QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: mol.106.023259v1 70/1/16    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Related articles in MolPharm Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by McLaughlin, J. T. Articles by Rosenberg, R. L. Search for Related Content PubMed PubMed Citation Articles by McLaughlin, J. T. Articles by Rosenberg, R. L.

Role of the Outer -Sheet in Divalent Cation Modulation of 7 Nicotinic Receptors

Departments of Pharmacology (J.T.M., J.F., A.D.S., R.L.R.) and Cell & Molecular Physiology (R.L.R.), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

Received February 7, 2006; accepted March 13, 2006

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
-7 Nicotinic acetylcholine receptors (AChRs) exhibit a positive modulation by divalent cations similar to that observed in other AChRs. In the chick 7 AChR, this modulation involves a conserved glutamate in loop 9 (Glu¹⁷²) that undergoes agonist-dependent movements during activation. From these observations, we hypothesized that movements of the nearby -sheet formed by the 7, 9, and 10 strands may be involved in agonist activation and/or divalent modulation. To test this hypothesis, we examined functional properties of cysteine mutations of the 7 and 10 strands, alone or in pairs. We postulated that reduced flexibility or mobility of the 7/9/10-sheet as a result of introduction of a disulfide bond between the strands would alter activation by agonists. Using a nondesensitizing 7 mutant background (L²⁴⁷T), we identified one mutant pair, K¹⁴⁴C + T¹⁹⁸C, that exhibited a unique characteristic: it was fully activated by divalent cations (Ca²⁺, Ba²⁺, or Sr²⁺) in the absence of acetylcholine (ACh). Divalent-evoked currents were blocked by the 7 antagonist methyllycaconitine and were abolished when Glu¹⁷² was mutated to glutamine. When the K¹⁴⁴C + T¹⁹⁸C pair was expressed in wild-type 7 receptors, activation required both ACh and divalent cations. We conclude that the introduction of a disulfide bond into 7/9/10 lowers the energetic barrier between open and closed conformations, probably by reducing the torsional flexibility of the -sheet. In this setting, divalent cations, acting at the conserved glutamate in loop 9, act as full agonists or requisite coagonists.

Our understanding of ligand binding has been advanced greatly by crystal structures of several invertebrate ACh-binding proteins (AChBPs; Brejc et al., 2001; Celie et al., 2005a,b). These soluble proteins share sequence homology with the LBD of nicotinic receptor subunits (24% sequence identity with the 7 subunit), and they assemble as homopentamers around a central cavity. The AChBP contains a core of 10 -strands, 1 -helix, and 10 variable regions linking secondary structural elements (Brejc et al., 2001). The -strands form two major antiparallel sheet structures referred to as the inner (1, 2, and 6) and outer (7, 9, and 10) sheets (Unwin, 2005). The structure also displays a clustering of conserved aromatic residues in proximity to both the subunit interface and the loop between strands 9 and 10 (the "C loop"). Crystal structures of AChBPs in complex with nicotine, carbamoylcholine, or the peptide antagonist -conotoxin PnIA have identified specific ligand-receptor contacts and confirmed that ligand binding involves the contraction of the C loop (Celie et al., 2005a,b).

Several current models attempt to interpret functional data in terms of possible conformational steps leading from agonist binding and C-loop contraction to channel opening. In one model, agonist binding leads to a rotation of the inner -sheet of one or more subunits, followed by similar movement of the second-channel transmembrane domain (M2) (Horenstein et al., 2001; Unwin, 2005). A "conformational wave" theory suggests that these or similar steps occur in sequential order from LBD domain to ICD domain (Grosman et al., 2000). More explicit models link agonist binding to channel opening via interactions between several pairs of residues (Lee and Sine, 2005; Mukhtasimova et al., 2005). In these models, electrostatic or hydrogen bonds between residues in the C-loop, the outer -sheet, and the inner -sheet propagate the signal of agonist binding to the interface between the LBD and the transmembrane helices that form the ICD. They provide a more detailed description of mechanisms proposed previously of coupling between binding and channel gating (Miyazawa et al., 2003) and are similar to a model proposed for activation of the related GABA_A receptor (Kash et al., 2004).

Our approach to examining receptor activation pathways uses cysteine scanning to track conformational changes based on rates of thiol modification (Karlin and Akabas, 1998). We demonstrated that a region of the 7 AChR known to be involved in modulation by divalent cations, loop 9, undergoes agonist-dependent conformational movements (Lyford et al., 2003). Based on this result and on our homology model of the 7 LBD, we postulated that a disulfide bond between strands would reduce the flexibility or mobility of the outer (7/9/10) sheet and could lead to alterations in receptor activation. If movements of the receptor around Glu¹⁷² (in loop 9) induced by agonist are due to global movements of the outer -sheet, then an introduced disulfide should have a significant effect on activation.

