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Nicotinic Receptors in Nervous and Immune Systems: Identification and Functional Roles

Victor I. Tsetlin1*, Irina V. Shelukhina1, Igor E. Kasheverov1, Yuri N. Utkin1 and Marina V. Skok2
  • 1 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Department of Molecular Basis of Neurosignaling, Russia
  • 2 Palladin Instituteof Biochemistry, Department of Molecular Immunology, Ukraine

Nicotinic Receptors in Nervous and Immune Systems: Identification and Functional Roles

Victor I. Tsetlin1, Irina V. Shelukhina1, Igor E. Kasheverov1, Yuri N. Utkin1, and Marina V. Skok2

1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russian Federation, 2Palladin Institute of Biochemistry, Kiev, Ukraine.
*Correspondence: Victor I. Tsetlin , 1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str. 16/10, Moscow 117997, Russia


Abstract

Nicotinic acetylcholine receptors (nAChR) are present in neuromuscular junctions, brain and other tissues, including skin and the immune system cells (see reviews Changeux , 2012, Wessler et al., 2008). They are involved in normal functions ranging from muscle contraction and regulation of immune processes to cognition. nAChR malfunctioning, due to mutations, disturbances in the expression levels or to wrong interactions are associated with such autoimmune diseases as myasthenia gravis (Dineley 2007, Lindstrom et al., 2008), as well as with different channelopathies, psychiatric and neurodegenerative diseases (Steinlein et al., 2008). Since nAChRs are composed of 5 identical or different subunits, the important task is identification of the individual receptor subtypes in normal physiological processes and at pathologies. Here we present a short summary of our work in this direction. Antibodies against the nAChR domains or respective synthetic peptides are useful tools in the nAChR field, in particular helping to isolate one or another nAChR subtype (see, for example Tsetlin et al, 2007). However, due to high homology of nAChR subunits, antibodies cannot give a reliable answer when used in Western blots analysis for localization of distinct nAChR subtypes in tissues. This limitation was demonstrated by a joint paper from several key laboratories showing that none of the existing antibodies could clearly differentiate the tissue from the wild-type mouse and that of the mouse with a knock-out of the respective nAChR subunit (Moser et al., 2007). More specific and reliable in this respect are protein and peptide neurotoxins selectively inhibiting distinct nAChR subtypes (Tsetlin et al., 2009). α-Bungarotoxin (α-Bgt) from the Bungarus multicinctus krait venom was indispensable for isolating the muscle-type nAChR from the Torpedo electric ray 4 decades ago did the same about decade ago for purifying from the Lymnaea stagnalis mollusk the acetylcholine-binding protein (AChBP), an excellent model for the ligand-binding domains of all types of nAChRs (see review Rucktooa et al., 2009) Radioactive and fluorescent derivatives of α-Bgt and other homologous α-neurotoxins also have a high affinity for the neuronal α7 nAChR. Our interests are focused mainly on this nAChR subtype which is present both in the nervous and immune systems. Using Alexa-α Bgt we reliably identified α7 nAChR in the mouse dorsal root ganglion. (Shelukhina et al., 2009). A certain per cent of mouse dorsal root ganglion (DRG) neurons was stained. The staining was prevented by pretreatment with α-cobratoxin (which interacts both with the α7 and muscle-type nAChRs), but not by preincubation with a short-type α-neurotoxin having high affinity only for the muscle-type nAChR. Moreover, no staining was observed with slices from the α7 knock-out mouse. Thus, our results undoubtedly proved the presence of α7 nAChR on the mouse DRG. At present we are trying to characterize in more detail such α7 nAChR-containing neurons and to clarify whether they are involved in nociception.
Protein and peptide neurotoxins proved very useful for localizing distinct nAChRs built of neuronal subunits outside the nerve cells. First of all it concerns the immune cells and here radioiodinated αBgt and tritiated epibatidine are used for estimation of the levels of the α7 and α4β2 nAChRs, respectively. With these tools it was shown in B-lymphocyte-derived cell lines these two receptor subtype are expressed and snake venom neurotoxins, antagonists of the α7 nAChR, inhibited cell proliferation but increased antibody production (Skok et al., 2003). These receptors are involved in regulation of lymphocyte development and control B lymphocyte survival (Skok et al, 2006). The involvement of α7 nAChR in lymphocyte survival was confirmed with antibodies against the α7 nAChR extracellular ligand-binding domain (Lykhmus et al., 2010). Interestingly, application of α-conotoxin specific for α9 (α10) homooligomeric nAChRs revealed their relatively high expression in mouse B lymphocytes (Koval et al., 2011). Finally, fluorescent and radioactive derivatives of snake venom α-neurotoxins in combination with such methods as electron microscopy, measurements of calcium currents and application of α7 knock-out mice were used to prove the presence of α7 nAChR on the outer membrane of the mitochondria (Gergalova et al., 2012). Interestingly, α7 nAChR agonists decreased both the proapoptotic release of cytochrome c and the intramitochondrial accumulation of Ca2+ ions indicating that these effects are not realized through the inducing of potent Ca2+ currents typical for neuronal α7 nAChR.
