of 3 in functional ^WT7 nAChR complexes, we sought a direct proof by injecting appropriate 3 mutants in combination with ^WT7 cDNAs. We reasoned that particular mutations in the M2 membrane-spanning segment of the 3 subunit should yield ACh responses whose parameters would clearly establish co-assembly with 7. We injected oocytes with ^WT7 and 3 bearing the mutations V250T or S243C. The first mutant aligns with L247T in the 7 subunit, whereas the second aligns with S240C in the 5 subunit, both previously shown to be exposed in the nAChR channel (2, 23). We observed similar functional and pharmacological receptor profiles in oocytes injected with ^V250T3 and ^WT7 cDNAs, as compared with oocytes injected with ^WT7 cDNA alone ([9/3], not shown). We coinjected the ^S243C3 mutant and ^WT7 into oocytes to test whether the MTS-derived thiol reagents MTSET, MTSEA, and MTSES were able to inhibit I_ACh, as reported elsewhere for muscle and neuronal nAChRs mutated to cysteine in the homologous residues (2, 22, 23). We found that MTSET, MTSEA, and MTSES (1, 2.5, and 10 mM, respectively) were all unable to affect I₁₀₀ ([8/3], not shown) in the same experimental conditions as described previously (2, 23). On the other hand, oocytes injected with ^S243C3 and ^L247T7 exhibited a profile similar to that of oocytes injected with wild-type 3 and ^L247T7, but MTSET irreversibly inhibited I_ACh by ~65%, indicating that the ^S243C3 mutant is functionally expressed in oocytes and readily assembles with ^L247T7 subunits.

Surface Expression of nAChRs upon Injection of Mixed 3 and ^WT7 cDNAs-- Measurements of plasma-membrane toxin binding were carried out in oocytes coinjected with ^WT7 and 3, and the results compared with those obtained upon injecting ^WT7 alone. It was found that the level of -Bgt binding in the coinjected oocytes (^WT7/3 ratio 1:3) decreased by 20%, whereas the I_ACh peak amplitude decreased by ~90% of control. Remarkably, a significant number of these oocytes bound large amounts of radiolabeled toxin but had null current responses to 2 mM ACh (Fig. 5). This finding indicates that -Bgt receptors readily assemble in the plasma membrane of coinjected oocytes and that they cannot be activated by ACh.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   ACh-activated currents are not proportional to plasma membrane -Bgt binding in oocytes injected with a mixture of WT7 and 3 cDNAs. Histograms (representative of three experiments) comparing IACh amplitude (ACh, 2 mM) with -[125I]Bgt surface binding. Oocytes were injected with WT7 cDNA or with mixtures of WT7 and 3 cDNAs in the ratios 1:3 and 1:6. Binding of -[125I]Bgt was determined after recording IACh and is expressed as the ratio of the radioactivity from injected over uninjected cells. Each bar represents the mean ± S.E. of 11-16 individually assayed cells. Inset, relationship between IACh evoked by 2 mM ACh and plasma membrane levels of -Bgt in oocytes injected with WT7 and 3 cDNAs (ratio 1:3). Note that six oocytes displaying considerable toxin binding have null responses to ACh.

