The Journal of Neuroscience, November 23, 2005, 25(47):10905-10912; doi:10.1523/JNEUROSCI.3805-05.2005
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Cellular/Molecular
Stoichiometry of the 
910 Nicotinic Cholinergic Receptor
Paola V. Plazas,1
Eleonora Katz,1,2
María E. Gomez-Casati,1
Cecilia Bouzat,3 and
A. Belén Elgoyhen1
1Institute of Investigations in Genetic Engineering and Molecular Biology, National Council of Scientific and Technical Investigations, and 2Department of Physiology, Molecular and Cellular Biology, Faculty of Exact and Natural Sciences, University of Buenos Aires, Buenos Aires 1428, Argentina, and 3Institute of Biochemical Investigation of Bahía Blanca, National University of the South, National Council of Scientific and Technical Investigations, Bahía Blanca F-8000FWB, Argentina
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Abstract
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The 
9 and 10 nicotinic cholinergic subunits assemble to form the receptor that mediates synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea. They are the latest vertebrate nicotinic cholinergic receptor (nAChR) subunits that have been cloned, and their identification has established a distant early divergent branch within the nAChR gene family. The 10 subunit serves as a "structural" component leading to heteromeric 910 nAChRs with distinct properties. We now have probed the stoichiometry of recombinant 910 nAChRs expressed in Xenopus oocytes. We have made use of the analysis of the population of receptors assembled from a wild-type subunit and its partner 9 or 10 subunit bearing a reporter mutation of a valine to threonine at position 13' of the second transmembrane domain (TM2). Because the mutation increased the sensitivity of the receptor for acetylcholine (ACh) but mutations at different subunits were not equivalent, the number of 9 and 10 subunits could be inferred from the number of components in compound concentration-response curves to ACh. The results were confirmed via the analysis of the effects of a mutation to threonine at position 17' of TM2. Because at this position the mutations at different subunits were equivalent, the stoichiometry was inferred directly from the shifts in the ACh EC50 values. We conclude that the recombinant 910 receptor is a pentamer with a (9)2(10)3 stoichiometry.
Key words: nicotinic receptors; stoichiometry; ligand-gated channels; mutagenesis; cholinergic receptors; cochlea
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Introduction
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Nicotinic acetylcholine receptors (nAChRs) are members of the "Cys-loop" family of neurotransmitter-gated ion channels (Le Novere and Changeux, 1995
). Receptors in the family are formed by five homologous subunits oriented around a central ion channel similar to barrel staves (Karlin, 2002). In vertebrates, nine nonmuscle subunits (2-10) and three 
subunits (2-4) have been cloned. The reported combinations of subunits that form functional vertebrate nAChRs are expanding and now include receptors assembled from single subunits (7, 8, 9) (Couturier et al., 1990; Elgoyhen et al., 1994; Gotti et al., 1994), receptors that contain multiple subunits both with (252, 352, 354, 452) (Conroy et al., 1992; Vernallis et al., 1993; Conroy and Berg, 1998; Balestra et al., 2000) and without (78, 910) supplemental subunits (Gotti et al., 1994; Elgoyhen et al., 2001), receptors with single and multiple subunits (324, 334) (Colquhoun and Patrick, 1997; Groot-Kormelink et al., 1998; Boorman et al., 2000), and receptors with multiple and subunits (3245) (Gerzanich et al., 1998) as well as heteromeric nAChRs formed via pairwise combinations of 2, 3, 4, or 6 with either the 2 or 4 subunits (Boulter et al., 1987; Goldman et al., 1987; Deneris et al., 1988; Duvoisin et al., 1989; Gerzanich et al., 1997). Thus, the number of potential molecular forms of nicotinic receptors is very large. Determining the stoichiometry of each association has become challenging in most cases.
The 9 and 10 nicotinic cholinergic subunits assemble to form the receptor that mediates synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea, one of the few certified examples of postsynaptic function for a nonmuscle nAChR (Elgoyhen et al., 2001). The 9 and 10 are the latest vertebrate nAChR subunits that have been cloned, and their identification has established a distant, distinct, and peculiar early divergent branch within the nAChR gene family (Elgoyhen et al., 1994, 2001; Rothlin et al., 1999; Le Novere et al., 2002). Whereas recombinant homomeric 9 receptors are functional, 10 homomeric receptors are not. The 10 subunit serves as a structural component leading to heteromeric 910 nAChRs with 100- to 1000-fold bigger macroscopic currents, particular desensitization kinetics, current-voltage dependency, and sensitivity to extracellular Ca2+ (Elgoyhen et al., 1994, 2001; Sgard et al., 2002; Weisstaub et al., 2002). It is believed that the native receptor of hair cells is a heteromer assembled from both 9 and 10, because no detectable responses are recorded from hair cells that transcribe only the gene coding for the 9 subunit (Katz et al., 2004).