In this report, we test this idea by examining the effect of introduced disulfide bonds between the 7 and 10 strands. We used our homology model of the 7 extracellular domain to identify sites for disulfide engineering, selecting residues with the carbons separated by an appropriate distance for disulfide formation (<6 Å). Using this criterion, Asn¹⁴² was paired with Thr¹⁹⁸ and Thr²⁰⁰, whereas Lys¹⁴⁴ was paired with Asp¹⁹⁶ and Thr¹⁹⁸. Several additional pairs that were predicted to be too distant to form a disulfide (Asn¹⁴²/T²⁰² and Lys¹⁴⁴/Thr²⁰⁰) were also included. We examined functional properties of both single cysteine mutations and paired mutations using a nondesensitizing AChR background (L²⁴⁷T) (Eddins et al., 2002a,b; Lyford et al., 2003). The unique phenotype of one pair, 7-K¹⁴⁴C/T¹⁹⁸C, confirmed the importance of the outer -sheet in the coupling of agonist binding to channel opening and suggested that cholinergic agonists and divalent cations initiate similar conformational transitions in the process of receptor activation.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Reagents. Ethyl trimethyl methane thiosulfonate (MTSET) was obtained from Toronto Research Chemical (Toronto, ON, Canada). Gentamicin was from Invitrogen (Carlsbad, CA). All other reagents were obtained from either Fluka (Ronkonkoma, NY) or Sigma (St. Louis, MO).

Site-Directed Mutagenesis. We used an 7 receptor containing two mutations (C¹¹⁵A, L²⁴⁷T) as our "background" for mutations described in this study. We mutated the lone unpaired cysteine in the extracellular domain (Cys¹¹⁵) to allow more straightforward interpretation of MTSET exposure experiments. We observed no functional effect of this mutation on receptor expression or ACh response. We included the mutation in the M2 transmembrane domain leucine 247 (L²⁴⁷T; L9'T) because of its large current amplitudes and nondesensitizing kinetics compared with wild-type 7 receptors (Revah et al., 1991). All mutations were introduced into chick 7 cDNA by site-directed mutagenesis using the QuikChange method (Stratagene, La Jolla, CA) as described previously (Eddins et al., 2002b) and were confirmed by DNA sequencing.

Xenopus laevis Oocyte Maintenance and Expression. Oocytes were surgically removed and prepared from female X. laevis in accordance with the University of North Carolina Institutional Animal Care and Use Committee guidelines as described previously (Eddins et al., 2002a). cRNA was prepared using the T7 RNA polymerase and mMessage mMachine kit (Ambion, Austin, TX) as described by the manufacturer. Oocytes were injected with 20 ng of cRNA and incubated at 18°C in ND96 (5 mM HEPES, 96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, and 1.8 mM CaCl₂, pH 7.5) for 2 to 5 days before use.

Data Collection and Analysis. Oocytes were superfused in normal extracellular solution containing a reduced Ca²⁺ concentration (ESLC; 96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 0.1 mM CaCl₂, and 10 mM HEPES, pH 7.5). Two-electrode voltage clamp was performed with a GeneClamp 500B controlled by pCLAMP6 software (Axon Instruments, Sunnyvale, CA). Electrodes were filled with 3 M KCl contacting Ag-AgCl wires and had resistances of 0.5 to 2.0 M. Currents were recorded at a constant holding potential of –60 mV. Currents were low-pass-filtered at 50 or 125 Hz and were sampled at 250 Hz. Agonist dose-response curves were obtained as described previously (Eddins et al., 2002a), and data were fit to the Hill equation using Origin software (OriginLab Corp., Northampton, MA).

Modification by MTSET. MTSET was prepared daily in water and stored on ice. Stock solution was diluted to the appropriate working concentration in ESLC immediately before each application. Oocytes were exposed to MTSET by continuous flow for 15 to 60 s, followed by a 5-min wash. To measure accessibility of each cysteine mutant, currents evoked by a submaximal concentration of ACh were compared before and after MTSET exposure. We also determined complete ACh dose-response relationships before and after MTSET for those mutants which showed sensitivity to the modifier.

