Structure/Function Studies of the P2X Receptor
thesisposted on 28.05.2010, 15:33 by Helen Digby
P2X receptors are a novel family of ligand-gated ion channels that open in response to the binding of extracellular ATP. There are no crystal structures available for the P2X receptor family and they share little homology with other ATP-binding proteins. For this reason, experimental evidence has been relied upon to determine the topology of the P2X receptor family and infer function. The family presents numerous drug targets for the treatment of cystic fibrosis, regulation of blood pressure, pain and irritable bowel syndrome to name a few (Khakh & North, 2006). A better understanding of the structure of P2X receptors gained through mutagenesis and more recently bioinformatic techniques will enable better drug design and development. The amino acid sequence of a protein determines its secondary structure which in turn dictates the tertiary or 3D structure. Protein function is dependent on protein structure and for this reason structural studies can give important insight into protein function. Alanine-replacement of conserved glycine residues in the P2X receptor family has been studied to determine the impact of the flexible nature of glycine in the extracellular segment of the P2X1 receptor. A key functional residue, glycine-250 (P2X1 receptor numbering), was identified and its predicted location at the C-terminal end of an a-helix confirms such a role. However, it is not possible to predict the tertiary structure of a protein based on amino acid sequence alone; the so-called ‘protein-folding problem’. Therefore, this thesis has relied upon sequence analysis and protein structure prediction methods as tools for extracting structural information from P2X receptor sequences. In particular, searching for structural templates on which to model both the putative ligand-binding segment of the P2X1 receptor and the newly defined cysteine-rich domain of the extracellular segment. Published homology models for the rat P2X4 receptor using class II aminoacyl-tRNA synthetases as structural templates exist. Such models have been validated by mutagenesis studies and residues thought to be important in ATP-binding at the P2X4 receptor have been identified. These residues have been aligned to the hP2X1 receptor sequence and the corresponding residues mutated to cysteine. It is clear that this P2X4 receptor model does not directly translate as a model of ATP-binding at the hP2X1 receptor due to inconsistencies in mutagenesis data and the unreliable nature of the original homology model. In contrast, a model of ATP-binding at the P2X1 receptor based on experimental data does provide an interesting insight into those residues involved in ATP-binding at the P2X4 receptor thus enhancing the published homology models and validating the P2X1 receptor models of ATP-binding.