CNR - Institute of Neuroscience CNR
Institute of Neuroscience
 

Project

Neuronal nicotinic receptors: from structure to function

Ionotropic neuronal nicotinic acetylcholine receptors (nachrs) are a heterogeneous class of cationic channels that are widely distributed in the nervous system and whose opening is controlled by the endogenous neurotransmitter acetylcholine (ach). they consist of five subunits, and their different subunit compositions generate distinct receptor subtypes with particular functional responses to ach and drugs. the activation of nachrs targets cells, and may mediate fast synaptic transmission (e.g. in autonomous ganglionic neurons and restricted brain areas), or modulate the release of almost all neurotransmitters. the receptors are involved in a large number of physiological functions in the central and peripheral nervous systems, and changes in their number and/or function are associated with various pathophysiological conditions. they are also the targets of nicotine (the most widely used drug of abuse), whose complex activities in the nervous system are due to its ability to mimic the activity of ach. the different effects of nicotinic drugs is determined by the functional features and location of the nachr subtypes with which they interact in specific neuronal systems.

We are interested in identifying and characterising the nAChR subtypes expressed in different areas of the CNS, and studying their involvement in physiological functions and various pathological conditions, including Alzheimer’s disease and Parkinson’s disease, some forms of epilepsy, depression, autism, and schizophrenia.

Subunit composition, stoichiometry and physiological role of native subtypes

We have established the subunit composition of nAChR subtypes in various areas of the CNS of several vertebrate species using subunit-specific antibodies, immunoprecipitation and immunopurification techniques, and tissues from wild-type, knock-ou (Ko) and lesioned animals [reviewed in Gotti et al., 2006a; Grady et al., 2009].

 

In particular, we have identified the nAChR subtypes expressed in the dopaminergic neurons of the nigrostriatal and mesolimbic pathways [reviewed in Gotti et al., 2006] and, in collaboration with Prof Zoli of the University of Modena, we have recently demonstrated that α6β2* receptors play an important role in controlling locomotion and the development of some long-lasting adaptations associated with nicotine abuse [Gotti et al., 2010]. We are also studying the in vivo role of the α6β2* subtype by using RNA interference to silence the expression of a6-containing receptors in rat brain.

In addition to differences in their subunit composition, nAChR subtypes may have different subunit stoichiometries even when they have the same subunit composition. Using functional and biochemical techniques, we have recently shown that cortical and thalamic nAChRs in heterozygous α4+/- and β2+/- mice show different relative expressions of the α4 and β2 subunits, and that this correlates with differences in the functional properties of native nAChRs [Gotti et al., 2008].

Overall, these findings support the conclusion that α4β2 nAChRs with different stoichiometries are expressed in native tissues

nAChR and neurotransmitter receptor regulation by long-term exposure to nicotine and nicotinic drugs

Studies of the brains of tobacco smokers and animals have shown that long-term exposure to nicotine often triggers an increase in the number of nAChRs (up-regulation). This particular effect may be due to the fact that nicotine acts as both an agonist and a time-averaged antagonist.

We and others have shown that the most up-regulated receptor is the α4β2 subtype, but there is considerable disagreement concerning the in vivo regulation of the number and function of the other native subtypes [Moretti et al., 2010]. We are studying whether different methods of nicotine administration lead to different brain nicotine levels and/or kinetics that can affect the number and function of the nAChR subtypes in different ways.

Over the last few years, various studies of different brain areas have shown that the effects of nicotine on synaptic connections within neuronal networks can outlast nAChR stimulation and sensitisation, and alter the properties of the neuronal network by modulating excitatory and inhibitory neurons [Cunotte et al., 2012]. This can lead to changes in overall network activity that ultimately determine the altered cognitive performance caused by nicotine.

We are studying whether chronic treatment with nicotine and other nicotinic compounds also affects the number, subunit composition, or location of other neurotransmitter receptors, such as ionotropic and metabotropic glutamate receptors and G protein-coupled D1, D2 and D3 dopamine receptors.

Trafficking of the α3β4 subtype

Like many other multimeric ion channels, nAChR subtypes undergo particularly stringent quality control processes on their way to the plasma membrane in order to ensure their correct folding, subunit assembly and function.

To study the assembly of pentameric receptors, their exit from the endoplasmic reticulum membrane and their arrival at the plasma membrane, we transfected HeLa cells with nicotinic receptor subunits and, in this reconstituted system, studied the trafficking of a specific subtype of nicotinic receptor consisting of α3 and β4 subunits, its degradation by the proteasome, and its regulation by nicotine.

Recent human genetic studies have revealed a link between these subunits (whose genes cluster with that of the accessory subunit α5), nicotine dependence phenotypes, and lung cancer. In particular, it was found that a number of single-nucleotide polymorphisms (SNPs) are associated with smoking-related behaviours. One of these SNPs leads to an asparagine-to-aspartic acid substitution in the nicotinic receptor α5 subunit at amino acid position 398

In our reconstituted system, we will investigate the role of the α5 subunit in the trafficking of the α3β4 subtype, and whether the presence of the polymorphism in position 398 affects its expression and/or trafficking.

nAChRs as new pharmacological targets

The discovery of new and selective nicotinic drugs is very important because of the preferential involvement of particular nAChR subtypes in many different diseases. Using traditional medicinal chemistry and the powerful new approach of combining 3D ligand binding site modelling with virtual drug discovery screening, we have generated a number of ligands starting from the structure of epibatidine, cytisine and nicotine, in collaboration with the Institute of Medical Chemistry of the Universities of Milan and Genoa [reviewed in Gotti et al., 2006b; Bolchi et al., 2011, Dellanoce et al., 2011].

