Elsevier

Neuropharmacology

Volume 60, Issue 1, January 2011, Pages 159-171
Neuropharmacology

Cysteine accessibility analysis of the human alpha7 nicotinic acetylcholine receptor ligand-binding domain identifies L119 as a gatekeeper

https://doi.org/10.1016/j.neuropharm.2010.07.014Get rights and content

Abstract

A large number of structurally diverse ligands have been produced to selectively target α7 nicotinic acetylcholine receptors (nAChRs). We applied the method of scanning cysteine accessibility mutations (SCAM) to the ligand-binding domain of the α7 nAChR to identify subdomains of particular importance to the binding and subsequent activation by select agonists. We evaluated the activity of four structurally distinct α7 agonists on wild-type human α7 and 44 targeted mutants expressed in Xenopus oocytes. Responses were measured prior and subsequent to the application of the sulfhydryl reagent methanethiosulfonate ethylammonium (MTSEA). One mutant (C116S) served as a Cys-null control, and the additional mutants were made in the C116S background. In many cases, the insertion of free cysteines into the agonist-binding site had a negative effect on function, with 12 of 44 mutants showing no detectable responses to ACh, and with only 19 of the 44 mutants showing sufficiently large responses to permit further study. Several of the cysteine mutations, including W55C, showed selectively reduced responses to the largest agonist tested, 2-methoxy,4-hydroxy-benzylidene anabaseine. Interestingly, although homology models suggest that most of the introduced cysteine mutations should have had good solvent accessibility, application of MTSEA had no effect or produced only modest changes in the agonist response profile of most mutants. Consistent with previous studies implicating W55 to play important roles in agonist activation, MTSEA treatment further decreased the functional responses of W55C to all the test agonists. While the cysteine mutation at L119 itself had relatively little effect on receptor function, treatment of L119C receptors with MTSEA or alternative cationic sulfhydryl reagents profoundly decreased activation by all agonists tested, suggesting a general block of gating. The homologous mutation in heteromeric nAChRs produced similar results, provided that the mutation was placed in the beta subunit complementary surface of the ligand-binding domain. Structural models locate the L119 residue directly across the subunit interface from the C-loop of the primary face of the binding domain. Our data suggest that a covalent modification of L119C by MTSEA or other cationic reagents might block the binding of even small agonists such as TMA through electrostatic interactions. Reaction of L119C with small non-polar reagents increases activation by small agonists but can block the access of large ligands such as benzylidene anabaseines to the ligand-binding domain.

Introduction

Nicotinic acetylcholine receptors (nAChRs) are members of a large superfamily of ligand-gated ion channels that are all characterized by several key structural similarities, including a signature disulfide-constrained loop that is thought to mediate the intramolecular conformational changes linking ligand binding and ion channel activation (Millar and Gotti, 2009). This “Cys-loop” superfamily also includes GABA and glycine receptors, which mediate inhibitory neurotransmission in the central nervous system (CNS). Although nAChRs are associated more with neuromodulation and presynaptic functions than with synaptic transmission, important roles in behavior and cognition can be ascribed to nAChRs in the brain (Gotti et al., 2006).

There are two main classes of nAChRs in the brain and peripheral nervous system. One class consists of homomeric pentamers of the α7 subunit, while the other class consists of heteromeric pentamers containing both alpha-type and non-alpha (beta) subunits. Homomeric α7 receptors are also found in many non-neuronal cells (Gahring and Rogers, 2005, Wessler and Kirkpatrick, 2008), where they have been shown to mediate multiple kinds of signal transduction (Arredondo et al., 2006, de Jonge et al., 2005, Marrero and Bencherif, 2009, Parrish et al., 2008).

The ligand-binding domain (LBD) of nAChRs for acetylcholine (ACh) is at the interface between subunits, a surface containing primary elements contributed by one subunit and complementary elements contributed by the adjacent subunit. In heteromeric neuronal nAChRs, α subunits (α2, α4, or α6) have evolved to contain the special subdomains of the primary face of the LBD, associated with three structural elements referred to as the A-, B-, and C-loops (Sine, 2002). Certain non-alpha subunits (β2 and β4 among the neuronal nAChR subunits, and δ, γ, and ε among the muscle nAChR subunits) have lost the specialized features of the primary LBD surface, but contain specializations associated with the complementary LBD surface, associated with three structural elements referred to as the D, E, and F loops (Sine, 2002). Alpha7 receptors are considered a primordial nAChR form of the Cys-loop receptors (Le Novere et al., 2002) and retain structural elements of both the primary and complementary surfaces of the LBD. Consequently, while heteromeric nAChR are limited to two LBDs in each pentamer at the interface between dissimilar subunits, data suggest that α7 receptors contain five potential LBDs, one at each α7–α7 interface.

Since numerous studies have supported a role for α7 receptors in cognitive and neuroprotective processes in the CNS and also as regulators of peripheral inflammation, the development of α7-selective agonists has been of interest to many scientists and pharmaceutical groups. There is a large amount of structural diversity in the compounds identified as α7-selective agonists and partial agonists due to the fact that at least three distinct structural motifs can be associated with the selective activation of α7 nAChR (Horenstein et al., 2008). The smallest molecule which can activate α7 (and other neuronal nAChR) is the tetramethyl ammonium ion (TMA, Fig. 1A), yet the α7 LBD can also evidently accommodate vastly larger molecules such as the selective partial agonist 2-methoxy-4-hydroxy-benzylidene anabaseine (2MeO4OHBA, also 4OH-GTS-21). Although numerous homology models of the α7 LBD have been published, they have been largely based on the distantly related snail ACh binding protein (AChBP) and so provide only limited insight into the binding of diverse ligands to the wild-type receptor. Therefore, we have applied the method of scanning cysteine accessibility mutations (SCAM) to the LDB of α7 nAChR.

