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Determinants of Zinc Potentiation on the 4 Subunit of Neuronal Nicotinic Receptors

Departments of Molecular & Cellular Pharmacology (B.H., K.B.M., S.E.R., D.E., A.H.M., C.W.L.) and Biochemistry & Molecular Biology (A.M.), Miller School of Medicine, University of Miami, Miami, Florida

Received June 9, 2005; accepted September 27, 2005

	Abstract

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We have shown previously that the function of neuronal nicotinic acetylcholine receptors can be modulated by zinc. This modulation varies from potentiation to inhibition, depending on receptor subunit composition and zinc concentration, with the 42 and 44 receptors displaying the most dramatic potentiation. In this study, we used site-directed mutagenesis to identify glutamate 59 and histidine 162 on the rat 4 subunit as potential mediators of zinc potentiation. By modeling the extracellular domain of the receptor pentamer, we locate these residues to two subunit-subunit interfaces that alternate with the two acetylcholine-binding interfaces. Substitution of a cysteine at either position allows additional reduction of zinc potentiation upon treatment with the methanethiosulfonate reagents N-biotinoylaminoethyl methanethiosulfonate (MTSEA-biotin) and [2-(trimethylammonium)ethyl] methanethiosulfonate. Mutagenesis and methanethiosulfonate treatment are most effective at position 162, and the presence of zinc hinders the reaction of MTSEA-biotin with the substituted cysteine at this position, suggesting that 4His162 participates in forming a coordination site for zinc. Mutagenesis and methanethiosulfonate treatment are less effective at position 59, suggesting that whereas 4Glu59 may be near the zinc coordination site, it may not be participating in coordination of the zinc ion. It is noteworthy that the position of 4Glu59 within the neuronal nAChR is identical to that of a residue that lines the benzodiazepine-binding site on GABA_A receptors. We suggest that the zinc potentiation sites on neuronal nAChRs are structurally and functionally similar to the benzodiazepine-binding sites on GABA_A receptors.

Ionic zinc is found in neurons throughout the brain, with highest concentrations in the cerebral cortex and limbic areas (Frederickson et al., 2000). Zn²⁺ is packaged in synaptic vesicles and, upon neuronal stimulation, is released in a calcium-dependent manner (Assaf and Chung, 1984; Howell et al., 1984). Estimates of extracellular [Zn²⁺] during neuronal activity vary from 1040 µM (Li et al., 2001) to 100300 µM (Assaf and Chung, 1984; Vogt et al., 2000). Zn²⁺ modulates the function of neuronal nAChRs (Palma et al., 1998; Garcia-Colunga et al., 2001; Hsiao et al., 2001) and other ligand-gated ion channels (Huang, 1997), suggesting that Zn²⁺ is a modulator of synaptic activity. Indeed, synaptically released Zn²⁺ modulates synaptic activity in the hippocampus (Vogt et al., 2000; Ueno et al., 2002).

Some neuronal nAChRs, such as 42 and 44, are potentiated by micromolar Zn²⁺ and inhibited by millimolar Zn²⁺ (Hsiao et al., 2001). Others, such as 32 (Hsiao et al., 2001) and 7 (Palma et al., 1998), are only inhibited by Zn²⁺. Results with chimeric receptor subunits, and the sensitivity of potentiation to diethylpyrocarbonate and changes in pH, indicate that extracellular histidines participate in forming the potentiating Zn²⁺ binding site(s) (Hsiao et al., 2001). In this study, we combined conventional mutagenesis, the substituted cysteine accessibility method (SCAM), and protein modeling to identify residues that are determinants of Zn²⁺ potentiation. Our results suggest that neuronal nAChRs bind ACh and Zn²⁺ at alternating subunit-subunit interfaces.

	Materials and Methods

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Molecular Biology. Mutations were introduced into rat neuronal nAChR subunits using the GeneEditor in vitro site-directed mutagenesis system (Promega, Madison, WI). All mutations were confirmed by sequencing. Capped cRNA encoding wild-type and mutant subunits was generated using mMessage mMachine kits (Ambion, Austin, TX).

Preparation of Oocytes and cRNA Injection. Oocytes were surgically removed from mature Xenopus laevis frogs (Nasco, Fort Atkinson, WI). The care and use of X. laevis frogs in this study were approved by the University of Miami Animal Research Committee and met the guidelines of the National Institutes of Health. Follicle cells were removed by treatment with Collagenase B (Roche Diagnostics, Indianapolis, IN) for 2 h at room temperature. Stage V oocytes were injected with 0.5 to 10 ng of each cRNA (at a molar ratio of 1:1) in 15 to 50 nl of water and incubated at 18°C in Barth's saline (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 0.3 mM CaNO₃, 0.41 mM CaCl₂, 0.82 mM MgSO₄, 15 mM HEPES, pH 7.6, and 100 µg/ml gentamicin) for 2 to 7 days.

