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Nicotinic acetylcholine receptors (nAChRs) are drug targets for neurological diseases and disorders, but selective targeting of the large number of nAChR subtypes is challenging. Marine cone snail α‐conotoxins are potent blockers of nAChRs and some have been engineered to achieve subtype selectivity. This engineering effort would benefit from rapid computational methods able to predict mutational energies, but current approaches typically require high‐resolution experimental structures, which are not widely available for α‐conotoxin complexes. Herein, five mutational energy prediction methods were benchmarked using crystallographic and mutational data on two acetylcholine binding protein/α‐conotoxin systems. Molecular models were developed for six nAChR subtypes in complex with five α‐conotoxins that were studied through 150 substitutions. The best method was a combination of FoldX and molecular dynamics simulations, resulting in a predictive Matthews Correlation Coefficient (MCC) of 0.68 (85 % accuracy). Novel α‐conotoxin mutants designed using this method were successfully validated by experimental assay with improved pharmaceutical properties. This work paves the way for the rapid design of subtype‐specific nAChR ligands and potentially accelerated drug development. [ABSTRACT FROM AUTHOR]

Neuropathic pain is a complex and debilitating syndrome for which there are few effective pharmacological treatments. Opioid-based medications are initially effective for acute pain, but tolerance to their analgesic effects quickly develops, and long-term use often leads to physical dependence and addiction. Furthermore, neuropathic pain is generally resistant to non-steroidal anti-inflammatory drugs. Other classes of medications including antidepressants, antiepileptics and voltage-gated calcium channel inhibitors are only partially effective in most patients, may be associated with significant side effects and have few disease-modifying effects on the underlying pathology. Medications that act through new mechanisms of action, and particularly ones that have disease-modifying properties, would be highly desirable. In the last decade, a potential new target for the treatment of neuropathic pain has emerged: the α9-containing nicotinic acetylcholine receptor (nAChR). Recent studies indicate that antagonists of α9-containing nAChRs are analgesic in animal models of neuropathic pain. These nerve injury models include chronic constriction injury, partial sciatic nerve ligation, streptozotocin-induced diabetic neuropathy and chemotherapeutic-induced neuropathy. This review details the history and state of the field regarding the role that α9-containing nAChRs may play in neuropathic pain. An alternative hypothesis that α-conotoxins exert their therapeutic effect through blocking N-type calcium channels via activation of GABAB receptors is also reviewed. Understanding how antagonists of α9-containing nAChRs exert their therapeutic effects may ultimately result in the development of medications that not only treat but also prevent the development of neuropathic pain states.Linked Articles: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc. [ABSTRACT FROM AUTHOR]

Nicotinic acetylcholine receptors (nAChRs) are actively being investigated as therapeutic targets for the treatment of pain and inflammation, but despite more than 30 years of research, there are currently no FDA‐approved analgesics that are specific for these receptors. Much of the initial research effort focused on the α4β2 nAChR subtype, but more recently, additional subtypes have been identified as promising new leads and include α6β4, α7, and α9‐containing nAChRs. This Review will focus on the distribution of these nAChRs in the cell types involved in neuropathic pain and inflammation and the activity of currently available nicotinic ligands. [ABSTRACT FROM AUTHOR]

The α6-containing nicotinic acetylcholine receptors (nAChRs) have recently been implicated in diseases of the central nervous system (CNS), including Parkinson's disease and substance abuse. In contrast, little is known about the role of α6* nAChRs in the peripheral nervous system (where the asterisk denotes the possible presence of additional subunits). Dorsal root ganglia (DRG) neurons are known to express nAChRs with a pharmacology consistent with an α7, α3β4*, and α4β2* composition. Here we present evidence that DRG neurons also express α6* nAChRs. We used RT-PCR to show the presence of α6 subunit transcripts and patch-clamp electrophysiology together with subtype-selective α-conotoxins to pharmacologically characterize the nAChRs in rat DRG neurons. α-Conotoxin BuIA (500 nM) blocked acetylcholine-gated currents (IACh) by 90.3 ± 3.0%; the recovery from blockade was very slow, indicating a predominance of αxβ4* nAChRs. Perfusion with either 300 nM BuIA[T5A;P6O] or 200 nM MII[E11A], α-conotoxins that target the α6β4* subtype, blocked IACh by 49.3 ± 5 and 46.7 ± 8%, respectively. In these neurons, IACh was relatively insensitive to 200 nM ArIB[V11L;V16D] (9.4±2.0% blockade) or 500 nM PnIA (23.0±4% blockade), α-conotoxins that target α7 and α3β2*/α6β2* nAChRs, respectively. We conclude that α6β4* nAChRs are among the subtypes expressed by DRG, and to our knowledge, this is the first demonstration of α6β4* in neurons outside the CNS. [ABSTRACT FROM AUTHOR]