We have made a step forward in providing novel information on the architecture of the 910 nAChR. We have used reporter mutations in transmembrane region 2 (TM2) of the subunits to infer the number of each subunit type that comprises the recombinant receptor expressed in Xenopus oocytes. We conclude that the receptor is a pentamer with a (9)2(10)3 stoichiometry.
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Materials and Methods
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Generation of mutant receptors. Site-directed mutagenesis of the 9 and 10 rat cDNAs, subcloned in a modified pGEMHE vector (Elgoyhen et al., 1994, 2001), was performed with the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Mutations were confirmed by sequence analysis to verify that only the desired nucleotide changes were present. The numbering that was used is the one that has been adopted to allow for comparison of homologous amino acids from different types of neurotransmitter-gated channels, as reported previously (Plazas et al., 2005). Position 1' corresponds to the start of the TM2 region.
Expression of recombinant receptors in Xenopus laevis oocytes. Capped cRNAs were transcribed in vitro from linearized plasmid DNA templates with the use of the mMessage mMachine T7 transcription kit (Ambion, Austin, TX). The maintenance of Xenopus laevis as well as the preparation and cRNA injection of stage V and VI oocytes has been described in detail previously (Katz et al., 2000). Typically, oocytes were injected with 50 nl of RNase-free water containing 0.01-1.0 ng of cRNAs and maintained in Barth's solution at 17°C.
Electrophysiological recordings were performed 2-6 d after cRNA injection under two-electrode voltage clamp with a GeneClamp 500 amplifier (Molecular Devices, Union City, CA). Both voltage and current electrodes were filled with 3 M KCl and had resistances of 
1-2 M
. Data acquisition was performed with the use of a Digidata 1200 and pClamp 7.0 software (Molecular Devices). Data were analyzed with Clampfit from the pClamp 6.1 software. During electrophysiological recordings, the oocytes were superfused continuously (10 ml/min) with normal frog saline composed of the following (in mM): 115 NaCl, 2.5 KCl, 1.8 CaCl2, and 10 HEPES buffer, pH 7.2. Drugs were applied in the perfusion solution of the oocyte chamber. To minimize activation of the endogenous Ca2+-sensitive chloride current (Elgoyhen et al., 2001), we performed all experiments in oocytes incubated with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA-AM; 100µM) for 3-4 h before the electrophysiological recordings. Oocytes were clamped at a holding potential of -70 mV.
Concentration-response curves were fit with the following equation, using a nonlinear least square method:
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where x is the number of fitted components and can vary from one to four, I is the peak current at a given concentration of ACh (A), Imax is the maximum current, EC50 is the concentration of ACh yielding a half-maximum response, and n is the Hill coefficient. The best fit was estimated with the F test from the GraphPad Prism 4.00 for Windows (GraphPad Software, San Diego, CA).
Materials. ACh chloride was bought from Sigma (St. Louis, MO). BAPTA-AM (Molecular Probes, Eugene, OR) was stored at -20°C as aliquots of a 100 mM solution in dimethyl sulfoxide, thawed, and diluted 1000-fold into saline solution shortly before incubation of the oocytes.
All experimental protocols were performed in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication 80-23), revised in 1978.
