Cysteine accessibility analysis of the human alpha7 nicotinic acetylcholine receptor ligand-binding domain identifies L119 as a gatekeeper
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.
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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)
- et al.
Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the alpha subunit
Neuron
(1994) - et al.
Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures
Neuron
(2004) - et al.
Functional significance of aromatic amino acids from three peptide loops of the alpha 7 neuronal nicotinic receptor site investigated by site-directed mutagenesis
FEBS Lett.
(1991) - et al.
Brain nicotinic acetylcholine receptors: native subtypes and their relevance
Trends Pharmacol. Sci.
(2006) - et al.
Reversal of agonist selectivity by mutations of conserved amino acids in the binding site of nicotinic acetylcholine receptors
J. Biol. Chem.
(2007) - et al.
Convergence of alpha 7 nicotinic acetylcholine receptor-activated pathways for anti-apoptosis and anti-inflammation: central role for JAK2 activation of STAT3 and NF-kappaB
Brain Res.
(2009) - et al.
Diversity of vertebrate nicotinic acetylcholine receptors
Neuropharmacology
(2009) The classification of amino acid conservation
J. Theor. Biol.
(1986)- et al.
Identification of acetylcholine receptor channel-lining residues in the M1 segment of the α-subunit
Biochemistry
(1995) - et al.
Acetylcholine receptor channel structure probed in cysteine-substitution mutants
Science
(1992)