The alpha7* (*denotes the possible presence of additional subunits) nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the vertebrate nervous system and implicated in neuropsychiatric disorders that compromise thought and cognition. In this report, we demonstrate that the recently developed fluorescent ligand Cy3-ArIB[V11L;V16A] labels alpha7 nAChRs in cultured hippocampal neurons. However, photobleaching of this ligand during long image acquisition times prompted us to develop a new derivative. In photostability studies, this new ligand, Alexa Fluor 546-ArIB[V11L;V16A], was significantly more resistant to bleaching than the Cy3 derivative. The classic alpha7 ligand alpha-bungarotoxin binds to alpha1* and alpha9* nAChRs. In contrast, Alexa Fluor 546-ArIB[V11L;V16A] potently (IC(50) 1.8 nM) and selectively blocked alpha7 nAChRs but not alpha1* or alpha9* nAChRs expressed in Xenopus oocytes. Selectivity was further confirmed by competition binding studies of native nAChRs in rat brain membranes. The fluorescence properties of Alexa Fluor 546-ArIB[V11L;V16A] were assessed using human embryonic kidney-293 cells stably transfected with nAChRs; labeling was observed on cells expressing alpha7 but not cells expressing alpha3beta2, alpha3beta4, or alpha4beta2 nAChRs. Further imaging studies demonstrate that Alexa Fluor 546-ArIB[V11L;V16A] labels hippocampal neurons from wild-type mice but not from nAChR alpha7 subunit-null mice. Thus, Alexa Fluor 546-ArIB[V11L;V16A] represents a potent and selective ligand for imaging alpha7 nAChRs.

Homomeric alpha7 nicotinic acetylcholine receptors are a well-established, pharmacologically distinct subtype. The more recently identified alpha9 subunit can also form functional homopentamers as well as alpha9alpha10 heteropentamers. Current fluorescent probes for alpha7 nicotinic ACh receptors are derived from alpha-bungarotoxin (alpha-BgTx). However, alpha-BgTx also binds to alpha9* and alpha1* receptors which are coexpressed with alpha7 in multiple tissues. We used an analog of alpha-conotoxin ArIB to develop a highly selective fluorescent probe for alpha7 receptors. This fluorescent alpha-conotoxin, Cy3-ArIB[V11L;V16A], blocked ACh-evoked alpha7 currents in Xenopus laevis oocytes with an IC(50) value of 2.0 nM. Observed rates of blockade were minute-scale with recovery from blockade even slower. Unlike FITC-conjugated alpha-BgTx, Cy3-ArIB[V11L;V16A] did not block alpha9alpha10 or alpha1beta1deltaepsilon receptors. In competition binding assays, Cy3-ArIB[V11L;V16A] potently displaced [(125)I]-alpha-BgTx binding to mouse hippocampal membranes with a K(i) value of 21 nM. Application of Cy3-ArIB[V11L;V16A] resulted in specific punctate labeling of KXalpha7R1 cells but not KXalpha3beta2R4, KXalpha3beta4R2, or KXalpha4beta2R2 cells. This labeling could be abolished by pre-treatment with alpha-cobratoxin. Thus, Cy3-ArIB[V11L;V16A] is a novel and selective fluorescent probe for alpha7 receptors.