In oocytes coinjected with 7 and 3 at the ratio 1:6, the relative level of nAChR surface expression established by -Bgt binding remained relatively high, whereas ACh responses all but disappeared (Fig. 5), confirming that the heteromeric ^WT73 receptor binds -Bgt but is insensitive to ACh.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The 5, 6, and 3 neuronal nAChR subunits were long believed incapable of assembling with other subunits to form functional receptors. However, recent evidence demonstrates that 5 assembles with 42 and that 6 assembles with 2 and 4 to form functional receptors in reconstituted systems (2, 6, 31-33). Here we give evidence that 3 assembles with an 7 mutant to form a heteromeric nAChR exhibiting functional and pharmacological profiles drastically different from those of oocytes expressing the homomeric mutant 7 nAChR. As compared with oocytes expressing the ^L247T7 receptor, oocytes injected with a mixture of the 3 and ^L247T7 cDNAs exhibit the following distinctive properties. (i) The ACh-activated currents decay much faster, (ii) the I-V relationship becomes nonlinear, (iii) the channel open time, 4.7 ms on average, is much shortened (11 ms for ^L247T7 channels) (27), (iv) the channel mean slope conductance, 19 pS, is markedly lowered (44 and 58 pS for the two ^L247T7 conductance classes) (20, 27), (v) the apparent binding affinity and the cooperativity are both reduced, and (vi) serotonin acts as a weak agonist/antagonist, being a potent agonist in oocytes expressing ^L247T7 nAChR (18). Taken together, these findings provide convincing evidence that the 3 subunit coassembles with ^L247T7 to form a functional heteromeric receptor.

The ^L247T73 channels share functional profiles matching those of most of the nicotinic heteromeric receptors studied in oocytes. For instance, (i) channel conductance (19 pS) is similar to that described for chick 44, 42, 32, and 34 nAChRs in Xenopus oocytes (18-24 pS conductance) (2, 34, 35), (ii) a strong inward rectification develops with oocyte depolarization as for most of the heteromeric nicotinic receptors (34), (iii) the time course of nAChR desensitization compares to that described for other heteromeric receptors and is very different from that of the homomeric 7 receptor (7). In addition, its pharmacological behavior appears similar to the profiles described for other heteromeric receptors, with both 5HT and MLA behaving as antagonists, as they do for other heteromeric receptors (36, 37).

The only striking difference in the profile of ^L247T73 nAChR versus the other heteromeric nAChRs described to date is in the sensitivity to -Bgt, a potent blocker of both the ^L247T73 and the homomeric 7 nAChR that is essentially ineffective on all the heteromeric neuronal nAChRs that have been described (reviewed in Ref. 38). This result is in contrast to the general assumption that -Bgt sensitivity is restricted to the homomeric nAChRs and suggests that the -Bgt binding activity detected in native preparations does not prove that homomeric nAChRs are expressed but merely reveals the presence of nAChRs containing the 7 or 8 subunits.

An open question raised by our data is the subunit composition of the heteromeric ^L247T73 nAChRs. Upon injection of an equimolar mixture of ^L247T7 and 3 cDNAs, one would expect the assembly of a variety of pentameric receptor species made up of ^L247T7 and 3 subunits, with probability n(1/2)⁵ for each of the compositions likely to be functional (     ₅^L247T7, n = 1;      ₄^L247T7₁3, n = 5;      ₃^L247T7₂3, and      ₂^L247T733, n = 10). On the assumption that the inward current elicited by 5HT is because of the activation of the homomeric 7 nAChR, whereas the heteromeric receptors are blocked by 5HT, the estimated homomeric population as a fraction of the I_5HT/I_ACh is ~5% that of the total nAChR expressed in oocytes injected with the cDNA mixture, in excellent agreement with the theoretical value. The remaining 95% of the nAChR-channel responses result from the activation of ^L247T73 populations of uncertain subunit composition. Single channel recordings showing a unique channel conductance class and a unique exponential open time component, together with the reduced Hill coefficient of the whole-cell dose-response relationships all suggest that substituting one or more 3 subunits into the ^L247T7 receptor reduces the number of binding sites for the transmitter and affects cooperativity, channel conductance populations, and channel kinetics. Given that data obtained under voltage-clamp conditions with the cDNAs mixture are not so scattered as to suggest inhomogeneous channel populations, it could be that formation of nAChR of the standard heteromeric stoichiometry,      ₂^L247T7₃3, is favored. As shown in another context (4), 3 is thought to enhance the assembly of 42 and 44 dimers into pentamers of ₂4₁2₁3₁4 composition. We speculate that 3 may likewise enhance pentamer assembly by associating with an ^L247T73 dimer to make a trimer, thus facilitating pentamer formation with available dimers. This predicts that an excess of 3 subunits would sharply curtail the amount of assembled pentamers by sequestering all of the ^L247T73 dimers into      ₁^L247T7₂3 trimers incapable of functional assembly. The sharp reduction of mean I_ACh we observe at elevated 3/7 ratios is in qualitative agreement with this model.