Electrophysiology and Data Analysis. Current responses were measured under two-electrode voltage clamp, at a holding potential of –70 mV, using TEV-200 voltage clamp units (Dagan, Minneapolis, MN). Micropipettes were filled with 3 M KCl and had resistances of 0.3 to 2.0 M. Current responses, filtered (8-pole, Bessel low pass) at 20 Hz (–3 db) and sampled at 100 Hz, were captured, stored, and analyzed using a Digidata 1322A (Molecular Devices, Sunnyvale, CA) in conjunction with either a Macintosh G3 computer running Axograph 4.6 software (Molecular Devices) or a Pentium III PC running pClamp 8 (Molecular Devices). Oocytes were perfused at room temperature (20–25°C), in a chamber constructed from Tygon tubing (inner diameter, 0.125 inches), with perfusion solution (115 mM NaCl, 1.8 mM CaCl₂, 2.5 mM KCl, 0.0001 mM atropine, and 10 mM HEPES, pH 7.2). Perfusion was continuous (except during MTSEA-biotin applications) at a rate of 4 ml/min. ACh alone and in combination with Zn²⁺ was applied diluted in perfusion solution. Some experiments were conducted using an OpusXpress 6000A Parallel Oocyte Voltage Clamp system running OpusXpress 1.1 and Clampfit 9.1 software (Molecular Devices). In these experiments, all perfusion and application of ACh and Zn²⁺ was handled by the OpusXpress system. All Zn²⁺-containing solutions were freshly prepared from a 1 M stock of Zn(CH₃COO)₂. In previous work, no difference was seen between the effects of Zn(CH₃COO)₂-and ZnCl₂-containing solutions (Hsiao et al., 2001). X. laevis oocytes express a Ca²⁺-activated Cl^– channel that can contribute to whole-cell current responses when Ca²⁺ permeable channels, such as neuronal nAChRs, are activated. In previous work (Hsiao et al., 2001), we found that the extent of zinc potentiation did not vary across a range of holding potentials (–90 to –40 mV). Because the Cl^– channel current amplitude varies dramatically over this voltage range, our results led us to conclude that the Ca²⁺-activated Cl^– channel does not underlie or affect measurement of zinc potentiation of neuronal nAChRs expressed in X. laevis oocytes. For this reason, we have chosen not to attempt to inhibit the Ca²⁺-activated Cl^– channel.

Wild-type and mutant 44 receptors displayed little or no desensitization in response to the low ACh concentrations used here, allowing measurement of Zn²⁺ potentiation as follows. Control current in response to ACh was determined from a 1-s average beginning 29 s after initiation of agonist application and compared with a 1-s average of baseline current immediately before ACh application. Current levels during Zn²⁺ coapplication were determined from a 1-s average beginning 29 s after initiation of Zn²⁺ application and compared with the control current. Potentiation of wild-type 44 and all mutants (with one exception, see below) was determined with 1 µM ACh, which lies between the EC₁ and EC₆ for each receptor. We have previously found that the extent of zinc potentiation of wild-type 44 does not vary across this portion of the ACh dose-response curve (Hsiao et al., 2001). For one mutant (4-2E59C 4C75S), 1 µM ACh was the EC_0.2. For this receptor, we used 5 µM ACh (the EC₂) to test potentiation.

Wild-type and mutant 42 receptors displayed substantial desensitization upon exposure to ACh, requiring measurement of potentiation or inhibition as described previously (Hsiao et al., 2001). In brief, the initial 30-s ACh response in the absence of Zn²⁺ was fit to a single exponential decay function. This fit was projected over the next 30 s during which both ACh and Zn²⁺ were coapplied. The degree of modulation was measured by taking a 1-s average 29 s after initiation of Zn²⁺ application and comparing it with a 1-s average of the projected response to ACh alone during the same time period. Thus, both Zn²⁺ and control values were taken 59 s after the initiation of the experiment. Potentiation of wild-type 42 and all mutants was determined with 10 µM ACh, which lies between the EC₁₄ and EC₂₄ for each receptor. We have previously found that the extent of zinc potentiation of wild-type 42 does not vary across this portion of the ACh dose-response curve (Hsiao et al., 2001). A similar extent of potentiation was also seen at the lower ACh concentration of 3 µM (the EC₃) (Hsiao et al., 2001).

In the SCAM experiments in Fig. 4, A to C, Zn²⁺ potentiation was measured before and after a 2-min incubation with 2 mM MTSEA-biotin (Toronto Research Chemicals, Inc., North York, ON, Canada). MTSEA-biotin was diluted from a dimethyl sulfoxide stock solution into perfusion solution immediately before application. The final DMSO concentration of 0.5% had no effect on ACh responses or zinc potentiation (data not shown). After the incubation, oocytes were rinsed for 5 min with perfusion solution before measuring Zn²⁺ potentiation. The high concentration of MTSEA-biotin and relatively long duration of the incubation were chosen to ensure saturation. Indeed, application of fresh reagent for a further 5-min incubation failed to cause any further loss of potentiation (data not shown). In some experiments, 1 mM MTSET was used.

