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Laboratory of artificial modulation of Neurotransmitter Receptor Function

Centro Emergente, financed by FAPESP from 2002-2007: Artificial Modulation of Neuronal Differentiation and Neurotransmitter Receptors by Synthetic Oligonucleotides Acting on Gene and Protein levels.

Principal Investigator: Dr. Henning Ulrich

Our laboratory is interested in studying the function of neurotransmitter receptors that control and integrate communication in the nervous system. Disturbed receptor function is implicated in many diseases of the nervous system, such as epilepsy and neurodegeneration, as well as in alterations of the neuronal development. Pathological conditions in the nervous system can result both from overactivity or inhibition of receptor function. In the former case, it is therapeutically desirable to discover an inhibitor of receptor function, i. e. in the case of the NMDA receptor-induced pathology which results in cell death during stroke. In the latter case it is desirable to evolve a compound that protects the receptor from binding of inhibitors and therefore from inhibition, such as in the case of cocaine-induced inhibition of the nicotinic acetylcholine receptor.

A simple theory for this inhibition is: 

(i) The receptors exist in two conformations, one biologically active and the other inactive.

(ii) Non-competitive inhibitors bind with higher affinity to the inactive conformation than to the active one, shifting the equilibrium towards the inactive form.  
(iii) Compounds that bind to the same site as do the allosteric inhibitors, but with the same affinities to the active and inactive protein conformations, do not inhibit the protein-mediated reaction but do prevent the binding of inhibitors. 
 

Fig.1-Mechanism of receptor inhibition as suggested for the inhibition of the nicotinic acetylcholine receptor. In this cyclic inhibition mechanism, R denotes the receptor, L the ligand, for example acetylcholine, I the inhibitor and A  the activator. K refers to the dissociation constant of L from the receptor; KI the dissociation constant of I from the closed-channel form of the receptor, KI the dissociation constant of I from the open-channel form of the receptor, KA represents the dissociation constant of the RNA aptamer from the closed-channel form of the receptor and KA denotes the dissociation constant of the activator (i. E. RNA aptamer; see below, evolved by using the SELEX technique) from the open-channel form of the receptor. F is the channel-opening equilibrium constant, FA the channel opening equilibrium constant in presence of RNA aptamer and FI the channel-opening equilibrium constant in the presence of an inhibitor.

 

 
1. Participation of purinergic P2X receptors in neuronal differentiation and the modulation of receptor function by synthetic oligonucleotides

 

Purinergic P2X receptors mediating ATP-induced signal transduction as a co-transmission to glutamate and acetylcholine are located in pre- and post synaptic membranes of nerve-nerve and nerve-muscle junctions and are inhibited by various therapeutic agents, animal toxins and abused drugs. Beside being involved in the transmission of stimuli from one to another neurons, these receptors have been shown to be involved in the regulation of developmental processes and are expressed in cells that undergo neuronal differentiation.

 

Pluripotent P19-embryonal carcinoma cells will be used as a model system to investigate the neuronal determination and differentiation in vitro, in analogy to what happens in vivo during embryogenesis. Neurons that appear in culture after 4 to 5 days the differentiation stimulation, express the same neuron-specific cell surface markers as the ones found on the frontal brain (Figure 2). 

 

Specific antagonists and agonists (RNA aptamers) stabilizing  subtypes of open and closed channel forms of P2X  receptors will be evolved from combinatorial SELEX (systematic evolution of ligands by exponential enrichment) libraries and tested for their ability to inhibit neuronal differentiation in vitro. At the same way, RNA interference (RNAi) by synthetic double-stranded RNA complementary to these targeted receptors will be used as a strategy to generate functional knockouts during different stages of differentiation.

 

The kinetics of channel opening and closing as well as the mechanism of inhibition is determined using electrophysiology with microsecond time resolution by combination of the whole-cell recording together with fast ligand application (called cell-flow technique) and laser-pulse activation of biologically inert, caged ATP. Using these techniques compounds are identified that stabilize either open or closed forms of channels of P2X neurotransmitter receptors. This information will be used for a mechanism-based discovery of nuclease-resistant RNA aptamers that bind to distinct sites on the receptors with high affinity and either act as agonists or inhibitors. These novel compounds will be tested for their possible ability to regulate purinergic receptor function during development and help the understanding of neurotransmitter receptor function in normal and pathological conditions.

 

2. The characterization of toxin-binding sites of the nicotinic acetylcholine receptor and the search for combinatorially synthesized compounds and natural products (toxins) that protect receptor function against inhibition by toxins and abused drugs

 

Abused drugs, many pharmaceutical useful compounds and toxins effect the central or peripheral nervous system and are therefore the subject of intensive investigation. These compounds may either act as inhibitors by displacing the agonist from its binding site on the receptor (competitive inhibition) or bind to a different site on the receptor and act either as inhibitor or activator.

 

 This suggests the possibility of finding compounds, that bind to the inhibitory site of the receptor without inducing the subsequent protein transition that leads to inactive receptor forms. The existence of such molecules has been proved by Ulrich et al. (1998) and Hess et al. (2000) who isolated RNA aptamers from a combinatorial RNA library that counteract inhibition of the nAChR by cocaine.  Various toxins isolated from snake venom of Bothrops jararaca, spiders and jelly-fish, as well as biological active compounds from combinatorial libraries will be characterized regarding their mechanism of action on the nicotinic acetylcholine receptor. The aim of the study is to find compounds that stabilize active receptors and protect receptor function by displacing abused drugs from the receptor.

 

Job vacancies for post-docs and graduate students

Laboratório de Neurociências

Instituto de Química
Universidade de São Paulo
Av. Prof. Lineu Prestes, 748, Butantã
CEP: 05508-000 - São Paulo - SP - Brasil

Tel: (11) 3091-8512 / 3091-9622

Bloco 9S, sala 964

Contato: henning@iq.usp.br

 

 

 

 

 

 

 

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conteúdo: henning @ iq.usp.br