A key feature of the homomeric 7 nAChR is its high Ca⁺² permeability, as compared with heteromeric receptors (39). We note a reduction in the null potential from 17 mV in homomeric ^L247T7 to 10 mV in ^L247T73, suggesting a reduction of Ca⁺² entry through the heteromeric channel, with consequent impairment of the Ca⁺²-activated Cl component of the ACh response. Because the ^L247T7 receptor inactivates slowly even in the presence of an intracellular Ca⁺² chelator (39), the faster decay of the ACh-evoked currents in the heteromer is unlikely to result from an enhanced Cl component and appears to be an intrinsic property of the receptor. This argument is strengthened by the observation that the heteromer maintains its inactivation kinetics at or close to the Cl reversal potential.

We have no evidence that 3 can assemble with ^WT7 to form a functional heteromeric receptor in oocytes, although we attempted the co-assembly of 7 not only with wild-type but also with mutant 3 subunits that should have detectably altered receptor properties. Our data argue that oocytes block the assembly of functional 73 wild-type heteromers, while allowing the assembly of functional ^L247T73 and ^L247T7^S243C3 receptors. The large plasma membrane binding of -Bgt coupled to the small ACh responses in oocytes injected with a mix of ^WT7 and 3 cDNAs is interpreted as reflecting the expression of a major population of nonfunctional heteromeric ^WT73 nAChRs (of undetermined stoichiometry) and of a minor population of homomeric ^WT7 nAChRs. Although it is possible that the heteromeric ^WT73 nAChRs could be activated by an unidentified ligand, their insensitivity to the natural transmitter, together with the reduced expression of functional receptors upon increasing the amount of injected 3, indicate that co-assembly of the 3 subunit negatively regulates the function of nicotinic receptors formed by ^WT7. It is possible that ^WT7 is prevented from functionally assembling with 3 by post-translational processes inherent to the Xenopus oocyte expression system and that in native systems the same constraints do not develop. There is a large variability in the nicotinic responses attributed to the expression of the 7 nAChRs in native systems (38, 40), indicating that 7 may indeed assemble with other subunits capable of modifying receptor parameters.

In conclusion, our findings provide conclusive evidence that 3 is capable of forming functional receptors in combination with other subunits, in this case an 7 mutant. Remarkably, the co-assembly of 3 with ^WT7 appears to form "silent" heteromeric receptors insensitive to ACh. This, together with a recent demonstration that the ^V273T3 mutant participates in the assembly of functional receptors with the 3 and 4 subunits (17), adds further members to the repertoire of nicotinic receptors and increases the potential regulatory complexity of the neural cholinergic system.

	FOOTNOTES

^* This work was supported by grants from Ministero Università Ricerca Scientifica e Tecnologica (to F. E.), by grants from the Swiss National Science Foundation (to M. B.), and by a Telethon fellowship (222/bi (to E. P.)).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom reprint requests should be addressed. Biochemistry/Sciences II, 30, quai Ernest Ansermet, 1211 Geneva 4, Switzerland. Fax: 41 22 702 6483; E-mail: marc.ballivet{at}biochem.unige.ch.

	ABBREVIATIONS

The abbreviations used are: nAChR, neuronal nicotinic acetylcholine receptor; MLA, methyllycaconitine; -Bgt, -bungarotoxin; 5HT, 5-hydroxytryptamine; WT, wild type; MTS, methanethiosulfonate; MTSEA, MTS ethylamine hydrobromide; MTSET, MTS ethyltrimethylammonium bromide; MTSES, sodium MTS ethylsulfonate; PP, pipette potential; pS, picosiemens.

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