View larger version (16K): [in this window] [in a new window]   Fig. 4. SCAM analysis at the Zn2+ potentiation site. A, potentiation of the ACh (1 µM) response of an 4-24C75S (pseudo–wild-type) expressing oocyte by 100 µM Zn2+ before and after a 2-min incubation with 2 mM MTSEA-biotin (scale, 100 nA, 10 s). B, potentiation of the ACh (1 µM) response of an 4-2H162C,4C75S-expressing oocyte by 100 µM Zn2+ before and after a 2-min incubation with 2 mM MTSEA-biotin (scale, 100 nA, 10 s). C, potentiation of the 4E59C, 4H61C, and 4H162C mutants (each expressed within the context of the pseudo–wild-type receptor, 4-24C75S) by 100 µM Zn2+, measured after a 2-min incubation with 2 mM MTSEA-biotin, is plotted as a percentage of pretreatment potentiation (mean ± S.E.M., n = 3–5). Significant differences from pretreated: *, p < 0.05; ***, p < 0.001. D, reaction rates for MTSEA-biotin at 4-2H162C 4C75S in the presence and absence of 100 µM Zn2+. Potentiation of the response to 1 µM ACh was measured before and after successive 5-s applications of 1 µM MTSEA-biotin. Results are presented as the ratio of post-treatment potentiation to the initial, pretreatment potentiation. Values are the mean ± S.E.M. from three to six oocytes and are fit to an exponential decay equation (see Materials and Methods).

Both ACh and Zn²⁺ dose-response curves were fit according to the equation I = I_max/[1 + (EC₅₀/X)^nH] where I represents the current response at a given concentration of ACh or Zn²⁺, X; I_max is the maximal response; EC₅₀ is the concentration of ACh or Zn²⁺ yielding a half-maximal response; n_H is the Hill coefficient. Zinc inhibition data (see Results) was fit according to the equation: I = I_max/[1 + (X/IC₅₀)^nH] where I represents the current response at a given metal concentration, X, I_max is the maximal current, IC₅₀ is the concentrations of metal yielding half-maximal inhibition, and n_H is the Hill coefficient. Data presented in Fig. 3B was fit to a more complex equation that included both a potentiating and an inhibitory site: I = I_min + (I_max – I_min){[1/(1 + (EC₅₀/X)^nH)] – [1/(1 + (IC₅₀/X)^mH)]}, where I represents the current response at a given metal concentration, X, I_min is the minimal current, I_max is the maximal current, EC₅₀ and IC₅₀ are the concentration of metal yielding half-maximal potentiation and inhibition, respectively, and n_H and m_H are the Hill coefficients for potentiation and inhibition, respectively.

View larger version (22K): [in this window] [in a new window]   Fig. 3. A, the effects of a range of zinc concentrations on wild-type 44 (), 4E59A4 (), 4H61N4 (), 4H162G4(), and 44H469Y () receptors are plotted as a percentage of the response to 1 µM ACh alone. Values are the mean ± S.E.M. from 3 to 6 oocytes and are fit to a Hill equation (see Materials and Methods). B, the potentiating and inhibiting phases of the action of zinc on 4H162G4 receptors is shown. Values are the mean ± S.E.M. from three oocytes and are fit to a dual-site Hill equation (see Results for an explanation).

Molecular Modeling. Sequence alignment of the amino-terminal extracellular region of the rat 4 and 4 neuronal nAChR subunits with the AChBP was performed using the Alignx module of Vector NTI 5 (InforMax, Inc., North Bethesda, MD). The alignments between the AChBP and 4 and 4 had identity values of 20.4 and 19.4%, respectively. Although the sequence identity between the AChBP monomer and amino-terminal extracellular domains of various nAChR subunits is relatively low, the presence of highly conserved ACh binding residues in the AChBP (Brejc et al., 2001), and the nicotinic pharmacology of the AChBP (Smit et al., 2001) suggests that homology modeling of neuronal nAChR extracellular domains using the AChBP structure is appropriate (Le Novere et al., 2002).

Three-dimensional models were constructed using the program Modeller 6 (Sali and Blundell, 1993) on a Silicon Graphics Indigo2 Extreme workstation. The script "model" was used with neuronal nAChR subunit/AChBP alignments. Disulfide bonds in the AChBP template structure were explicitly included during homology model refinement. The amino-terminal extracellular domain sequences of the 4 and 4 subunits were modeled using the AChBP pentamer structure (Brejc et al., 2001) (Protein Data Bank code 1I9B [PDB] ) to get initial coordinates for an 44 pentamer (subunit ordering of ). Ten models were produced with energy refinement handled within the program. Conditions were optimized such that resulting structures exhibited energies in line with current published nicotinic receptor homology models (Le Novere et al., 2002; Everhart et al., 2003). The lowest energy structure was then inspected visually and with Procheck (Laskowski et al., 1993) for inappropriate stereochemistry (clashing side chains, disallowed torsion angles, etc.). In only one case did a residue require manual adjustment using the O software package (Jones et al., 1991). Further minimization was then carried out using the CNS software package (Brünger, 1998), with 20 cycles of conjugate gradient. The CNS minimized structure was then reanalyzed with Procheck to ensure stereochemical soundness. The images in Figs. 1B and 6 were produced using Ribbons (Carson, 1997). Coordinates for the final 44 model may be obtained at http://chroma.med.miami.edu/pharm/faculty_Luetje.html.

View larger version (60K): [in this window] [in a new window]   Fig. 1. A, the effect of Zn2+ varies with receptor subunit composition. ACh induced current responses of oocytes expressing the indicated neuronal nAChRs before, during, and after coapplication of 100 µM Zn2+. ACh concentrations for each receptor are 32 = 4 µM (EC10), 34 = 17 µM (EC2), 42 = 10 µM (EC14), 44 = 1 µM (EC2). Scale bars, 250 nA, 10 s. B, two subunits, 4 (blue) and 4 (green), taken from our homology model of the pentameric 44 receptor extracellular domain are shown. Residues tested in this study are indicated in red. 4Cys594 and 4His469 are not shown because they are not within the homology model. C, alignment of the N-terminal extracellular domains (preceding transmembrane I) of 4, 3, 4, and 2, and the C-terminal extracellular tails (after transmembrane IV) of 4–1, 4–2, 3, 4, and 2. Residues tested in this study are indicated in red.

