Anales RANF

P.05 UNCOVERING ‘HIDDEN’ CONFORMATIONS OF ATP-GATED P2X RECEPTORS USING BIOCHEMICAL AND BIOINFORMATICS METHODS A.Stavrou 1 , R.Evans 1 , R.Schmid 1 1 . University of Leicester, Leicester, UK Static structures of P2X receptors are available, but the extent of additional transitional states is unclear. Previous work on P2X1 suggests that the upper vestibule of the receptor is accessible to MTS-compounds that is not predicted by the apo homology model (Roberts et al. Proc. Natl. Acad. Sci USA 109; 4663-67, 2012). This work has been extended with the larger MTS-TAMRA to fluorescently label residues in hP2X1, which indicate that the upper vestibule is even more accessible than previously thought. Cysteine accessibility studies using MTS-TPAE and MTS-TAMRA confirms that access to residues inside the upper vestibule is size-dependent. These biochemical data are difficult to reconcile with structural information derived from homology models based on the closed and open states of zfP2X4.We have therefore generated further P2X1 homology models to include antagonist-bound and desensitized state models derived from more recent hP2X3 and paP2X7 and used HOLE to identify any internal cavities or tunnels which might be accessible to MTS molecules. The models allowed us to compare the differences in the relaxation of the subunits and perform measurements between selected residues that defined the cavities. Molecular dynamics simulations were used to further sample these models for more accessible states and variability in cavities. From the combination of our biochemical data and molecular modelling a picture emerges where dynamically accessible conformations of the P2X1 receptor allow bigger MTS-compounds access to the upper vestibule. This might point to further, looser, conformations states of the P2X proteins that cannot be captured using crystallographic work.