Oocyte Antibody Binding Assay. Oocytes injected with cRNA were incubated 2 to 5 days before assay. They were fixed with 2% paraformaldehyde in HEPES-buffered saline (20 mM HEPES and 100 mM NaCl, pH 7.4) for 20 min at 22°C, rinsed with 3 changes of ND96, and then incubated 30 min in ND96 plus 3% BSA. Oocytes were then incubated overnight at 4°C in ND96, 3% BSA, plus a 1:500 dilution of anti-7 antiserum. The antiserum (Ab1382) was raised in rabbits against a peptide corresponding to the N-terminal sequence of rat 7 AChR (EFQRRLYKELVKNYN). After six washes in ND96, the oocytes were incubated for 2 h at 22°C in ND96/BSA plus 2 nM ¹²⁵I-labeled goat anti-rabbit IgG (PerkinElmer Life and Analytical Sciences, Boston, MA). After five washes with ND96, bound secondary antibody was determined using a gamma counter. Nonspecific binding was measured to either uninjected oocytes or to injected oocytes incubated with primary antibody preincubated with excess antigenic peptide.

Structural Model of 7. A model of the chick 7 nicotinic receptor extracellular domain based on the coordinates of the Lymnea ACh binding protein was constructed as described previously (Lyford et al., 2003). Images of the model were generated using an open-source version of Pymol (DeLano Scientific, South San Francisco, CA).

	Results

Top Abstract Materials and Methods Results Discussion References

 
Single Cysteine Mutations in 7 and 10 Are Well-Tolerated. Our model of the chick 7 LBD suggests that the side chain of Asn¹⁴² is positioned in close proximity to those of Thr¹⁹⁸ and Thr²⁰⁰. Likewise, Lys¹⁴⁴ is positioned near Asp¹⁹⁶ and Thr¹⁹⁸. The distance between -carbons of each of these pairs is 4.5 Å (Fig. 1). These five residues (and Thr²⁰²) were mutated individually to cysteine and tested for functional expression in X. laevis oocytes. Table 1 shows a summary of the dose-response data for these mutations. Five of the six substitutions caused modest rightward shifts in the acetylcholine dose-response curve relative to the parent genotype, C¹¹⁵A/L²⁴⁷T. Only the K¹⁴⁴C mutant caused an increase of more than 6-fold in the ACh EC₅₀ values. Peak currents for each mutant were also comparable with those measured in C¹¹⁵A/L²⁴⁷T 7 AChRs.

View larger version (40K): [in this window] [in a new window]   Fig. 1. Structural model of an 7 subunit (from Eddins et al., 2002). The sequence is depicted as two major regions defined by inner and outer -sheets. The inner -sheet (1, 2, and 6) is shown in yellow. The outer -sheet is shown in blue (7, 9, and 10). Residues used in disulfide engineering are highlighted with native side chains superimposed over ribbon figure. These include Asn142 and Lys144 in the 7 strand and Asp196, Thr198, Thr200, and Thr202 in 10. The conserved glutamate essential for modulation by divalent cations, Glu172, is shown in red. This model is based on the HEPES-bound conformation of the AChBP (Brejc et al., 2001) and thus is likely to represent an activated or desensitized conformation.

We examined the solvent accessibility of the introduced cysteines by measuring ACh responses before and after exposure to the thiol modifier MTSET. Both N¹⁴²C and K¹⁴⁴C displayed significant leftward shifts in their ACh dose-response profiles, demonstrating that both introduced cysteines were accessible to the modifying agent. MTSET also had a significant effect on D¹⁹⁶C, causing a 70-fold increase in the EC₅₀ value of ACh. In a previous study involving cysteine substitution of this conserved aspartate in the Torpedo californica AChR, modification caused either inhibition or constitutive activation depending on the specific modifier used (Sullivan and Cohen, 2000). Exposure of T¹⁹⁸C or T²⁰⁰C to MTSET caused consistent shifts in the ACh dose-response curves, but these did not reach the level of statistical significance. We conclude that N¹⁴²C, K¹⁴⁴C, and D¹⁹⁶C are clearly accessible to the surrounding aqueous solvent.