View larger version (50K): [in this window] [in a new window]   Fig. 6. Neuronal nAChRs bind zinc and ACh at alternating subunit-subunit interfaces. A, zinc-binding residues at a 4-4 interface. Residues 4Glu59 and 4His162 are shown. B, the extracellular domain of the 44 pentamer is shown viewed from above. 4 subunits are green, 4 subunits are blue. Side chains of several residues that are conserved features of ACh binding sites (4-Tyr93, Trp149, Tyr190, Tyr197, and 4-Trp59) are shown in purple. Side chains of 4Glu59 and 4His162 are shown in red.

	Results

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Amino acid residues that most commonly form Zn²⁺-binding sites include histidines, free cysteines, aspartates, and glutamates (Glusker, 1991). Our previous work indicated the involvement of extracellular histidine residues (Hsiao et al., 2001), so we used site-directed mutagenesis to test the role of the seven histidine residues unique to the extracellular portions of 4 and 4. We also examined the two unique extracellular free cysteine residues. Our screen included five residues within the amino-terminal extracellular domain of 4 (His2, His61, His104, His109, His162), two residues within this region of 4 (Cys75, His157), one residue within the carboxyl-terminal extracellular portion of 4 (Cys594), and one residue within the carboxyl-terminal extracellular portion of 4 (His469). At some positions, the residue was changed to the residue present at that position in 3 or 2, as appropriate. For 4His2, 4His104, and 4Cys75, the residues present in 3 and 2 might also be Zn²⁺-coordinating (glutamate, aspartate, and aspartate, respectively). Thus, in addition to testing 4H2E and 4H104D mutants, we also tested the alanine mutants 4H2A and 4H104A. For 4Cys75, we tested a serine mutant. To test the role of 4Cys594, we used a naturally occurring splice variant (4-2) that lacks this C-terminal cysteine (Goldman et al., 1987). The positions of these residues within an alignment of 3, 4, 2, and 4 are shown in Fig. 1C. To visualize the positions of these residues within the receptor structure, we generated a homology model of the extracellular domain of the (4)₂(4)₃ pentamer using the AChBP (Brejc et al., 2001) as a template structure (see Materials and Methods). Many of these residues are located at the non–ACh-binding subunit-subunit interfaces within the pentamer (Fig. 1B).

We used the 44 receptor as our primary target to allow analysis of 4 and 4 residues in the same receptor. The 44 receptor is also convenient because it displays the greatest degree of zinc potentiation, expresses well in oocytes, and shows little or no desensitization in response to low ACh concentrations. The role of critical residues in 4 was also confirmed with 42 (see below), a major nAChR subtype in the mammalian central nervous system (Corringer et al., 2000). Mutant 4 and 4 subunits were coexpressed in X. laevis oocytes with wild-type 4 or 4 subunits, respectively. ACh dose-response curves, constructed for each mutant receptor, showed that the mutations had little or no effect on ACh sensitivity (Table 1). We used 100 µM Zn²⁺ to test the effect of each mutation because 100 µM Zn²⁺ lies at the peak of the biphasic potentiation-inhibition curve for 44 (Hsiao et al., 2001). Thus, reductions in maximal potentiation or shifts in the curve would be detected. Compared with wild-type 44 receptors, some mutant receptors (such as 4H162G4) displayed reduced potentiation by 100 µM Zn²⁺ (45 ± 4% of wild-type), whereas others (such as 44C75S) displayed potentiation similar to that of wild-type 44 (Fig. 2A). In addition to 4H162G, the 4H61N, and 4H469Y mutations also caused significant decreases in potentiation by 100 µM Zn²⁺ (Fig. 2B, Table 2). The 4-24 receptor (which lacks the C-terminal cysteine) displayed an increase in potentiation, suggesting potential involvement in the allosteric pathway. However, because we are focused on identifying the zinc potentiation site, we chose not to pursue this possibility. In our model of the 44 extracellular domain, the side chains of 4Asp195 and 4Glu59 are near the side chain of 4His162 (Fig. 1B). Because Asp195 is conserved in both the 2 and 4 subunits, we substituted an alanine at this position. We also prepared a 4E59A mutant. The 4E59A4 receptor displayed significantly reduced potentiation by 100 µMZn²⁺, whereas the 44D195A receptor did not differ from wild-type 44 (Fig. 2B). The results of this mutagenesis screen identify 4Glu59, 4His61, 4His162, and 4His469 as candidates for involvement in forming the zinc potentiation site.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Mutation analysis to identify candidate Zn2+ coordinating residues. A, current responses of oocytes expressing wild-type 44 (left), 44C75S (center), or 4H162G4 (right) to 1 µM ACh before, during, and after coapplication of 100 µM Zn2+. Scale bars: 44 and 4H162G4, 200 nA and 20 s; 44C75S, 100 nA and 20 s. B, potentiation of mutant receptors by 100 µM Zn2+. Results are presented as the ratio of mutant receptor potentiation to the potentiation of wild-type receptor in the same batch of oocytes (mean ± S.E.M., n = 3–14). The ACh concentration for all receptors is 1 µM. Significant differences in Zn2+ potentiation compared with wild-type 44 are indicated (***, p < 0.001; **, p < 0.01; *, p < 0.05).

