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Defining the putative inhibitory site for a selective negative allosteric modulator of human ?4?2 neuronal nicotinic receptors.

ABSTRACT: Neuronal nicotinic receptors (nAChRs) have been implicated in several diseases and disorders such as autism spectrum disorders, Alzheimer's disease, Parkinson's disease, epilepsy, and nicotine addiction. To understand the role of nAChRs in these conditions, it would be beneficial to have selective molecules that target specific nAChRs in vitro and in vivo. Our laboratory has previously identified a novel allosteric site on human ?4?2 nAChRs using a series of computational and in vitro approaches. At this site, we have identified negative allosteric modulators that selectively inhibit human ?4?2 nAChRs, a subtype implicated in nicotine addiction. This study characterizes the allosteric site via site-directed mutagenesis. Three amino acids (Phe118, Glu60, and Thr58) on the ?2 subunit were shown to participate in the inhibitory properties of the selective antagonist KAB-18 and provided insights into its antagonism of human ?4?2 nAChRs. SAR studies with KAB-18 analogues and various mutant ?4?2 nAChRs also provided information concerning how different physiochemical features influence the inhibition of nAChRs through this allosteric site. Together, these studies identify the amino acids that contribute to the selective antagonism of human ?4?2 nAChRs at this allosteric site. Finally, these studies define the physiochemical features of ligands that influence interaction with specific amino acids in this allosteric site.

SUBMITTER: Henderson BJ

PROVIDER: S-EPMC3447398 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Defining the putative inhibitory site for a selective negative allosteric modulator of human α4β2 neuronal nicotinic receptors.

Henderson Brandon J BJ González-Cestari Tatiana F TF Yi Bitna B Pavlovicz Ryan E RE Boyd R Thomas RT Li Chenglong C Bergmeier Stephen C SC McKay Dennis B DB

ACS chemical neuroscience 20120525 9

Neuronal nicotinic receptors (nAChRs) have been implicated in several diseases and disorders such as autism spectrum disorders, Alzheimer's disease, Parkinson's disease, epilepsy, and nicotine addiction. To understand the role of nAChRs in these conditions, it would be beneficial to have selective molecules that target specific nAChRs in vitro and in vivo. Our laboratory has previously identified a novel allosteric site on human α4β2 nAChRs using a series of computational and in vitro approaches ...[more]

PMID: 23019495

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Project description:Homomeric ?7 nicotinic acetylcholine receptors (nAChR) have an intrinsically low probability of opening that can be overcome by ?7-selective positive allosteric modulators (PAMs), which bind at a site involving the second transmembrane domain (TM2). Mutation of a methionine that is unique to ?7 at the 15' position of TM2 to leucine, the residue in most other nAChR subunits, largely eliminates the activity of such PAMs. We tested the effect of the reverse mutation (L15'M) in heteromeric nAChR receptors containing ?4 and ?2, which are the nAChR subunits that are most abundant in the brain. Receptors containing these mutations were found to be strongly potentiated by the ?7 PAM 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS) but insensitive to the alternative PAM 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea. The presence of the mutation in the ?2 subunit was necessary and sufficient for TQS sensitivity. The primary effect of the mutation in the ?4 subunit was to reduce responses to acetylcholine applied alone. Sensitivity to TQS required only a single mutant ? subunit, regardless of the position of the mutant ? subunit within the pentameric complex. Similar results were obtained when ?2L15'M was coexpressed with ?2 or ?3 and when the L15'M mutation was placed in ?4 and coexpressed with ?2, ?3, or ?4. Functional receptors were not observed when ?1L15'M subunits were coexpressed with other muscle nAChR subunits. The unique structure-activity relationship of PAMs and the ?4?2L15'M receptor compared with ?7 and the availability of high-resolution ?4?2 structures may provide new insights into the fundamental mechanisms of nAChR allosteric potentiation. SIGNIFICANCE STATEMENT: Heteromeric neuronal nAChRs have a relatively high initial probability of channel activation compared to receptors that are homomers of ?7 subunits but are insensitive to PAMs, which greatly increase the open probability of ?7 receptors. These features of heteromeric nAChR can be reversed by mutation of a single residue present in all neuronal heteromeric nAChR subunits to the sequence found in ?7. Specifically, the mutation of the TM2 15' leucine to methionine in ? subunits reduces heteromeric receptor channel activation, while the same mutation in neuronal ? subunits allows heteromeric receptors to respond to select ?7 PAMs. The results indicate a key role for this residue in the functional differences in the two main classes of neuronal nAChRs.

Project description:Acetylcholine-based neurotransmission is regulated by cationic, ligand-gated ion channels called nicotinic acetylcholine receptors (nAChRs). These receptors have been linked to numerous neurological diseases and disorders such as Alzheimer's disease, Parkinson's disease, and nicotine addiction. Recently, a class of compounds has been discovered that antagonize nAChR function in an allosteric fashion. Models of human ?4?2 and ?3?4 nicotinic acetylcholine receptor (nAChR) extracellular domains have been developed to computationally explore the binding of these compounds, including the dynamics and free energy changes associated with ligand binding. Through a blind docking study to multiple receptor conformations, the models were used to determine a putative binding mode for the negative allosteric modulators. This mode, in close proximity to the agonist binding site, is presented in addition to a hypothetical mode of antagonism that involves obstruction of C loop closure. Molecular dynamics simulations and MM-PBSA free energy of binding calculations were used as computational validation of the predicted binding mode, while functional assays on wild-type and mutated receptors provided experimental support. Based on the proposed binding mode, two residues on the ?2 subunit were independently mutated to the corresponding residues found on the ?4 subunit. The T58K mutation resulted in an eight-fold decrease in the potency of KAB-18, a compound that exhibits preferential antagonism for human ?4?2 over ?3?4 nAChRs, while the F118L mutation resulted in a loss of inhibitory activity for KAB-18 at concentrations up to 100 µM. These results demonstrate the selectivity of KAB-18 for human ?4?2 nAChRs and validate the methods used for identifying the nAChR modulator binding site. Exploitation of this site may lead to the development of more potent and subtype-selective nAChR antagonists which may be used in the treatment of a number of neurological diseases and disorders.

Dataset Information

Defining the putative inhibitory site for a selective negative allosteric modulator of human ?4?2 neuronal nicotinic receptors.

Publications

Defining the putative inhibitory site for a selective negative allosteric modulator of human α4β2 neuronal nicotinic receptors.

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