SCAM has been used to successfully identify portions of the Torpedo nAChR subunits which contribute to the ion conduction pathway (Akabas and Karlin, 1995, Akabas et al., 1994, Akabas et al., 1992, Zhang and Karlin, 1996), residues associated with the binding of agonists and competitive antagonists (Gay et al., 2008, McLaughlin et al., 1995, Spura et al., 1999, Sullivan et al., 2002), and domain changes associated with positive allosteric modulation (Barron et al., 2009). The method involves systematically substituting cysteines, one at a time, for each of the residues in the domains of interest. The accessibility of the cysteine residues can be probed with a small, positively charged, sulfhydryl reagent such as methanethiosulfonate ethylammonium (MTSEA), which has a diameter of ≈6 Å, smaller than nicotine or anabaseine, and much smaller than the benzylidene anabaseines. Alternatively, sulfhydryl reagents which are larger or with specific functional groups, varying in charge or H-bonding properties, can be used. With this approach we have identified important functional subdomains of the α7 agonist-binding site and associated portions of the receptor, the accessibilities of which regulate agonist binding and receptor activation.

Section snippets

nAChR clones and mutants

The wild-type human nAChR clones were provided by Dr. Jon Lindstrom (Univ. Pennsylvania, Philadelphia, PA). Mutations to cDNA clones were introduced using the QuikChange kit from Stratagene according to the manufacturer’s instructions. The mutations were confirmed with automated fluorescent sequencing.

Preparation of RNA

After linearization and purification of cloned cDNAs, RNA transcripts were prepared in vitro using the appropriate mMessage mMachine kit from Ambion Inc. (Austin, TX).

Expression in Xenopus oocytes

Mature (>9 cm) female Xenopus

Construction and characterization of the cysteine-null C116S α7 pseudo-wild-type

There are four conserved cysteine residues in the extracellular domain of every nicotinic alpha subunit which are required for function, two of which form a disulfide bond and stabilize this eponymous element of every protein in the Cys-loop ligand-gated ion channel superfamily. The other pair of conserved cysteines is a vicinal pair in the C-loop of the primary face of the agonist-binding site, and this is a defining feature of the nicotinic alpha subunits. The vicinal cysteines are also

Acknowledgements

We thank Adolph Chiu for assistance in preparation of the α7 homology model used to illustrate mutation sites discussed in this study. We also acknowledge and thank Chad Brodbeck, Dolan Abu-Aouf, Lisa Jacobs, Lynda Cortes, Casie Lindsly, Sara Braley, and Shehd Abdullah Abbas Al Rubaiy for technical assistance. This work was supported by the National Institutes of Health Grant R01 GM057481.

References (42)

  • J. Arredondo et al.

    Receptor-mediated tobacco toxicity: cooperation of the Ras/Raf-1/MEK1/ERK and JAK-2/STAT-3 pathways downstream of alpha7 nicotinic receptor in oral keratinocytes

    FASEB J.

    (2006)
  • S.C. Barron et al.

    An allosteric modulator of alpha7 nicotinic receptors, N-(5-Chloro-2,4-dimethoxyphenyl)-N’-(5-methyl-3-isoxazolyl)-urea (PNU-120596), causes conformational changes in the extracellular ligand binding domain similar to those caused by acetylcholine

    Mol. Pharmacol.

    (2009)
  • K. Brejc et al.

    Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors

    Nature

    (2001)
  • D. Colquhoun

    Binding, gating, affinity and efficacy: the interpretation of structure-activity relationships for agonists and of the effects of mutating receptors

    Br. J. Pharmacol.

    (1998)
  • W.J. de Jonge et al.

    Stimulation of the vagus nerve attenuates macrophage activation by activating the Jak2-STAT3 signaling pathway

    Nat. Immunol.

    (2005)
  • C. Dowell et al.

    Alpha-conotoxin PIA is selective for alpha6 subunit-containing nicotinic acetylcholine receptors

    J. Neurosci.

    (2003)
  • T. Dunckley et al.

    Mutational analysis of roles for extracellular cysteine residues in the assembly and function of human alpha 7-nicotinic acetylcholine receptors

    Biochemistry

    (2003)
  • J. Dundas et al.

    CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues

    Nucleic Acids Res.

    (2006)
  • L.C. Gahring et al.

    Neuronal nicotinic acetylcholine receptor expression and function on nonneuronal cells

    AAPS J.

    (2005)
  • E.A. Gay et al.

    Aromatic residues at position 55 of rat alpha7 nicotinic acetylcholine receptors are critical for maintaining rapid desensitization

    J. Physiol.

    (2008)
  • T. Grutter et al.

    An H-bond between two residues from different loops of the acetylcholine binding site contributes to the activation mechanism of nicotinic receptors

    EMBO J.

    (2003)
  • Cited by (0)

    View full text