In Fig. 3A, we examined the effects of a range of zinc concentrations on wild-type and mutant 44 receptors. The 4E59A, 4H61N, 4H162G, and 4H469Y mutations each reduced maximal potentiation by Zn²⁺, but none of the mutations increased the EC₅₀ for zinc potentiation, as might be expected if the mutations were damaging a zinc-binding site (Table 2). However, a simple rightward shift in the zinc dose-response curve could be expected only if zinc potentiation were occurring in isolation. We have previously shown that zinc both potentiates and inhibits these receptors at separate sites. At and above 300 µM Zn²⁺, receptor inhibition of the 44 receptor becomes apparent, leading to a distinctly biphasic Zn²⁺ dose-response curve (Hsiao et al., 2001). If the mutations were causing damage to the potentiation site but leaving the inhibition site intact, a rightward shift in the potentiation curve could be obscured.

To investigate this issue in more detail, we examined the effects of a wider range of Zn²⁺ concentrations on the 4H162G4 mutant (Fig. 3B). Obtaining accurate values from fitting biphasic zinc dose-response data to a two-site equation is difficult because of the close proximity of potentiating and inhibiting phases (Hsiao et al., 2001). However, in this earlier study, we found that diethylpyrocarbonate treatment eliminates potentiation, without affecting inhibition, of the 44 receptor. We fit these data to a single-site inhibition equation to obtain an IC₅₀ for Zn²⁺ inhibition of 362 ± 70 µM (see Materials and Methods). We then fit the biphasic zinc dose-response data for wild-type 44 in our earlier study to a two-site equation (see Materials and Methods) using this IC₅₀ value as a constant. This allowed us to estimate parameters for zinc potentiation of the wild type 44 receptor (EC₅₀ = 110 ± 33 µM, n_H = 0.96 ± 0.04, maximal potentiation = 1400 ± 250%). The EC₅₀ value is 4-fold greater and the maximal potentiation value 2-fold greater than what we obtain when we fit the potentiating phase of the data to a single site equation. This suggests that the inhibiting phase does indeed partially obscure the potentiation phase. Because zinc inhibition occurs at a separate class of site (Hsiao et al., 2001), it is unlikely to be affected by mutations at the zinc potentiation site. This allows us to use the inhibition parameters obtained from fitting the postdiethylpyrocarbonate treatment wild-type 44 receptor when fitting the data obtained for the 4H162G4 mutant. If we also assume that maximal potentiation is not changing, we find that upon fitting to a two-site equation, the EC₅₀ for zinc potentiation of the 4H162G4 mutant is greater (267 ± 7 µM) than the value for wild-type 44. This analysis suggests, but does not prove, that the 4H162G4 receptor is indeed less sensitive to zinc potentiation than wild-type 44. To prove this conclusively using dose-response analysis, we would need to be able to examine potentiation in the absence of inhibition. However, we have been unable to eliminate, or even damage, the inhibition of nAChRs by zinc (Hsiao et al., 2001). None of the mutations we have examined reduces inhibition by high concentrations of zinc (data not shown). Thus, we turned to other approaches to provide additional information about the role of candidate residues in forming the zinc potentiation site.

First, we examined the effect of double mutant combinations, reasoning that simultaneous mutation of two residues involved in mediating zinc potentiation should yield an effect greater than with either single mutation. Because the 4H162G mutant displayed the greatest loss of Zn²⁺ potentiation, we used this receptor as a point of reference. Each of the other three mutations was examined as a double mutant with 4H162G. The 4H61N and 4H469Y mutations failed to alter potentiation of the 4H162G 4 receptor. Potentiation of these double mutants by 100 µM Zn²⁺ was 107 ± 7 and 99 ± 7% of the potentiation of the 4H162G single mutant, respectively. This result suggests that these residues might not play important roles in mediating Zn²⁺ potentiation. In contrast, receptors formed by the double mutant 4E59A,H162G displayed significantly decreased potentiation compared with the potentiation of 4H162G (81 ± 5% of the single mutant potentiation, p < 0.05, n = 13). It is noteworthy that potentiation was not completely eliminated. Receptors formed by the 4E59A,H162G double mutant retained a modest ability to be potentiated by Zn²⁺ (36% of wild-type; see Discussion).

As an alternative approach to testing the role of candidate residues in forming a zinc potentiation site, we turned to SCAM (Karlin and Akabas, 1998). In SCAM analysis, a cysteine is placed at the position of interest and function is then measured before and after reaction with a methanethiosulfonate (MTS) reagent. Although free cysteines can participate in coordination of Zn²⁺, they often do not substitute effectively for other coordinating residues (Paoletti et al., 2000), probably because of the stringent spatial requirements for the relevant atoms (S, N, and O) to be able to coordinate the zinc ion. The result of our double-mutant experiments (see above) suggest that even if a substituted cysteine failed to directly participate in Zn²⁺ coordination (thus constituting a single mutant), the functional damage to the site would be partial. A further reduction of Zn²⁺ potentiation after reaction with an MTS reagent would then suggest a physical proximity to the zinc ion and would strengthen the case for direct coordination. As the MTS reagent for most of our experiments, we used MTSEA-biotin, a large, relatively membrane impermeant MTS reagent that has been used to characterize various sites on GABA_A receptors (Teissere and Czajkowski, 2001; Wagner and Czajkowski, 2001). To avoid the potential for confounding effects of the MTS reagent acting at other free cysteines, the 4-2 4C75S mutant receptor, which lacks extracellular free cysteine residues, was used as a "pseudo–wild-type" background (Karlin and Akabas, 1998) in which to test each cysteine mutant. This pseudo–wild-type receptor displayed ACh sensitivity and Zn²⁺ potentiation similar to that of true wild-type 44 and was unaffected by MTSEA-biotin treatment (Tables 1 and 2; Fig. 4, A and C).

