Arik J. Hone

Nicotinic acetylcholine receptors in dorsal root ganglion neurons include the α6β4* subtype

The α6‐containing nicotinic acetylcholine receptors (nAChRs) have recently been implicated in diseases of the central nervous system (CNS), including Parkinsons 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.—Hone, A. J., Meyer, E. L., McIntyre, M., McIntosh, J. M. Nicotinic acetylcholine receptors in dorsal root ganglion neurons include the α6β4∗ subtype. FASEB J. 26, 917–926 (2012). www.fasebj.org

αConotoxin Arenatus IB[V11L,V16D] Is a Potent and Selective Antagonist at Rat and Human Native α7 Nicotinic Acetylcholine Receptors

A recently developed α-conotoxin, α-conotoxin Arenatus IB-[V11L,V16D] (α-CtxArIB[V11L,V16D]), is a potent and selective competitive antagonist at rat recombinant α7 nicotinic acetylcholine receptors (nAChRs), making it an attractive probe for this receptor subtype. α7 nAChRs are potential therapeutic targets that are widely expressed in both neuronal and non-neuronal tissues, where they are implicated in a variety of functions. In this study, we evaluate this toxin at rat and human native nAChRs. Functional α7 nAChR responses were evoked by choline plus the allosteric potentiator PNU-120596 [1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea] in rat PC12 cells and human SH-SY5Y cells loaded with calcium indicators. α-CtxArIB[V11L,V16D] specifically inhibited α7 nAChR-mediated increases in Ca2+ in PC12 cells. Responses to other stimuli, 5-I-A-85380 [5-iodo-3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride], nicotine, or KCl, that did not activate α7 nAChRs were unaffected. Human α7 nAChRs were also sensitive to α-CtxArIB[V11L,V16D]; acetylcholine-evoked currents in Xenopus laevis oocytes expressing human α7 nAChRs were inhibited by α-CtxArIB[V11L,V16D] (IC50, 3.4 nM) in a slowly reversible manner, with full recovery taking 15 min. This is consistent with the time course of recovery from blockade of rat α7 nAChRs in PC12 cells. α-CtxArIB[V11L,V16D] inhibited human native α7 nAChRs in SHSY5Y cells, activated by either choline or AR-R17779 [(2)-spiro[1-azabicyclo[2.2.2]octane-3,59-oxazolidin]-29-one] plus PNU-120596. Rat brain α7 nAChRs contribute to dopamine release from striatal minces; α-CtxArIB[V11L,V16D] (300 nM) selectively inhibited choline-evoked dopamine release without affecting responses evoked by nicotine that activates heteromeric nAChRs. This study establishes that α-CtxArIB[V11L,V16D] selectively inhibits human and rat native α7 nAChRs with comparable potency, making this a potentially useful antagonist for investigating α7 nAChR functions.

Phosphocholine – an agonist of metabotropic but not of ionotropic functions of α9-containing nicotinic acetylcholine receptors

We demonstrated previously that phosphocholine and phosphocholine-modified macromolecules efficiently inhibit ATP-dependent release of interleukin-1β from human and murine monocytes by a mechanism involving nicotinic acetylcholine receptors (nAChR). Interleukin-1β is a potent pro-inflammatory cytokine of innate immunity that plays pivotal roles in host defence. Control of interleukin-1β release is vital as excessively high systemic levels cause life threatening inflammatory diseases. In spite of its structural similarity to acetylcholine, there are no other reports on interactions of phosphocholine with nAChR. In this study, we demonstrate that phosphocholine inhibits ion-channel function of ATP receptor P2X7 in monocytic cells via nAChR containing α9 and α10 subunits. In stark contrast to choline, phosphocholine does not evoke ion current responses in Xenopus laevis oocytes, which heterologously express functional homomeric nAChR composed of α9 subunits or heteromeric receptors containing α9 and α10 subunits. Preincubation of these oocytes with phosphocholine, however, attenuated choline-induced ion current changes, suggesting that phosphocholine may act as a silent agonist. We conclude that phophocholine activates immuno-modulatory nAChR expressed by monocytes but does not stimulate canonical ionotropic receptor functions.

