Anales RANF

P1. S TRUCTURE OF P URINE R ECEPTORS AND E CTONUCLEOTIDASES P.01 IDENTIFICATION OF P2X7 RECEPTOR EXTRACELLULAR VESTIBULE RESIDUES CONTRIBUTING TO RECEPTOR STRUCTURE AND CHANNEL GATING M. Rupert 1 , M. Jindrichova 1 , A. Mokdad 1 , V. Tvrdonova 1 , A. Bhattacharya 1 , H. Zemkova 1 1 Department of Cellular and Molecular Neuroendocrinology, Institute of Physiology Academy of Sciences of the Czech Republic, Prague, Czech Republic. Extracellular vestibule, unexpectedly discovered for the first time in crystal structure of zfP2X4 receptor, exhibits multiple roles in P2X receptor function: it is important for ion entry to the channel pore, and vestibule widening upon ATP binding is important for channel gating. Extracellular vestibule is present also in the P2X7 receptor, but its role has not yet been studied. To address this task, we have substituted several conserved and/or unique residues in this region (residues K49, Y51, Q52, F322, G323, G326, K327, F328 and Q332) with alanine, and expressed wild type and mutated receptor in HEK293T cells. These positions have been selected based on previous studies where they have been identified as important for function and structure of P2X1 and P2X4 receptors. Western blotting and electrophysiology showed that four of nine residues (Y51, Q52, G323, and F328) are important for P2X7 trafficking and substitution of three residues (K49, G326 and K327), including receptor-specific residue Q332, did not alter or only slightly reduced receptor function. Alanine substitution of non-conserved F322, that is present also in the P2X4, dramatically increased P2X7 receptor sensitivity to agonist and enhanced the rate of receptor-mediated dye uptake. These results revealed that clusters of conserved residues above TM1 (Y51 and Q52) and TM2, (F328) play a role in receptor expression in the plasma membrane. Conserved residue G323 is important for P2X7 receptor trafficking and vestibule structure. Aromatic residue at position 322, in the top of extracellular vestibule just between extracellular and central vestibules, is important for pore opening. Overall, our data demonstrate that transition between extracellular and central vestibule represents a promising new target for the allosteric control of P2X7 receptor sensitivity and channel gating.