We prepared E59C, H61C, and H162C mutants, each within the context of the pseudo–wild-type receptor. Each mutation resulted in significantly reduced Zn²⁺ potentiation (61 ± 5, 63 ± 9, and 77 ± 2% of pseudo–wild-type potentiation, respectively), similar to what was seen with the E59A, H61N, and H162G mutations. MTSEA-biotin treatment (2 mM, 2 min) had no significant effect on the ACh responses of any of the mutant receptors (data not shown). MTSEA-biotin also had no effect on Zn²⁺ potentiation of the 4H61C receptor (Fig. 4C), again suggesting that 4His61 may not play an important role in mediating Zn²⁺ potentiation. In contrast, MTSEA-biotin treatment significantly reduced potentiation of the 4E59C and 4H162C receptors, suggesting that 4Glu59 and 4His162 are located at or near the Zn²⁺-binding site (Fig. 4, B and C). However, as was the case for the double mutant, these MTSEA-biotin treated mutant receptors retained some ability to be potentiated by Zn²⁺. In Fig. 4, we used 1 µM ACh to test potentiation of the pseudo–wild-type receptor, as well as the 4H61C and 4H162C receptors. However, we used the equipotent concentration of 5 µM ACh to test potentiation of the 4E59C (see Materials and Methods). We also tested the ability of MTSEA-biotin to reduce zinc potentiation of the 4E59C using 1 µM ACh, finding post-treatment potentiation to be 76 ± 4% of pretreatment potentiation (p < 0.05, n = 6). Zinc potentiation could also be reduced by MTSET application (1 mM, 2 min). Although the trimethylammonium ethyl group deposited by MTSET is substantially smaller than the biotinylaminoethyl group deposited by MTSEA-biotin, it does have a positive charge that would be likely to interfere with Zn²⁺ binding. Post-treatment potentiation of 4-2H162C 4C75S by 100 µM Zn²⁺ was 71 ± 3% of pretreatment potentiation (n = 6, p < 0.001) and for 4-2E59C 4C75S, the value was 83 ± 3% of pretreatment potentiation (n = 3, p < 0.05). The effects of MTSEA-biotin and MTSET on zinc potentiation of the 4E59C mutant were substantially less than the effects on the 4H162C mutant. This may be because substitution of a cysteine for 4Glu59 had a greater effect on zinc potentiation than did substitution of 4His162 (61 ± 5 and 77 ± 2% remaining potentiation, respectively). Thus, there is less remaining potentiation to be affected by the MTS reagent. However, it is also possible that although 4Glu59 may be near the site of zinc binding, it might not be directly participating in coordination of the zinc ion.

SCAM can also be used to obtain information about the relative accessibility of a site under different conditions (Karlin and Akabas, 1998). By alternating short exposures to low concentrations of MTS reagent with functional measurements, a reaction rate (and thus a relative measure of accessibility) can be determined (Pascual and Karlin, 1998). In Fig. 4D, we use this method to examine the relative accessibility for MTSEA-biotin at position 162 of 4 in the presence and absence of Zn²⁺. Oocytes expressing 4-2H162C 4C75S were exposed to 1 µM MTSEA-biotin in 5-s increments. After each application, the oocytes were rinsed, and the extent of potentiation by 100 µM Zn²⁺ was determined. The decline in Zn²⁺ potentiation upon repeated exposure to MTSEA-biotin was then fit to an exponential decay function. In the absence of Zn²⁺, the half-time of the reaction was 8.7 s, yielding a rate of 79,000 ± 14,000 M^–1s^–1. In the presence of 100 µM Zn²⁺, the halftime of the reaction was 21.3 s, yielding a rate of 32,000 ± 10,000 M^–1s^–1. Despite the change in reaction rate, the extent of the effect was the same in the presence and absence of Zn²⁺ (the fit plateau was 0.69 ± 0.05 and 0.65 ± 0.02, respectively). The significant decrease in reaction rate (p < 0.01) indicates that the site is less accessible to MTSEA-biotin when Zn²⁺ is present and suggests that Zn²⁺ is competing with MTSEA-biotin for occupation of the site.

In addition to potentiation of the ACh response, zinc potentiates the response of neuronal nAChRs to nicotine. Potentiation of 44 by coapplication of 100 µM Zn²⁺ with 1 µM nicotine was 444 ± 43% of the response to nicotine alone. The 4H162G and 4E59A mutant receptors each displayed significantly reduced potentiation by 100 µM Zn²⁺ (50 ± 5% of wild-type 44, p < 0.05, and 88 ± 4% of wild-type 44, p < 0.01, respectively).