Positional Scanning Mutagenesis of α-Conotoxin PeIA Identifies Critical Residues That Confer Potency and Selectivity for α6/α3β2β3 and α3β2 Nicotinic Acetylcholine Receptors

Background: Ligands that selectively target α6β2* nAChRs are needed. Results: An analog of α-conotoxin PeIA was synthesized that was >15,000-fold more potent at inhibiting α6/α3β2β3 receptors than the closely related α3β2 subtype. Conclusion: PeIA analogs can be synthesized that distinguish between α6/α3β2β3 and α3β2 receptors. Significance: Selective ligands will facilitate the identification of α6β2* receptors in tissues that co-express α3β2* receptors. The nicotinic acetylcholine receptor (nAChR) subtype α6β2* (the asterisk denotes the possible presence of additional subunits) has been identified as an important molecular target for the pharmacotherapy of Parkinson disease and nicotine dependence. The α6 subunit is closely related to the α3 subunit, and this presents a problem in designing ligands that discriminate between α6β2* and α3β2* nAChRs. We used positional scanning mutagenesis of α-conotoxin PeIA, which targets both α6β2* and α3β2*, in combination with mutagenesis of the α6 and α3 subunits, to gain molecular insights into the interaction of PeIA with heterologously expressed α6/α3β2β3 and α3β2 receptors. Mutagenesis of PeIA revealed that Asn11 was located in an important position that interacts with the α6 and α3 subunits. Substitution of Asn11 with a positively charged amino acid essentially abolished the activity of PeIA for α3β2 but not for α6/α3β2β3 receptors. These results were used to synthesize a PeIA analog that was >15,000-fold more potent on α6/α3β2β3 than α3β2 receptors. Analogs with an N11R substitution were then used to show a critical interaction between the 11th position of PeIA and Glu152 of the α6 subunit and Lys152 of the α3 subunit. The results of these studies provide molecular insights into designing ligands that selectively target α6β2* nAChRs.

A novel fluorescent α‐conotoxin for the study of α7 nicotinic acetylcholine receptors

Homomeric α7 nicotinic acetylcholine receptors are a well‐established, pharmacologically distinct subtype. The more recently identified α9 subunit can also form functional homopentamers as well as α9α10 heteropentamers. Current fluorescent probes for α7 nicotinic ACh receptors are derived from α‐bungarotoxin (α‐BgTx). However, α‐BgTx also binds to α9* and α1* receptors which are coexpressed with α7 in multiple tissues. We used an analog of α‐conotoxin ArIB to develop a highly selective fluorescent probe for α7 receptors. This fluorescent α‐conotoxin, Cy3‐ArIB[V11L;V16A], blocked ACh‐evoked α7 currents in Xenopus laevis oocytes with an IC50 value of 2.0 nM. Observed rates of blockade were minute‐scale with recovery from blockade even slower. Unlike FITC‐conjugated α‐BgTx, Cy3‐ArIB[V11L;V16A] did not block α9α10 or α1β1δε receptors. In competition binding assays, Cy3‐ArIB[V11L;V16A] potently displaced [125I]‐α‐BgTx binding to mouse hippocampal membranes with a Ki value of 21 nM. Application of Cy3‐ArIB[V11L;V16A] resulted in specific punctate labeling of KXα7R1 cells but not KXα3β2R4, KXα3β4R2, or KXα4β2R2 cells. This labeling could be abolished by pre‐treatment with α‐cobratoxin. Thus, Cy3‐ArIB[V11L;V16A] is a novel and selective fluorescent probe for α7 receptors.

Comparative functional expression of nAChR subtypes in rodent DRG neurons.

We investigated the functional expression of nicotinic acetylcholine receptors (nAChRs) in heterogeneous populations of dissociated rat and mouse lumbar dorsal root ganglion (DRG) neurons by calcium imaging. By this experimental approach, it is possible to investigate the functional expression of multiple receptor and ion-channel subtypes across more than 100 neuronal and glial cells simultaneously. Based on nAChR expression, DRG neurons could be divided into four subclasses: (1) neurons that express predominantly α3β4 and α6β4 nAChRs; (2) neurons that express predominantly α7 nAChRs; (3) neurons that express a combination of α3β4/α6β4 and α7 nAChRs; and (4) neurons that do not express nAChRs. In this comparative study, the same four neuronal subclasses were observed in mouse and rat DRG. However, the expression frequency differed between species: substantially more rat DRG neurons were in the first three subclasses than mouse DRG neurons, at all developmental time points tested in our study. Approximately 70–80% of rat DRG neurons expressed functional nAChRs, in contrast to only ~15–30% of mouse DRG neurons. Our study also demonstrated functional coupling between nAChRs, voltage-gated calcium channels, and mitochondrial Ca2+ transport in discrete subsets of DRG neurons. In contrast to the expression of nAChRs in DRG neurons, we demonstrated that a subset of non-neuronal DRG cells expressed muscarinic acetylcholine receptors and not nAChRs. The general approach to comparative cellular neurobiology outlined in this paper has the potential to better integrate molecular and systems neuroscience by uncovering the spectrum of neuronal subclasses present in a given cell population and the functionally integrated signaling components expressed in each subclass.

