Estudo do Segmento Consenso Adjacente ao Domínio TM2 e parte do Domínio TM2 do Receptor P2X7: Dinâmica Molecular e Atividade de Canal Iônico. / Study of the segment adjacent consensus to domain TM2 and part of domain TM2 of receiver P2X7: molecular dynamics and activity of ionic canal.

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

The purinergic receptor P2X7 (P2X7R) is a membrane protein which forms channels in the presence of extracellular ATP. Once opened, the channels allow the transport of cations through the cellular membrane, being expressed preferentially in immune cells. Among all purinergic receptors, P2X7R has distinct characteristics from other P2X (P2XR) members. For example, this receptor has the longest C-terminal and poses the ability to form a cytolytic pore allowing molecules up to 900 Da to cross the membrane under sustained ATP activation. Although, great effort has been done to elucidate the mechanism of pore formation, no conclusive data has been published so far. The same is valid for the molecular structure of the receptor. However, some authors suggest that the segment M2 of P2XRs, more specifically the -helix transmembrane segment (TM2), is not structured as -helix and this region of the protein would be responsible for the receptor selectivity. It was also demonstrated, through bioinformatic modeling, that P2XRs were configured preferentialy in -sheet and turns. In this context, we assessed the segment M2 and a region of this segment composed by a consensus residues adjacent (within all P2XRs subtypes) to the TM2 of hP2X7R and part of it (ADSEG peptide) using bioinformatic programs. The bioinformatics predictions confirmed the data described on the literature where the M2 segment was not configured only as -helix, but also as -sheet. Related to the ADSEG peptide the bioinformatic predictions and molecular dynamic (MD) results were confirmed experimentally by circular dichroism (CD) showing that this peptide was also configured preferentially as -sheet. In subsequent analysis, we used artificial lipid bilayer and patch-clamp to test whether ADSEG or recombinant P2X7R would form channels or a cytolytic pore. The recombinant protein of P2X7R failed to form channel in the artificial lipid bilayer. The incorporation of the peptides in the artificial lipid bilayer was able to form channels with conductance of 25.1 pS  0.2 (KCl 150 mM) and 14.0 pS  0.1 (NaCl 150 mM). They also presented cationic selectivity, being more permeable to KCl (PK+/PCl- = 9/1) than NaCl (PNa+/PCl- = 4/1), and they were voltage-independent between -90 to +90 mV. The open lifetime evaluation of these channels had shown a large opening duration (1.025 s  0.115 with 2.5 mM KCl) compatible with the P2X7R. Monovalent ions such as NMDG+ with high molecular weight sharply inhibited the conductance of these channels (12.4 pS to 4.7 pS). Using divalent cations such as Mg+ also have shown conductance inhibition (17.8 pS to 12.1 pS). A similar profile was observed when ADSEG was tested in the patch-clamp in HEK 293 cells showing conductance of 9.9 pS (150 mM KCl). Altogether, our results strongly suggest that ADSEG peptide, configured as -sheet, possibly play a key role in the structure of ionic channel of P2X7Rs. This segment might be involved in the channel selectivity and probably compose the channel wall formed by M2 segment. In the light of present knowledge, we show for the first time strong evidence that contests the paradigm of channel or the cytolytic pore of the P2X7R being formed only by -helix structures.

ASSUNTO(S)

lipid bilayers biologia molecular patch-clamp techniques técnicas de patch-clamp receptors purinergic p2 canais iônicos ion channels bicamadas lipídicas lipossomos receptores purinérgicos p2 liposomes

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