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Results
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Mutations of a conserved valine at position 13' of TM2 of the
9 or 10 subunits increase the sensitivity to ACh of the 910 nAChR We examined the subunit stoichiometry of the recombinant 910 nAChR by analysis of the shifts in the EC50 values in receptors carrying the V13'T reporter mutation. Although previous reports have used reporter mutations at the 9' position of TM2 to analyze the stoichiometry of neuronal nAChR or GABAA receptors (Chang et al., 1996; Boorman et al., 2000), the 13' position was chosen in the case of 910 because bigger shifts in the EC50 values were obtained (Plazas et al., 2005). We have described previously that the V13'T 910 double-mutant receptor displays a dramatic increase in ACh sensitivity, assessed as the decrease in its EC50 value (Plazas et al., 2005). Figure 1 shows representative responses and concentration-response curves to ACh when either 9 or 10 subunits were mutated and expressed with the wild-type partner subunit. The curves for the double-mutant and wild-type receptors are included for comparison. Henceforth, the mutated subunits will be designated 9* and 10*. Mutant receptors exhibited an increase in the sensitivity to ACh, which was evidenced by a leftward shift in the concentration-response curves, a decrease in the EC50 value, and no major changes in the Hill coefficients. The EC50 and Hill coefficient values included the following: 910, 14.6 ± 1.3 µM and 1.1 ± 0.1 (n = 5); 9*10, 0.4 ± 0.1 µM and 1.1 ± 0.1 (n = 5); 910*, 0.5 ± 0.1 µM and 1.4 ± 0.1 (n = 5); 9*10*, 0.2 ± 0.1 µM and 1.1 ± 0.1 (n = 5). In addition, as reported previously (Plazas et al., 2005), mutant receptors exhibited a decrease in the rate of desensitization.
Studies in heteromeric (muscle and neuronal) nAChRs have concluded that the effects of mutations at position 9'of TM2 of the different subunits on the ACh EC50 are nearly independent, equivalent, and multiplicative (Filatov and White, 1995; Labarca et al., 1995; Boorman et al., 2000). In those cases, the stoichiometry can be deduced directly from the comparison of the shifts in the EC50 values produced by the introduction of one mutated subunit at a time with that of fully mutated receptors (Boorman et al., 2000). On the contrary, the EC50 values obtained for single and double mutants of the 910 nAChR at 9' (Plazas et al., 2005) and 13' indicate that at these positions both types of subunits contribute in an asymmetric and nonmultiplicative manner to the shift in EC50 values. This can be inferred from the fact that receptors containing only the 9 or the 10 mutant subunits showed the same decrease in the EC50 values, despite the fact that they cannot be in the same proportion in the pentameric assembly of a nAChR. We therefore followed the strategy described by Chang et al. (1996) for GABAA receptors, in which the deduction of the stoichiometry is independent from asymmetries in the contribution to the shifts in EC50 values produced by mutating individual subunits.
Coexpression of wild-type and mutant V13'T receptor subunits
Predictions and assumptions
If we assume there is only one 9 subunit in the 910 nAChR, coexpression of both 9 and 9* along with wild-type 10 subunits would result in an ACh concentration-response curve with two components (Fig. 2A): one from the activation of 910 receptors and the other from the activation of 9*10 receptors. In addition, on the basis of the data derived from Figure 1, the ACh EC50 values of the two components would be 15 and 0.4 µM, respectively. Alternatively, if the 910 nAChR contained two 9 subunits, the ACh concentration-response curve resulting from the expression of 9, 9*, and 10 subunits would have three components (Fig. 2B): one component from receptors in which both 9 subunits are wild type (EC50, 15 µM), one component from receptors in which both 9 subunits are mutant (0.4 µM), and a third one with an intermediate EC50 value from receptors with one wild-type and one mutant 9 subunit. If the mutation of each 9 subunit has an equivalent effect, the shift in the EC50 value contributed by each 9 subunit would be the square root of the shift in EC50 value observed when both 9 subunits are mutant (38-fold). Thus, each 9* subunit would contribute a 6.1-fold increase in the ACh sensitivity, predicting an intermediate component with an EC50 value of 2.5 µM. Similar logic could be applied for receptors containing three or four 9* or for receptors assembled from 10-mutated subunits.
The conclusions to be drawn from these experiments are dependent on the assumption that mutations do not alter the normal stoichiometry of the 910 receptor and that the different possible subunit arrangements of mutant and wild-type subunits within the complex are functionally equivalent. Although we have no direct evidence to prove this, it seems unlikely that a point mutation in a residue that is presumed to face the lumen of the channel (Miyazawa et al., 2003) would alter subunit interactions so drastically as to result in an atypical stoichiometry. Possible currents derived from homomeric 9 receptors were not taken into consideration, because they are almost undetectable when coexpressing 9 with 10 and are being recorded from BAPTA-treated oocytes (Elgoyhen et al., 2001).