Functional 7 Receptors Are Formed by Select Pairs of Cysteine Mutants. We next constructed and tested six mutant pairs designed to position cysteines in close proximity in the 7 and 10 strands. Each of the 7 cysteine replacements was paired with three cysteine substitutions in 10; the model predicts that, in each case, at least two of these three pairs should be close enough to form a disulfide bond. We found that only two of the pairs, N¹⁴²C/T²⁰⁰C and K¹⁴⁴C/T¹⁹⁸C, form ACh-responsive channels when expressed in oocytes. ACh dose-response profiles of these mutants are shown in Fig. 2. Both double-mutant pairs displayed leftward shifts in the ACh EC₅₀ values relative to one or both of their respective single-mutant parental genotypes, suggesting some degree of phenotypic rescue.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Selected pairs of cysteine mutants express functional AChRs. A, ACh dose-response curves for C115A/L247T background phenotype (), N142C (), T200C (), and T200C/N142C (). B, C115A/L247T (), K144C (), T198C (), and T198C /K144C (). Data are normalized to the maximal ACh current for each oocyte. Pairing of these cysteine mutants causes provides at least partial recovery from the effect of the single mutations. Additional data on these and other cysteine mutants are summarized in Table 1.

The absence of functional receptors in four of six mutant pairs may be due to an interaction between the introduced cysteines that prevented ACh-evoked activation. On the other hand, it may have resulted from a perturbation in receptor translation, folding, assembly, or surface expression. To determine whether nonfunctional receptors containing cysteine pairs were expressed on the oocyte surface, we used an antibody binding assay (Table 1). All six of the mutant pairs expressed measurable surface antibody binding (1–10 fmol of antibody binding sites per oocyte). This indicates that the lack of ACh responsiveness in four of the six pairs of introduced cysteines was not caused by a defect in surface expression. For those cysteine pairs unresponsive to agonist, therefore, the specific combinations of mutations seem to disrupt agonist binding and/or receptor gating.

K¹⁴⁴C/T¹⁹⁸C 7 AChRs Are Activated by Both ACh and Divalent Cations. In our initial characterization of the K¹⁴⁴C/T¹⁹⁸C mutant, we observed basal currents in the absence of agonist that were higher than normal. These early experiments used a physiological Ca²⁺ concentration (1.8 mM). The basal currents were eliminated when we used a low-calcium (0.1 mM) extracellular recording solution (ESLC). Ba²⁺, Ca²⁺, or Sr²⁺ activated an inward current only in oocytes expressing the K¹⁴⁴C/T¹⁹⁸C 7 AChR. We measured the dose-response relationship for Ba²⁺-evoked current and found that maximal currents were similar in magnitude to those elicited by ACh (Fig. 3A). Currents elicited by Ca²⁺ or Sr²⁺ (data not shown) were similar to those observed when Ba²⁺ was used as the activating divalent. In addition, Ba²⁺-evoked currents were blocked by the 7-specific antagonist MLA (Fig. 3, B and C). These results show that the divalent-activated currents were carried by the K¹⁴⁴C/T¹⁹⁸C 7 AChR. Other mutations exhibited modest activation by Ba²⁺ ( 10–15%, see Table 1), but the K¹⁴⁴C/T¹⁹⁸C 7 AChR was the only phenotype in which divalents were full agonists. Divalents such as Zn²⁺, Mg²⁺, or Mn²⁺ did not elicit any current responses from the K¹⁴⁴C/T¹⁹⁸C 7 AChR (data not shown), suggesting that the activation by divalent we observed is likely to be distinct from that reported for a recently cloned member of the cysteine-loop receptor gene family (Davies et al., 2003).

View larger version (13K): [in this window] [in a new window]   Fig. 3. Divalent cations activate 7 K144C/T198C mutant receptors. The cysteine mutant pair K144C/T198C, when expressed in the L247T background, responds to bath-applied Ba2+ with currents similar to those observed in response to ACh. A, barium dose-response curve for K144C/T198C (). Neither of two single cysteines K144C () or T198C () show a similar response. Ba2+-evoked currents are normalized to the maximal ACh-evoked current of each receptor type. B, responses of K144C/T198C 7 receptors to 3 mM Ba2+ plus increasing concentrations of the 7-specific antagonist MLA. Blockade by MLA demonstrates that the Ba2+-evoked currents were due to activation of 7 AChRs. C, MLA inhibition plotted from fractional peak currents, as shown in B, yields an apparent IC50 value of 1.4 nM, comparable with inhibition observed for ACh-evoked currents.