We also examined the effect of mutating 4His162 and 4Glu59 in a different receptor subunit context: 42 (Fig. 5). The 42 is a major nAChR subtype in the central nervous system (Corringer et al., 2000). The effect of zinc on 42 receptors was substantial, with a maximum potentiation of approximately 2.5-fold achieved at 50 µM Zn²⁺ (Figs. 1A and 5A, and see Hsiao et al., 2001). The 4E59A and 4H162G mutations were each able to significantly reduce the extent of potentiation by 50 µM Zn²⁺ (Fig. 5B).

View larger version (19K): [in this window] [in a new window]   Fig. 5. Mutation of 4Glu59 or 4His162 decreases zinc potentiation of 42 receptors. A, current responses of oocytes expressing wild-type 42 (left) or 4H162G2 (right) to 10 µM ACh before, during, and after coapplication of 50 µMZn2+. Scale bars are 2 µA and 10 s. B, potentiation of mutant receptors by 50 µM Zn2+. Results are presented as the ratio of mutant receptor potentiation to the potentiation of wild type receptor in the same batch of oocytes (mean ± S.E.M., n = 13–15). Significant differences in Zn2+ potentiation compared with wild-type 42 are indicated (, p < 0.001).

	Discussion

Top Abstract Materials and Methods Results Discussion References

The Location of Zinc Potentiation Sites on Neuronal nAChRs. The positions of 4His162 and 4Glu59 within the receptor and the involvement of both and subunits in mediating Zn²⁺ potentiation (Hsiao et al., 2001) suggest that neuronal nAChRs bind Zn²⁺ at subunit-subunit interfaces that alternate with ACh-binding interfaces. This depends on our decision to model the receptor with a subunit arrangement of . Whether the fifth subunit is or is irrelevant. In either case, the receptor would have two ACh-binding and two zinc-binding interfaces. More important is the arrangement of the first four subunits in an alternating pattern. This arrangement seems to be required. The subunits of the AChBP are arranged in a rotationally symmetric manner, with each subunit supplying a principal face to one ACh binding site and a complementary face to another ACh binding site (Brejc et al., 2001; Celie et al., 2004). In heteromeric neuronal nAChRs, the principal residues are only present on the subunit, whereas the complementary residues are only present on the subunit (Corringer et al., 2000). Thus, both and subunits are required to form an ACh binding site. Regardless of whether the / stoichiometry is 2:3 or 3:2, only two nonadjacent interfaces will form ACh binding sites. An additional two nonadjacent interfaces would form Zn²⁺-binding sites.

Neuronal nAChRs also form as homopentamers of the 7 subunit (Corringer et al., 2000). The 7 subunit lacks both His162 and Glu59 (Le Novere and Changeux, 2001), and 7 homopentamers are thought to bind ACh at all five interfaces (Corringer et al., 2000). As would be expected, 7 homomers are not potentiated by Zn²⁺ (Palma et al., 1998). A novel Zn²⁺-activated channel (ZAC) has been reported (Davies et al., 2003). This homomeric receptor is structurally homologous to nAChRs. Whereas ZAC subunits have a histidine in a position similar to that of 4His162, no aspartates or glutamates are located near the position analogous to 4Glu59. It is unclear whether the ZAC binds Zn²⁺ at a site similar to the potentiation site we have identified on neuronal nAChRs.

We have identified at least one residue (4His162) as part of a pair of identical Zn²⁺ potentiation sites on neuronal nAChRs. Because Zn²⁺-binding sites can have four, five, or six coordination points (Glusker, 1991), additional residues remain to be identified. These receptors may also possess a second class of Zn²⁺ potentiation site. In our single-mutation experiments, only partial losses of potentiation were observed. This was not unexpected. However, even the double mutant 4E59A,H162G retained a modest ability to be potentiated by Zn²⁺ application (approximately one third of wild-type potentiation). In our SCAM experiments, we also did not completely eliminate potentiation. This contrasts with the complete elimination of potentiation upon diethypyrocarbonate treatment (Hsiao et al., 2001). Thus, neuronal nAChRs might possess at least three distinct classes of Zn²⁺ binding site: a major potentiation site accounting for roughly two thirds of the potentiation (identified here), a minor potentiation site accounting for roughly one third of the potentiation, and an inhibitory site. Glutamate, GABA and glycine receptors also possess multiple classes of Zn²⁺ modulatory sites (Paoletti et al., 2000; Laube et al., 2002; Hosie et al., 2003).

Zinc Potentiation Sites Have Structural Similarities to ACh Binding Sites. We compared the zinc potentiation site with other binding sites on Cys-loop receptors. The ACh binding site on nAChRs has been extensively characterized (Corringer et al., 2000). Glu59 is located within the "D-loop" region of 4. In an ACh-binding interface, the D-loop is supplied by the , , or subunits of muscle nAChRs or the subunits of neuronal nAChRs and contributes a critical tryptophan residue to the binding site, as well as several determinants of pharmacological diversity (Corringer et al., 2000; Celie et al., 2004). His162 is located within the "F-loop" region of 4. At ACh binding interfaces, this region is also supplied by the "non-" subunits of muscle and neuronal nAChRs and contains determinants of pharmacological diversity (Corringer et al., 2000). Thus, the Zn²⁺-binding site that we have identified has structural similarity to the ACh binding site.