We have also used complementary approaches ranging from chemistry to molecular and cell biology and morphology, biochemistry and electrophysiology, and from in vitro functional assays to behavioural studies, to identify the effects of newly generated drugs in an attempt to unravel the complexity of nAChR transmission in different neuronal pathways, and during the extreme life periods of development and aging.

Starting from the sequence of the alpha-Conus peptide isolated from cone snails, we have recently identified the amino acids in MII toxin that are important for defining subtype selectivity, and have developed new structural analogues with greater selectivity for the native α6* subtype [Pucci et al., 2011].

This line of research has led to the formulation of new compounds including one recently patented α7-specific compound (Europeean Patent No. 2038281) [Dellanoce et al., 2011; Bolchi et al., 2011].

Publications

  • Counotte DS, Goriounova NA, Moretti M, Smoluch MT, Irth H, Clementi F, Schoffelmeer AN, Mansvelder HD, Smit AB, Gotti C, Spijker S (2012) Adolescent nicotine exposure transiently increases high-affinity nicotinic receptors and modulates inhibitory synaptic transmission in rat medial prefrontal cortex. FASEB J. 26:1810-20.
  • Bolchi C, Gotti C, Binda M, Fumagalli L, Pucci L, Pistillo F, Vistoli G, Valoti E, Pallavicini M (2011) Unichiral 2-(2'-pyrrolidinyl)-1,4-benzodioxanes: the 2R,2'S diastereomer of the N-methyl-7-hydroxy analogue is a potent ?4?2- and ?6?2-nicotinic acetylcholine receptor partial agonist. J. Med. Chem. 54:7588-601.
  • Pucci L, Grazioso G, Dallanoce C, Rizzi L, De Micheli C, Clementi F, Bertrand S, Bertrand D, Longhi R, De Amici M, Gotti C (2011) Engineering of ?-conotoxin MII-derived peptides with increased selectivity for native ?6?2* nicotinic acetylcholine receptors. FASEB J. 25:3775-89.
  • Moretti M, Mugnaini M, Tessari M, Zoli M, Gaimarri A, Manfredi I, Pistillo F, Clementi F, Gotti C (2010) A comparative study of the effects of the intravenous self-administration or subcutaneous minipump infusion of nicotine on the expression of brain neuronal nicotinic receptor subtypes. Mol. Pharmacol. 78:287-96.
  • Gotti C, Guiducci S, Tedesco V, Corbioli S, Zanetti L, Moretti M, Zanardi A, Rimondini R, Mugnaini M, Clementi F, Chiamulera C, Zoli M (2010) Nicotinic acetylcholine receptors in the mesolimbic pathway: primary role of ventral tegmental area alpha6beta2* receptors in mediating systemic nicotine effects on dopamine release, locomotion, and reinforcement. J. Neurosci. 30:5311-25.
  • Grady SR, Moretti M, Zoli M, Marks MJ, Zanardi A, Pucci L, Clementi F, Gotti C (2009) Rodent habenulo-interpeduncular pathway expresses a large variety of uncommon nAChR subtypes, but only the alpha3beta4* and alpha3beta3beta4* subtypes mediate acetylcholine release. J. Neurosci. 29:2272-82.
  • Gotti C, Moretti M, Meinerz NM, Clementi F, Gaimarri A, Collins AC, Marks MJ (2008) Partial deletion of the nicotinic cholinergic receptor alpha 4 or beta 2 subunit genes changes the acetylcholine sensitivity of receptor-mediated 86Rb+ efflux in cortex and thalamus and alters relative expression of alpha 4 and beta 2 subunits. Mol. Pharmacol. 73:1796-807.
  • Gaimarri A, Moretti M, Riganti L, Zanardi A, Clementi F, Gotti C (2007) Regulation of neuronal nicotinic receptor traffic and expression. Brain Res Rev 55:134-43.
  • Gotti C, Zoli M, Clementi F (2006) Brain nicotinic acetylcholine receptors: native subtypes and their relevance. Trends Pharmacol. Sci. 27:482-91.
  • Gotti C, Riganti L, Vailati S, Clementi F (2006) Brain neuronal nicotinic receptors as new targets for drug discovery. Curr. Pharm. Des. 12:407-28.

Grants

European Community, Neurocypres and Eranet.

NIDA

PRIN

MIUR

Collaborations

  • G. Collo e C. Missale , Università di Brescia.
  • S. Fucile, Università la Sapienza, Roma.
  • R. Longhi, CNR Institute of Molecular Recognition, Milan, Italy.
  • F. Marchi, Università di Genova.
  • M.J. McIntosh, University of Utah, USA.
  • C. De Micheli, M. De Amici, M.Pallavicini, Università di Milano, Milan, Italy.
  • F. Sparatore, Università di Genova, Genoa, Italy.
  • M. Marks and S. Grady, University of Colorado, Boulder, CO, USA.
  • U. Maskos, Pasteur Institute, Paris France .
  • A.B. Smit and S. Spijker, Free University of Amsterdam, Amsterdam, The Netherlands.
  • J. H. Steinbach, Washington University, St. Louis, Missouri, Usa.
  • J. Stizel, University of Colorado, Boulder, CO, USA.
  • M. Zoli, University of Modena and Reggio Emilia, Modena, Italy.

 

PI photo

Cecilia Gotti

Contact information

email  E-mail

email  +39 02 5031 6974

Participating staff

Francesco Clementi

Milena Moretti

Irene Manfredi

Francesco Pistillo

Francesca Fasoli

Sara Colombo

Francesca Mazzo