Divalent-Activated Currents Are Abolished by the E¹⁷²Q Mutation. Previous studies showed that mutation of the conserved glutamate at 172 eliminated divalent modulation of 7 AChRs, demonstrating that this residue is a critical component of a divalent allosteric binding site (Galzi et al., 1996; Eddins et al., 2002a). To test whether this site was involved in the activation by divalent cations of K¹⁴⁴C/T¹⁹⁸C 7 AChR, we constructed 7 receptors with Glu¹⁷² mutated to glutamine in addition to K¹⁴⁴C and T¹⁹⁸C mutations. This construct displays an ACh dose-response relationship similar to that seen previously in the E¹⁷²Q mutant (Eddins et al., 2002b) but shows no divalent-activated current (Fig. 4). This result suggests that the introduction of the K¹⁴⁴C/T¹⁹⁸C cysteine pair enhances the endogenous allosteric modulation such that divalent binding to K¹⁴⁴C/T¹⁹⁸C 7 receptors can induce the conformational changes necessary for channel opening even in the absence of cholinergic agonists.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Activation of K144C/T198C by Ba2+ requires the conserved glutamate Glu172. Neutralization of the charge at Glu172 shifts the ACh doseresponse curve but eliminates activation by Ba2+. Normalized dose response relationships to ACh (A) or Ba2+ (B) for K144C/T198C () and K144C/T198C/E172Q (). Currents are normalized to peak responses of each receptor type to a maximal dose of ACh.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Activation of K144C/T198C in the wild-type background requires both ACh and divalent cations. A, current traces of wild-type 7 AChR (black) show potentiation by 1 mM (blue) or 10 mM (red) added Ba2+. B, ACh dose-response curves in low divalent (0.1 mM Ca2+, ) and in 0.1 mM Ca2+ plus 10 mM Ba2+ (, red). C, K144C/T198C in a wild-type 7 background: divalent-evoked currents are no longer observed, and ACh-evoked currents are substantially diminished (black trace). Current responses for coapplication of ACh and 1 mM (blue trace) or 10 mM (red trace) Ba2+. D, comparison of ACh dose responses in 0.1 mM Ca2+ () and 0.1 mM Ca2+ plus 10 mM Ba2+ (, red) for K144C/T198C in the wild-type 7 background.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The goal of these experiments was to test the role of the outer -sheet, formed by strands 7, 9, and 10, in the activation and allosteric modulation of 7 nicotinic receptors. The potential importance of this structural element was first suggested by its position between the ligand binding site and conserved regions near the transmembrane domain, such as the cysteine loop and the 1–2 linker (Brejc et al., 2001). Based on the loss of sensitivity to thiol modifiers, we show that it is possible to introduce disulfide bonds between the 7 and 10 strands in at least two positions (N¹⁴²C/T²⁰⁰C and K¹⁴⁴C/T¹⁹⁸C) without a loss of ACh-evoked receptor activation. We also show that (in a nondesensitizing 7 variant) the putative disulfide bond between K¹⁴⁴C and T¹⁹⁸C enhances receptor activation by converting allosteric modulators (e.g., Ca²⁺ or Ba²⁺) to full agonists.

Although the "modulation-activation" by divalent cations occurs around the conserved glutamate (Glu¹⁷²) of the 8–9 linker, it also involves conformational transitions at the ligand binding site, because the competitive antagonist MLA prevents activation by divalent cations. This effect is specific for the Lys¹⁴⁴ + Thr¹⁹⁸ pair, suggesting that both the stabilization provided by a disulfide and its position in the outer -sheet contribute to the phenotype. Our results are relevant to several recently proposed models for receptor activation and allow us to further refine hypotheses of the conformational transitions involved.

A report by Mukhtasimova et al. (2005) examined the role of several conserved residues surrounding agonist binding site in muscle AChRs, including the lysine homologous to 7 Lys¹⁴⁴. They used a combination of mutagenesis, single-channel electrophysiology, and ligand binding experiments to demonstrate an interdependence between Lys¹⁴⁴, a C-loop tyrosine (Tyr¹⁸⁷ in 7), and the conserved aspartate in 10 (Asp¹⁹⁶). They proposed a "salt-bridge switch" model in which a salt bridge between Lys¹⁴⁴ and Asp¹⁹⁶ is present in the absence of ligand. In this model, the binding of ACh leads to a contraction of the C-loop and moves Tyr¹⁸⁷ close to Lys¹⁴⁴, where it replaces the Asp¹⁹⁶ and forms a new salt bridge. Our findings that a covalent bond between substituted cysteine residues at positions Lys¹⁴⁴ and Thr¹⁹⁸ can exist in a receptor that is ACh-activated seems incompatible with this model. This discrepancy may arise from the differences in the AChR isoforms used in each study, because the 1 subunit of muscle shows significant sequence differences, particularly in the C loop, compared with the 7 AChR. It is likely that such differences will reflect variations in the subtle conformational changes underlying the activation of each receptor isoform.