Zinc Potentiation Sites Are Not Related to Calcium Potentiation Sites on Neuronal nAChRs. Neuronal nAChRs are also potentiated by extracellular Ca²⁺ (Mulle et al., 1992; Vernino et al., 1992). Critical glutamate residues mediating Ca²⁺ potentiation have been identified on 7 (Galzi et al., 1996). These residues are highly conserved among neuronal nAChRs and correspond to 4Glu45/4Glu49 (located between strands 1 and 2) and 4Glu175/4Glu179 (located just before strand 9). Both putative Ca²⁺ binding sites are located at the bottom (near the membrane) of the receptor structure shown in Fig. 1B, away from the Zn²⁺ potentiation site we have identified. In addition, Ca²⁺ potentiation of 44 is unaffected by the 4H162G and 4E59A mutations (data not shown). Thus, Zn²⁺ and Ca²⁺ act at separate sites on neuronal nAChRs.

Zinc Potentiation Sites Are Not Related to Zinc Binding Sites of Other Ligand-Gated Ion Channels. Other Cys-loop receptors also possess Zn²⁺-binding sites. On the glycine receptor 1 subunit, Asp80 participates in a Zn²⁺-potentiation site at subunit-subunit interfaces near the top (distal from the cell surface) of the receptor extracellular domain, whereas His107, His109, and Thr112 participate in an inhibitory site that faces into the vestibule (Laube et al., 2002). The Zn²⁺ site that we have identified on neuronal nAChRs is not structurally comparable with either of these sites. GABA_A receptors also possess multiple Zn²⁺-binding sites. For clarity, it is important to remember that GABA_A receptor subunits are equivalent to neuronal nAChR subunits, whereas GABA_A receptor subunits are equivalent to neuronal nAChR subunits. Thus, whereas neuronal nAChRs bind ACh at - interfaces, GABA receptors bind GABA at - interfaces (Smith and Olsen, 1995). In the 13 GABA_A receptor, 1Glu137, 1His141 and 3Glu182 participate in a Zn²⁺-binding inhibitory site located at - interfaces (non-GABA binding interfaces) near the bottom (proximal to the cell surface) of the receptor extracellular domain (Hosie et al., 2003). A separate class of Zn²⁺-binding inhibitory site is located within the ion channel. The Zn²⁺-potentiation site we have identified on neuronal nAChRs is not structurally comparable with either of these Zn²⁺-binding sites on GABA_A receptors. Glutamate-gated ion channels also possess zinc-binding sites, which have been characterized in detail (Paoletti et al., 2000). However, these receptors are structurally unrelated to nAChRs, precluding a comparison with the Zn²⁺-potentiation site on neuronal nAChRs.

Zinc Potentiation Sites Are Analogous to the Benzodiazepine-Binding Site on GABA_A Receptors. The most interesting comparison is with the binding site for benzodiazepines on GABA_A receptors. Whereas GABA binds at - interfaces, incorporation of a subunit allows benzodiazepines to bind at the - interface (Smith and Olsen, 1995). A series of residues on the 2 subunit (Tyr58, Phe77, Ala79, and Thr81) have been identified as components of the benzodiazepine-binding site (Kucken et al., 2000, 2003; Teissere and Czajkowski, 2001). Examination of aligned amino acid sequences (Le Novere and Changeux, 2001) and a model of the 122 GABA_A receptor extracellular domain (Kucken et al., 2003) reveals that the benzodiazepine-binding site residue 2Thr81 (Teissere and Czajkowski, 2001) is in a location identical to that of 4Glu59. Although 4Glu59 may not directly coordinate zinc, it is close to the zinc potentiation site on neuronal nAChRs. This suggests that the binding of agonists and modulators at alternating subunit-subunit interfaces is a general property of heteromeric Cys-loop receptors. The similar location of the zinc potentiation sites on neuronal nAChRs and the benzodiazepine binding site on GABA_A receptors, as well as the similar modulatory function of both sites, leads us to suggest that the Zn²⁺ potentiation sites on neuronal nAChRs are structurally and functionally similar to the benzodiazepine binding site on GABA_A receptors. This identifies the Zn²⁺ potentiation site on neuronal nAChRs as a promising target for future drug development.

	Footnotes

 
This work was supported by National Institutes of Health grants MH66038 and DA08102 (to C.W.L.) and GM069972 (to A.M.). A.M. was supported in part by award BM030 from the Florida Biomedical Research Foundation, and by American Heart Association, Florida/Puerto Rico Affiliate grant SDG-0130456B. B.H., K.B.M., D.E., and S.E.R. were supported in part by National Institutes of Health grant T32-HL07188. B.H. was supported in part by a PhRMA Foundation Medical Student Research Fellowship and is a Lois Pope LIFE Fellow.

B.H and K.B.M. contributed equally to this work.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.015164.

ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; AChBP, acetylcholine binding protein; SCAM, substituted cysteine accessibility method; MTSEA, N-biotinoylaminoethyl methanethiosulfonate; MTSET, [2-(trimethylammonium)ethyl] methanethiosulfonate; ACh, acetylcholine; MTS, methanethiosulfonate.

Address correspondence to: Dr. Charles W. Luetje, Molecular and Cellular Pharmacology, R-189, University of Miami, P.O. Box 016189, Miami, FL 33101. E-mail: cluetje{at}chroma.med.miami.edu

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