Coexpression of 9*, 9, and 10 reveals the presence of two 9 subunits in the 910 receptor complex
Figure 3A shows the results from experiments in which 9*, 9, and 10 subunits were coexpressed in the same oocyte in a 1:1:2 cRNA ratio. The resulting concentration-response curve was well adjusted by the sum of three Hill equations. Table 1 shows the values of the EC50 and the percentage of maximal responses for each component. The EC50 value of the first component, 0.2 ± 0.1 µM, would correspond to that determined for 9*10 receptors (0.4 ± 0.1 µM) (Fig. 1), and that of the third component, 9.3 ± 1.5 µM, would correspond to that of the 910 receptor (14.6 ± 1.3 µM) (Fig. 1). The existence of an intermediate component with an EC50 value of 1.1 ± 0.1 µM close to the predicted one (2.5 µM) indicates that the 910 nAChR must contain two 9 subunits. This intermediate component would result from receptors containing one 9 and one 9* subunit.
To preclude the possibility of multiple indistinguishable intermediate components (resulting from the sum of multiple stoichiometries) in the concentration-response curve of Figure 3A, we varied the ratio of wild-type and mutant cRNA (9:9*), maintaining an equal overall (9 + 9*):10 ratio. If the intermediate component of the ACh concentration-response curve consists of more than one component, the variation of the 9 to 9* ratio should change the relative fractions of the different receptor combinations underlying the putative multiple intermediate components and consequently their EC50 values. Figure 3, B and C, shows the concentration-response curves to ACh obtained when the ratio of 9 to 9* was 3:1 and 1:3, respectively. At a 1:3 cRNA injection ratio, the concentration-response curves consisted of three components. The EC50 value of the intermediate component (1.8 ± 0.2 µM) was within the same range as that obtained at a 1:1 ratio. Moreover, the relative fractions of these three components varied in a manner that would be expected for the different cRNA ratios (e.g., the amplitude of the 9*10 component increased with an increase in the ratio of 9* to 9 cRNA) (Table 1). At a 3:1 9 to 9* cRNA ratio, only two components were observed: one corresponding to the second component and the other to the third. Moreover, the relative fraction of the second component increased and that of the first component was below the level of detection, as expected from an increase in the ratio of 9 to 9* (Table 1). The EC50 value of the intermediate component (2.5 ± 0.5 µM) was within the same range as that obtained at the other cRNA ratios.
Coexpression of 9, 10, and 10* reveals the presence of three 10 subunits in the 910 receptor complex Figure 4A shows the results from experiments in which 9, 10, and 10* subunits were coexpressed in the same oocyte in a 2:1:1 cRNA ratio. The resulting concentration-response curve was well adjusted by the sum of four Hill equations. Table 2 shows the values of the EC50 and the percentage of maximal responses for each component. The EC50 value of the first component, 0.6 ± 0.1 µM, would correspond to that determined for 910* receptors (0.5 ± 0.1 µM) (Fig. 1), and that of the fourth component, 22.7 ± 1.3 µM, would correspond to that of the 910 receptor (14.6 ± 1.3 µM) (Fig. 1). The existence of two intermediate components would indicate that the 910 nAChR contains three 10 subunits. If we assume that 10 and 10* equally contribute to the shift in the EC50 value from 910 to 910* receptors (30-fold), each 10 subunit should contribute with a 3.1-fold increase in the sensitivity to ACh. This would predict two intermediate components: one component with an EC50 value of 4.7 µM for receptors with two 10 and one 10* and another component with an EC50 value of 1.5 µM for receptors with one 10 and two 10*. These values are similar to those obtained experimentally, 5.0 ± 0.4 and 2.2 ± 0.2 µM, respectively (Table 2).
To preclude the possibility of multiple indistinguishable intermediate components in the concentration-response curve of Figure 4A, we varied the ratio of wild-type and mutant cRNA (10:10*), maintaining an equal overall 9:(10 + 10*) ratio. If the intermediate components of the ACh concentration-response curve consist of more than one component each, the variation of the 10 to 10* ratio should change the relative fractions of the different receptor combinations underlying the putative multiple intermediate components and consequently their EC50 values. Figure 4, B and C, shows the concentration-response curves to ACh obtained when the ratio of 10 to 10* was 3:1 and 1:3, respectively. At a 1:3 cRNA injection ratio, the concentration-response curves consisted of four components. The EC50 values of the intermediate components (1.9 ± 0.1 and 4.9 ± 0.4 µM) were similar to those obtained at a 1:1 ratio. Moreover, the relative fractions of these four components varied in a manner that would be expected for the different cRNA ratios (e.g., the amplitude of the 910* component increased with an increase in the ratio of 10* to 10 cRNA) (Table 2). At a 3:1 10 to 10* cRNA ratio, only three components were observed: two corresponding to the intermediate components and the other to the fourth. Moreover, the relative fraction of the fourth component increased and that of the first component was below the level of detection, as expected from an increase in the ratio of 10 to 10* (Table 1). The EC50 values of the intermediate components (1.1 ± 0.3 and 4.2 ± 0.3 µM) were similar to those obtained at a 1:1 ratio.