α-Conotoxin PeIA[S9H,V10A,E14N] potently and selectively blocks α6β2β3 versus α6β4 nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) containing α6 and β2 subunits modulate dopamine release in the basal ganglia and are therapeutically relevant targets for treatment of neurological and psychiatric disorders including Parkinsons disease and nicotine dependence. However, the expression profile of β2 and β4 subunits overlap in a variety of tissues including locus ceruleus, retina, hippocampus, dorsal root ganglia, and adrenal chromaffin cells. Ligands that bind α6β2 nAChRs also potently bind the closely related α6β4 subtype. To distinguish between these two subtypes, we synthesized novel analogs of a recently described α-conotoxin, PeIA. PeIA is a peptide antagonist that blocks several nAChR subtypes, including α6/α3β2β3 and α6/α3β4 nAChRs, with low nanomolar potency. We systematically mutated PeIA and evaluated the resulting analogs for enhanced potency and/or selectivity for α6/α3β2β3 nAChRs expressed in Xenopus oocytes (α6/α3 is a subunit chimera that contains the N-terminal ligand-binding domain of the α6 subunit). On the basis of these results, second-generation analogs were then synthesized. The final analog, PeIA[S9H,V10A,E14N], potently blocked acetylcholine-gated currents mediated by α6/α3β2β3 and α6/α3β4 nAChRs with IC50 values of 223 pM and 65 nM, respectively, yielding a >290-fold separation between the two subtypes. Kinetic studies of ligand binding to α6/α3β2β3 nAChRs yielded a koff of 0.096 ± 0.001 min−1 and a kon of 0.23 ± 0.019 min−1 M−9. The synthesis of PeIA[S9H,V10A,E14N] demonstrates that ligands can be developed to discriminate between α6β2 and α6β4 nAChRs.

Alexa Fluor 546-ArIB[V11L;V16A]is a potent ligand for selectively labeling α7 nicotinic acetylcholine receptors

J. Neurochem. (2010) 114, 994–1006.

Alpha-conotoxin Arenatus IB[V11L,V16D] [corrected] is a potent and selective antagonist at rat and human native alpha7 nicotinic acetylcholine receptors.

A recently developed α-conotoxin, α-conotoxin Arenatus IB-[V11L,V16D] (α-CtxArIB[V11L,V16D]), is a potent and selective competitive antagonist at rat recombinant α7 nicotinic acetylcholine receptors (nAChRs), making it an attractive probe for this receptor subtype. α7 nAChRs are potential therapeutic targets that are widely expressed in both neuronal and non-neuronal tissues, where they are implicated in a variety of functions. In this study, we evaluate this toxin at rat and human native nAChRs. Functional α7 nAChR responses were evoked by choline plus the allosteric potentiator PNU-120596 [1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea] in rat PC12 cells and human SH-SY5Y cells loaded with calcium indicators. α-CtxArIB[V11L,V16D] specifically inhibited α7 nAChR-mediated increases in Ca2+ in PC12 cells. Responses to other stimuli, 5-I-A-85380 [5-iodo-3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride], nicotine, or KCl, that did not activate α7 nAChRs were unaffected. Human α7 nAChRs were also sensitive to α-CtxArIB[V11L,V16D]; acetylcholine-evoked currents in Xenopus laevis oocytes expressing human α7 nAChRs were inhibited by α-CtxArIB[V11L,V16D] (IC50, 3.4 nM) in a slowly reversible manner, with full recovery taking 15 min. This is consistent with the time course of recovery from blockade of rat α7 nAChRs in PC12 cells. α-CtxArIB[V11L,V16D] inhibited human native α7 nAChRs in SHSY5Y cells, activated by either choline or AR-R17779 [(2)-spiro[1-azabicyclo[2.2.2]octane-3,59-oxazolidin]-29-one] plus PNU-120596. Rat brain α7 nAChRs contribute to dopamine release from striatal minces; α-CtxArIB[V11L,V16D] (300 nM) selectively inhibited choline-evoked dopamine release without affecting responses evoked by nicotine that activates heteromeric nAChRs. This study establishes that α-CtxArIB[V11L,V16D] selectively inhibits human and rat native α7 nAChRs with comparable potency, making this a potentially useful antagonist for investigating α7 nAChR functions.

α9-containing nicotinic acetylcholine receptors and the modulation of pain

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.