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Probing the Allosteric Role of the ?5 Subunit of ?3?4?5 Nicotinic Acetylcholine Receptors by Functionally Selective Modulators and Ligands.

ABSTRACT: Nicotinic acetylcholine receptors regulate the nicotine dependence encountered with cigarette smoking, and this has stimulated a search for drugs binding the responsible receptor subtypes. Studies link a gene cluster encoding for ?3?4?5-D398N nicotinic acetylcholine receptors to lung cancer risk as well as link a second mutation in this cluster to an increased risk for nicotine dependence. However, there are currently no recognized drugs for discriminating ?3?4?5 signaling. In this study, we describe the development of homogeneous HEK-293 cell clones of ?3?4 and ?3?4?5 receptors appropriate for drug screening and characterizing biochemical and pharmacological properties of incorporated ?5 subunits. Clones were assessed for plasma membrane expression of the individual receptor subunits by mass spectrometry and immunochemistry, and their calcium flux was measured in the presence of a library of kinase inhibitors and a focused library of acetylcholine receptor ligands. We demonstrated an incorporation of two ?3 subunits in approximately 98% of plasma membrane receptor pentamers, indicating a 2/3 subunit expression ratio of ?3 to ?4 alone or to coexpressed ?4 and ?5. With prolonged nicotine exposure, the plasma membrane expression of receptors with and without incorporated ?5 increased. Whereas ?5 subunit expression decreased the cell calcium response to nicotine and reduced plasma membrane receptor number, it partially protected receptors from nicotine mediated desensitization. Hit compounds from both libraries suggest the ?5 and ?5-D398N subunits allosterically modify the behavior of nicotine at the parent ?3?4 nicotinic acetylcholine receptor. These studies identify pharmacological tools from two distinct classes of drugs, antagonists and modifiers that are ?5 and ?5-D398N subtype selective that provide a means to characterize the role of the CHRNA5/A3/B4 gene cluster in smoking and cancer.

SUBMITTER: Ray C

PROVIDER: S-EPMC5417700 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Probing the Allosteric Role of the α5 Subunit of α3β4α5 Nicotinic Acetylcholine Receptors by Functionally Selective Modulators and Ligands.

Ray Caroline C Soderblom Erik J EJ Bai Yushi Y Carroll F Ivy FI Caron Marc G MG Barak Larry S LS

ACS chemical biology 20170124 3

Nicotinic acetylcholine receptors regulate the nicotine dependence encountered with cigarette smoking, and this has stimulated a search for drugs binding the responsible receptor subtypes. Studies link a gene cluster encoding for α3β4α5-D398N nicotinic acetylcholine receptors to lung cancer risk as well as link a second mutation in this cluster to an increased risk for nicotine dependence. However, there are currently no recognized drugs for discriminating α3β4α5 signaling. In this study, we des ...[more]

PMID: 28045487

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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.

Dataset Information

Probing the Allosteric Role of the ?5 Subunit of ?3?4?5 Nicotinic Acetylcholine Receptors by Functionally Selective Modulators and Ligands.

Publications

Probing the Allosteric Role of the α5 Subunit of α3β4α5 Nicotinic Acetylcholine Receptors by Functionally Selective Modulators and Ligands.

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