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Figure 2. Predicted results for receptors composed of either one or two 9 subunits. A, With one 9 subunit in the receptor complex, the ACh concentration-response curve from oocytes expressing 9,9*, and 10 subunits would be composed of two components (solid line): one component from the activation of 9*10 receptors and the other from 910 receptors (dotted lines).B, With two 9 subunits in the receptor complex, the ACh concentration-response curve from oocytes expressing 9, 9*, and 10 subunits would be composed of three components (solidline): one component from activation of 9* 10 receptors, one from 9* 9 10 receptors, and the other from 9 10 receptors (dotted lines). The asterisk denotes the mutated subunit. Each arrow indicates the predicted EC50 for each component.
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Together, the results so far support the notion that the 910 receptor is assembled from two 9 and three 10 subunits to form a pentameric structure, as reported for other nAChRs (Karlin, 2002). Slight changes in the EC50 values of the different components at different cRNA ratios might result from the difficulty of extracting precise EC50 values from the compound concentration-response curves as the amplitudes of the components vary. The amplitude of the components in these wild-type and mutant coexpression studies did not follow a binomial distribution, so possible explanations are considered in the Discussion.
The stoichiometry of the 910 nAChR does not depend on the relative abundance of 9 and 10 subunits
Alternate stoichiometries have been reported for nAChRs depending on either the subunit availability or the experimental conditions (Zwart and Vijverberg, 1998; Nelson et al., 2003). To analyze whether the 910 stoichiometry can vary depending on the abundance of the different subunits, we injected 9*10 at a 4:1 ratio and 910* at a 1:4 ratio (Table 3). If the increase in availability of mutant subunits would increase the number of mutant subunits incorporated to the receptor and therefore alter the final stoichiometry, the EC50 values should be smaller than those obtained at a 1:1 cRNA ratio. As we observed in Table 3, the EC50 values obtained at this 4:1 cRNA ratio were similar to those obtained at a 1:1 ratio. This result indicates that at least at these cRNA ratios the number of 9 and 10 subunits in the receptor complex is fixed with a stoichiometry of (9)2(10)3.
Assembly of 910 receptors with a mutation at position 17' of TM2 supports the (9)2(10)3 stoichiometry
We have described previously that a 910 double-mutant receptor in which the isoleucine at position 17' of the TM2 of 9 and the methionine 17' of 10 were mutated to threonine displays an increase ACh sensitivity (Plazas et al., 2005). Figure 5 shows concentration-response curves to ACh when either 9 or 10 subunits were mutated and expressed with the wild-type partner subunit. The curves for the double-mutant and wild-type receptors are included for comparison. Mutant receptors exhibited an increase in the sensitivity to ACh, which was evidenced by a leftward shift in the concentration-response curves, a decrease in the EC50 values, and no major changes in the Hill coefficients. The EC50 and Hill coefficient values included the following: 910, 14.6 ± 1.3 µM and 1.1 ± 0.1 (n = 5); 9*10, 5.5 ± 0.7 µM and 1.2 ± 0.2 (n = 5); 910*, 2.9 ± 0.1 µM and 1.1 ± 0.2 (n = 9); 9*10*, 1.1 ± 0.1 µM and 1.5 ± 0.2 (n = 4). Thus, mutations in either 9 or 10 lead to a 2.7- and 5-fold decrease in the EC50 values, respectively. The expected shift for the double-mutant receptor if both types of subunits contribute independently to the ACh sensitivity would be 13.5-fold. Interestingly, this agrees with the experimentally calculated decrease in the EC50 value (13.3-fold). Therefore, it can be postulated that at this position both types of subunits contribute independently and symmetrically to ACh sensitivity. As reported by Boorman et al. (2000), in this case, the shifts in the EC50 values of mutants could be used to calculate the stoichiometry of the receptor. Because the shift in the EC50 of the double mutant was 13.3-fold, the predicted shift produced by each mutant subunit incorporated in the receptor should be 1.7-fold. Hence, the incorporation of two mutant subunits should produce a 2.9-fold shift and that of three mutant subunits a 4.9-fold shift in the EC50 values. These predicted values are similar to those experimentally obtained: 2.7- and 5-fold for 9*10 and 910* mutants. These results confirm those obtained by the analysis of compound concentration-response curves with V13'T-mutated receptors and support the deduced (9)2(10)3 stoichiometry.