Current models place the binding site for divalent cations at the interface between adjacent subunits, at which several conserved charged residues are found in close proximity (Le Novere et al., 2002). In addition to Glu¹⁷², the binding site may include the conserved glutamate (Glu⁴⁴) and aspartates (Asp⁴¹ and Asp⁴³) that structural models place in the region linking 1 and 2, the two major strands of the "inner" -sheet. Several recently proposed models for receptor activation have suggested a role for the 1–2 linker in the coupling of ligand binding to receptor activation (Kash et al., 2004; Law et al., 2005; Lee and Sine, 2005). A common theme in these proposed mechanisms is an interaction between components of the extracellular domain and the amino acid residues linking the second and third transmembrane helices (the M2–M3 linker). Our results suggest that the outer -sheet plays a role in transmitting the conformational transitions at the binding site to this or other mediators of channel gating.

Law et al. (2005) used molecular dynamics to model the gating of the human 7 receptor. They constructed a model using LBD coordinates from the ACh binding protein (Brejc et al., 2001) and ICD coordinates from T. californica AChR cryoelectron microscopy (Miyazawa and Unwin, 2003). They noted both outward motions of the C loop (corresponding to a relaxation to the closed state) and asymmetric movement of the transmembrane domains consistent with channel closing. Based on these observations, they suggest that in the process of activation, the outer -sheet transmits the gating signal from the binding site to the ICD via contacts with the conserved cysteine loop at the N-terminal of 7. Our data provide experimental evidence to support this model and allow us to propose a mechanism for the allosteric action of divalent cations in the process of AChR activation.

We found that introduction of a disulfide at a central location in the outer -sheet allowed divalent binding at one end of the sheet (the "bottom") to elicit the same conformational transition that agonist binding did at the other end (the "top"). This suggests that the outer -sheet may act as a lever that moves, with the disulfide at or near the fulcrum. In this way, binding at one end can elicit the same pivoting motions as those elicited by binding at the other. Compounds such as MLA can prevent activation either by direct competition with ACh or by a noncompetitive mechanism, as suggested by Law et al. (2005). We can explain the effect of introducing the K¹⁴⁴C/T¹⁹⁸C disulfide into the 7 wild type if we assume that the L²⁴⁷T mutation exerts a "relaxing" effect on the entire transduction pathway. In the L²⁴⁷T background, introducing the K¹⁴⁴C/T¹⁹⁸C disulfide allows either ACh or divalent cations to exert enough leverage on the outer -sheet to activate the channel. In the wild-type background, however, the stiffening of the outer -sheet by the introduction of the K¹⁴⁴C/T¹⁹⁸C disulfide creates a greater energetic barrier between closed and open states. This slows channel kinetics and leads to a requirement for binding of both ACh and divalent cations to efficiently drive the conformational transitions, leading to channel opening.

Lyford et al. (2003) provided evidence for an asymmetric rotation of subunits as part of agonist-evoked receptor activation. This is consistent with the model developed using molecular dynamics (Law et al., 2005) in which two subunits exhibit rotational motion during activation, but the others do not. Lee and Sine (2005) have proposed a more detailed activation scheme for the T. californica AChR in which the coupling of the binding site to the channel is mediated by an electrostatic interaction between residues of the inner and outer -sheets (Glu⁴⁵ and Arg²⁰⁹). The movement of the outer sheet we propose could represent a step in the activation pathway for the 7 AChR comparable with the Glu⁴⁵ + Arg²⁰⁹ interaction in the T. californica AChR that serves to link the outer and inner -sheets. It is noteworthy that the T. californica AChR does not show the same potentiation by divalent cations as the 7 AChR, and the conserved glutamate corresponding to the T. californica Glu⁴⁵ in 7 (Glu⁴⁴) is has been shown to participate in a formation of the divalent binding site (Galzi et al., 1996). Thus, the difference in divalent modulation between the two isoforms may be due to distinct mechanistic roles for this conserved glutamate.

In another recent report, Lummis et al. (2005) showed data suggesting that activation of another cysteine-loop receptor (5-hydroxytryptamine type 3) may involve cis-trans isomerization of a proline residue in the M2–M3 loop. Because this residue is not conserved throughout the cysteine-loop receptor family, their result implies that there must be different activation mechanisms used by different receptor isoforms (Czajkowski, 2005). This is consistent with another recent report showing that whereas electrostatic interactions are key to the movements linking binding and gating, these interactions are mediated by different sets of residues in different receptor isoforms (Xiu et al., 2005). Our results provide evidence for a critical role of the outer -sheet in the conformational mechanism linking agonist binding to channel gating in the 7 AChR. They also suggest, compared with other recent studies, that various AChR isoforms use different conformational transitions in the coupling of ligand binding to channel activation.