There is still a need in new tools for research on nAChRs In proteomic studies of snake venoms we found some new potential tools: from Naja kaouthia cobra venom a minor component was isolated built of two α-cobratoxins connected by two intermolecular disulfide bonds. This post-translational modification endowed the respective dimer with a capacity to block α3β2 nAChR (Osipov et al., 2008). In collaboration with Dutch crystallograpers the X-ray structure of the α-cobratoxin dimer was solved which also allowed us to establish the disposition of the intermolecular disulfides (Osipov et al., 2012). On the other hand, from the Azemiopis feae venom we isolated a linear peptideazemiopsin which blocks selectively the muscle-type nAChRs but, contrary to all earlier known peptide and protein inhibitors of nAChRs, does not contain disulfide, which considerably simplifies the synthesis of this potential myorelaxant (Utkin et al., 2012).
α-Conotoxins, relatively short neurotoxic peptides from Conus marine snails, are even more sophisticated tools for distinguishing between distinct nAChR subtypes. α-Conotoxins are widely used tools in structure-function and pharmacological studies on nAChRs. One group of α-conotoxins block only muscle-type nAChRs, while another group acts exclusively on neuronal nAChRs. Moreover, some from the latter group inhibit several neuronal subtypes, while certain representatives are more selective (see reviews Norton et al., 2006, Kasheverov et al., 2009). New α-conotoxin structures appeared both due to isolation of appropriate peptides from the Conus venoms and as a result of analyzing mRNA from the venom glands. However, it is still a challenging task to have new α-conotoxins with high affinity and exclusively rigid selectivity towards one particular nAChR subtype. In labeled (radioactive or fluorescent) form it would be suitable for visualizing the respective nAChRs. Such a task has been solved by MacIntosh and colleagues (Hone et al., 2010) when in α-conotoxin ArIB two substitutions were introduced by peptide synthesis and the resulting compound proved an excellent marker of the α7 nAChR. We set a similar task to get more potent α-conotoxins starting from the computer modeling of the respective complexes. α-Conotoxin PnIA was chosen because earlier the X-ray structure of its homolog PnIA[A10L, D14K] in complex with Aplysia californica AChBP was determined (Celie et al., 2005 ). We synthesized 15 analogues containing from 1 to 5 substitutions and through competition with radioiodinated αBgt analyzed their binding to Aplysia californica AChBP, Lymnaea stagnalis AChBP and α7 nAChR (Kasheverov et al., 2011). For the majority of analogues a decrease rather than increase in the affinity was observed which apparently reflects limitations of the computational approaches. However, among the positive results should be mentioned PnIA[L5D,P7R,A10L] with the about 1000-fold higher affinity for Aplysia californica AChBP than for Lymnaea stagnalis AChBP. Introduction of two additional substitutions into PnIA[A10L] increased 20-fold the affinity of the resulting PnIA[L5R,A10L,D14R] towards α7 nAChR. Moreover, this analog was iodinated which made possible to analyze its binding not through competition with radioiodinated αBgt (which is not easy because of the very high affinity and virtually irreversible binding of αBgt) but directly. This work also demonstrated the advantage of radioiodinated α-conotoxins for screening the compounds with moderate cholinergic activity.

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Keywords: nicotinic acetylcholine receptor, ligand binding, alpha-neurotoxin, alpha-conotoxin, radioligand analysis, Electrophysiology

Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.

Presentation Type: Abstract

Topic: Immune receptors and signaling

Citation: Tsetlin VI, Shelukhina IV, Kasheverov IE, Utkin YN and Skok MV (2013). Nicotinic Receptors in Nervous and Immune Systems: Identification and Functional Roles. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00426

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Received: 10 Apr 2013; Published Online: 22 Aug 2013.

* Correspondence: Prof. Victor I Tsetlin, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Department of Molecular Basis of Neurosignaling, Moscow, 117997, Russia, victortsetlin3f@gmail.com.OLD