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Discussion
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In the present work, we describe for the first time that the recombinant 910 nAChR is assembled from two 9 and three 10 subunits. As far as we are aware, this is the first description of the stoichiometry of a heteromeric recombinant nAChR that includes only subunits and might contribute to the emerging knowledge of the structure of nAChRs (Karlin, 2002).
To establish the stoichiometry of the recombinant 910 nAChR, we used a modification of the method described by Chang et al. (1996) for GABAA receptors, which infers the number of subunits from the number of components in compound concentration-response curves to ACh derived from experiments with receptors assembled from wild-type and mutant subunits. The strategy of using mutant subunits that confer different single-channel properties to assembled receptors was used originally to determine the stoichiometry of the main CNS nAChR, (4)2(2)3 (Cooper et al., 1991). In the present experiments, the proportion of each component within the total population of receptors (e.g., 910, 99*10, 9*10) that can be deduced from the percentage of maximal response for each component in the compound concentration-response curves deviated from that predicted by a binomial distribution. For example, at a wild-type to mutant ratio of 9:9* of 1:1 (Table 1), the percentage of maximal responses included 11.8, 28.8, and 58.1% for the 9*10, 99*10, and 910 components, respectively. If we assume that the proportion of each component was to follow a binomial distribution, the predicted percentage of maximal responses should have been 25, 50, and 25%, respectively. Moreover, because in the V13'T mutant channel opening is favored and desensitization is decreased when compared with wild-type receptors (Plazas et al., 2005), one would expect that components including mutant subunits should be favored over the wild-type component, a prediction that is opposite to the observed experimental results. One possible explanation could be that the assembly and/or trafficking to the membrane is less efficient for receptors that include mutant subunits. However, this does not rule out the prediction that, when assembled, subunit arrangements of mutant and wild-type receptors within the pentameric complex are equivalent and therefore that the stoichiometry can be deduced from the number of components in the compound concentration-response curves. Mutations in the TM2 are not expected to influence the correct assembly directly, because the sequence determinants for subunit assembly are not in the pore region (Gu et al., 1991; Yu and Hall, 1994; Kreienkamp et al., 1995). Moreover, our conclusions are strengthened by the agreement between the observed and predicted EC50 values. In the case of GABAA receptors that assemble from 9' mutant subunits, a similar deviation from the binomial distribution has been observed (Chang et al., 1996).
Two different approaches have shown independently that chick 42 nAChRs have a stoichiometry of (4)2(2)3 when expressed in Xenopus laevis oocytes from cRNAs or cDNAs injected at a 1:1 to ratio (Anand et al., 1991; Cooper et al., 1991). However, alternate stoichiometries might exist, depending on the experimental conditions. Thus, increasing the amount of transfected 2 cDNA to human kidney cells, as well as an overnight incubation in nicotine or an overnight culture at 29°C, results in an increase in receptors with a (4)3(2)2 stoichiometry. Moreover, when the rat 4:2 subunit ratio is varied from 1:1 to 1:9, nAChRs of more than one functional class are formed in oocytes (Zwart and Vijverberg, 1998). In addition, heterogeneity of neuronal nAChRs has been suggested from the observation that the occurrence of distinct single-channel conductances in oocytes expressing neuronal-type nAChRs depends on the : ratio (Papke et al., 1989). The existence of alternate stoichiometries for the same assembled subunits with distinct functional properties raises the possibility for an interesting mode of synaptic regulation of nicotinic signaling in the mammalian brain (Nelson et al., 2003).
Our results indicate that up to a 1:4 or a 4:1 ratio of 9 to 10, derived from the analysis of shifts in the EC50 values, only one functional class of receptor is formed with a fixed stoichiometry. We cannot preclude the possibility that, under different experimental conditions or at extreme differences in the rate of transcription of the genes coding for the 9 and 10 subunits, alternate stoichiometries might exist. However, because both 9 and 10 subunits are expressed at high levels in outer hair cells (Elgoyhen et al., 1994, 2001; Simmons and Morley, 1998; Morley and Simmons, 2002), it is unlikely that one subunit might be a limiting factor for the assembly of the more abundant representative form of the receptor. In addition, although the transcription of the gene coding for the 10 (but not the 9) gene undergoes drastic developmental changes before the onset of hearing (second postnatal week) in rats (Morley and Simmons, 2002; Katz et al., 2004), the properties of the native 910-containing inner hair cell receptor highly resemble those of heteromeric 910 expressed in Xenopus oocytes (Gomez-Casati et al., 2005).