	Footnotes

 
Supported by National Institute on Drug Abuse grant DA017882 (to R.L.R.).

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

doi:10.1124/mol.106.023259.

ABBREVIATIONS: LBD, ligand-binding domain; ACh, acetylcholine; AChR, nicotinic acetylcholine receptor; ICD, ion channel domain; MTSET, 2-trimethylammonioethylmethane thiosulfonate; ESLC, extracellular recording solution containing low Ca²⁺ concentration; ND96, X. laevis oocyte storage buffer; MLA, methyllycaconitine; AChBP, acetylcholine binding protein; BSA, bovine serum albumin.

¹ Current affiliation: Department of Cell and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina.

Address correspondence to: Dr. James T. McLaughlin, Department of Pharmacology, CB# 7365, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365. E-mail: jmclaughlin{at}unc.edu

	References

Top Abstract Materials and Methods Results Discussion References

 
Brejc K, van Dijk WJ, Klaassen RV, Schuurmans M, van Der Oost J, Smit AB, and Sixma TK (2001) Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature (Lond) 411: 269–276.[CrossRef][Medline]

Celie PH, Klaassen RV, van Rossum-Fikkert SE, van Elk R, van Nierop P, Smit AB, and Sixma TK (2005a) Crystal structure of acetylcholine-binding protein from Bulinus truncatus reveals the conserved structural scaffold and sites of variation in nicotinic acetylcholine receptors. J Biol Chem 280: 26457–26466.[Abstract/Free Full Text]

Celie PH, van Rossum-Fikkert SE, van Dijk WJ, Brejc K, Smit AB, and Sixma TK (2005b) Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41: 907–914.

Czajkowski C (2005) Neurobiology: triggers for channel opening. Nature (Lond) 438: 167–168.[CrossRef][Medline]

Davies PA, Wang W, Hales TG, and Kirkness EF (2003) A novel class of ligand-gated ion channel is activated by Zn²⁺. J Biol Chem 278: 712–717.[Abstract/Free Full Text]

Eddins D, Lyford LK, Lee JW, Desai SA, and Rosenberg RL (2002a) Permeant but not impermeant divalent cations enhance activation of non-desensitizing alpha₇ nicotinic receptors. Am J Physiol 282: C796–C804.

Eddins D, Sproul AD, Lyford LK, McLaughlin JT, and Rosenberg RL (2002b) Glutamate 172, essential for modulation of L²⁴⁷T alpha7 ACh receptors by Ca²⁺, lines the extracellular vestibule. Am J Physiol 283: C1454–C1460.

Galzi JL, Bertrand S, Corringer PJ, Changeux JP, and Bertrand D (1996) Identification of calcium binding sites that regulate potentiation of a neuronal nicotinic acetylcholine receptor. EMBO (Eur Mol Biol Organ) J 15: 5824–5832.[Medline]

Grosman C, Zhou M, and Auerbach A (2000) Mapping the conformational wave of acetylcholine receptor channel gating. Nature (Lond) 403: 773–776.[CrossRef][Medline]

Holmgren M, Liu Y, Xu Y, and Yellen G (1996) On the use of thiol-modifying agents to determine channel topology. Neuropharmacology 35: 797–804.[CrossRef][Medline]

Horenstein J, Wagner DA, Czajkowski C, and Akabas MH (2001) Protein mobility and GABA-induced conformational changes in GABA_A receptor pore-lining M2 segment. Nat Neurosci 4: 477–478.[Medline]

Karlin A (2002) Emerging structure of the nicotinic acetylcholine receptors. Nat Rev Neurosci 3: 102–114.[CrossRef][Medline]

Karlin A and Akabas MH (1998) Substituted-cysteine accessibility method. Methods Enzymol 293: 123–145.[CrossRef][Medline]

Kash TL, Kim T, Trudell JR, and Harrison NL (2004) Evaluation of a proposed mechanism of ligand-gated ion channel activation in the GABA_A and glycine receptors. Neurosci Lett 371: 230–234.[CrossRef][Medline]

Law RJ, Henchman RH, and McCammon JA (2005) A gating mechanism proposed from a simulation of a human alpha7 nicotinic acetylcholine receptor. Proc Natl Acad Sci USA 102: 6813–6818.[Abstract/Free Full Text]

Lee WY and Sine SM (2005) Principal pathway coupling agonist binding to channel gating in nicotinic receptors. Nature (Lond) 438: 243–247.[CrossRef][Medline]

Le Novere N, Grutter T, and Changeux JP (2002) Models of the extracellular domain of the nicotinic receptors and of agonist- and Ca²⁺-binding sites. Proc Natl Acad Sci USA 99: 3210–3215.[Abstract/Free Full Text]

Lester HA, Dibas MI, Dahan DS, Leite JF, and Dougherty DA (2004) Cys-loop receptors: new twists and turns. Trends Neurosci 27: 329–336.[CrossRef][Medline]

Lummis SC, Beene DL, Lee LW, Lester HA, Broadhurst RW, and Dougherty DA (2005) Cis-trans isomerization at a proline opens the pore of a neurotransmittergated ion channel. Nature (Lond) 438: 248–252.[CrossRef][Medline]

Lyford LK, Sproul AD, Eddins D, McLaughlin JT, and Rosenberg RL (2003) Agonist-induced conformational changes in the extracellular domain of 7 nicotinic acetylcholine receptors. Mol Pharmacol 64: 650–658.[Abstract/Free Full Text]

Miyazawa A, Fujiyoshi Y, and Unwin N (2003) Structure and gating mechanism of the acetylcholine receptor pore. Nature (Lond) 423: 949–955.[CrossRef][Medline]

Mukhtasimova N, Free C, and Sine SM (2005) Initial coupling of binding to gating mediated by conserved residues in the muscle nicotinic receptor. J Gen Physiol 126: 23–39.[Abstract/Free Full Text]

Revah F, Bertrand D, Galzi JL, Devillers-Thiery A, Mulle C, Hussy N, Bertrand S, Ballivet M, and Changeux JP (1991) Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor. Nature (Lond). 353: 846–849.[CrossRef][Medline]

Sine SM (2002) The nicotinic receptor ligand binding domain. J Neurobiol 53: 431–446.[CrossRef][Medline]

Sullivan DA and Cohen JB (2000) Mapping the agonist binding site of the nicotinic acetylcholine receptor. Orientation requirements for activation by covalent agonist. J Biol Chem 275: 12651–12660.[Abstract/Free Full Text]

Unwin N (2005) Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J Mol Biol 346: 967–989.[CrossRef][Medline]

Xiu X, Hanek AP, Wang J, Lester HA, and Dougherty DA (2005) A unified view of the role of electrostatic interactions in modulating the gating of cys loop receptors. J Biol Chem 280: 41655–41666.[Abstract/Free Full Text]

Related articles in MolPharm:

Potentiation of Acetylcholine Receptors by Divalent Cations

Jon Lindstrom
MolPharm 2006 70: 5-7. [Abstract] [Full Text]  

This article has been cited by other articles:

				S. P. Venkatachalan and C. Czajkowski A conserved salt bridge critical for GABAA receptor function and loop C dynamics PNAS, September 9, 2008; 105(36): 13604 - 13609. [Abstract] [Full Text] [PDF]

				E. A. Gay and J. L. Yakel Gating of nicotinic ACh receptors; new insights into structural transitions triggered by agonist binding that induce channel opening J. Physiol., November 1, 2007; 584(3): 727 - 733. [Abstract] [Full Text] [PDF]

				J. T. McLaughlin, J. Fu, and R. L. Rosenberg Agonist-Driven Conformational Changes in the Inner beta-Sheet of {alpha}7 Nicotinic Receptors Mol. Pharmacol., May 1, 2007; 71(5): 1312 - 1318. [Abstract] [Full Text] [PDF]

				H. Tsuneki, S. Kobayashi, K. Takagi, S. Kagawa, M. Tsunoda, M. Murata, T. Matsuoka, T. Wada, M. Kurachi, I. Kimura, et al. Novel G423S Mutation of Human {alpha}7 Nicotinic Receptor Promotes Agonist-Induced Desensitization by a Protein Kinase C-Dependent Mechanism Mol. Pharmacol., March 1, 2007; 71(3): 777 - 786. [Abstract] [Full Text] [PDF]

				J. Lindstrom Potentiation of Acetylcholine Receptors by Divalent Cations Mol. Pharmacol., July 1, 2006; 70(1): 5 - 7. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: mol.106.023259v1 70/1/16    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Related articles in MolPharm Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by McLaughlin, J. T. Articles by Rosenberg, R. L. Search for Related Content PubMed PubMed Citation Articles by McLaughlin, J. T. Articles by Rosenberg, R. L.