Extrapolating the stoichiometry of native receptors from the present results would be too far-fetched. However, the biophysical and pharmacological properties of native receptors mirror those of the recombinant 910 nAChR expressed in Xenopus oocytes (Elgoyhen et al., 1994, 2001; Gomez-Casati et al., 2005), thus arguing in favor of the hypothesis that native and recombinant receptors might have a similar stoichiometry. Moreover, cross-linking, affinity chromatography purification, or immunopurification experiments with receptors extracted from native tissue, like those performed for other Cys-loop receptors (Hucho et al., 1978; Einarson et al., 1982; Whiting et al., 1987; Langosch et al., 1988; Keyser et al., 1993), will result in a challenge in the case of 910 native nAChRs because of the limited number of hair cells per cochlea. Thus, the present experiments might be the best approximation to start elucidating the molecular arrangement of the 910 nAChR.
The order of subunits around the channel in the neuronal 42 nAChR is presumed to be 42422 by homology with the 
organization of the Torpedo californica and muscle nAChRs (Karlin et al., 1983; Karlin, 2002; Le Novere et al., 2002; Miyazawa et al., 2003). This provides two ACh-binding sites at specific interfaces of 4 and 2 subunits and allows the third 2, which is not involved in forming an ACh-binding site, to occupy a position equivalent to that of muscle or Torpedo californica subunits. The much stronger affinity labeling with competitive antagonists of 1 compared with that of the and subunits supports an asymmetric location of the binding site with respect to the interface. This has led to the proposal to refer to the 1 subunits as carrying the "principal component" and the or subunits as contributing to the "complementary component" of the nicotinic binding site (Le Novere et al., 2002). By comparison, the subunit would contribute to the complementary component in the neuronal nAChRs. By homology, and when we take into account that 9 nAChR subunits can form homomeric channels whereas 10 cannot and that 10 behaves as a structural subunit in the 910 heteromer to render a receptor with distinct properties (Elgoyhen et al., 2001), it is tempting to speculate an 91091010 arrangement within the pentamer. However, it has been reported that subunit chimeras constructed to contain the extracellular ligand-binding domain of the 9 or 10 subunits fused to the C-terminal domain of the 5-hydroxytryptamine type 3A subunit bind the nicotinic radioligand [3H]methyllycaconitine with specific high affinity (Baker et al., 2004). Therefore, either 9 or 10 or both could contribute to the main component of the binding site, resulting in alternative arrangements. Additional experiments, including the analysis of receptors formed from linked subunits (Baumann et al., 2001; Zhou et al., 2003), will be necessary to elucidate the arrangement of subunits within the 910 nAChR. Although the potential subunit arrangement and interaction underlying the binding of ACh and gating of this special receptor are yet to be elucidated, knowledge of the stoichiometry will facilitate structure-function studies tending to elucidate a model for the 910 receptor-channel complex.
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Footnotes
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Received Sep 8, 2005;
revised October 4, 2005;
accepted October 6, 2005.
This work was supported by an international research scholar grant from the Howard Hughes Medical Institute, National Institutes of Health Research Grant R03TW006247 from the Fogarty International Center and the National Institute on Deafness and Other Communication Disorders, the National Organization for Hearing Research, an H. F. Lenfest grant in Auditory Science, and a research grant from the National Agency of Scientific and Technological Promotion (ANPCyT) and University of Buenos Aires (Argentina) to A.B.E. This work was also supported by grants from ANPCyT, National Council of Scientific and Technical Investigations, and National University of the South (Argentina) to C.B.
Correspondence should be addressed to Dr. Ana Belén Elgoyhen, Institute of Investigations in Genetic Engineering and Molecular Biology, University of Buenos Aires, Vuelta de Obligado 2490, Buenos Aires 1428, Argentina. E-mail: elgoyhen{at}dna.uba.ar.
Copyright © 2005 Society for Neuroscience 0270-6474/05/2510905-08$15.00/0
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Abstract
Introduction
