Molecular basis of antagonist action at the P2X1 receptor
2012-03-26T11:36:56Z (GMT) by
P2X receptors are ATP-gated cation channels. P2X1 receptors are widely expressed throughout the body and have a range of functional roles, e.g. contraction of mesenteric arteries and regulation of blood clotting. The recent crystallisation of the zebra fish P2X4 receptor has provided a major advance in understanding the molecular basis of receptor properties. However, how agonists or antagonists are co-ordinated and the extent of the proposed ligand binding site have not been addressed at a structural level. A mutagenesis based approach was used to propose a model of the ATP binding site and has highlighted some residues involved in antagonist action at P2X receptors. The aim of this thesis was to investigate the molecular basis of antagonist action at the P2X1 receptor using site-directed mutagenesis and P2X receptor chimeras. The wild-type, mutant and chimaeric P2X receptors were expressed in Xenopus laevis oocytes and the currents were characterised using two electrode voltage clamp. Initially, suramin was shown to act as a competitive antagonist and PPADS as a non-competitive antagonist at the P2X1 receptor. The contribution of residues V74 to G96 to human P2X1 receptor properties were determined using cysteine scanning mutagenesis. This region contains a residue that has been shown to be important in suramin action at the P2X4 receptor (K78) but cysteine mutation of the residues V74 to G96 had either no effect or slightly increased antagonism by suramin or PPADS. Also, a further residue was found to be important in ATP potency (F92) and the use of partial agonists and modification with cysteine reactive methanethiosulfonate (MTS) reagents identified additional residues important in channel activation. Mapping these residues onto a homology model of the P2X1 receptor showed the depth of the agonist binding site and highlighted the importance of the rear/inner cavity of the binding pocket in the gating of the channel subsequent to agonist binding. The cysteine rich head region of the P2X receptor, which is adjacent to the proposed ATP binding pocket, is absent in the antagonist insensitive Dictyostelium receptors. P2X1 and P2X2 receptors have ~1400-fold difference in sensitivity to a suramin analogue NF449. Chimeras and point mutations in the cysteine rich head region were made between the P2X1 and P2X2 receptors and they identified the region between the third and fourth conserved cysteine residues of the P2X1 receptor as being important in conferring the difference in sensitivity. In particular, the positively charged residues at the base of the cysteine rich head region of the P2X1 receptor accounted for the highly selective antagonism of NF449 at the P2X1 receptor. Additionally, these residues were shown to play a role in the molecular basis of suramin and PPADS action at the P2X1 receptor. Reciprocal chimeras and mutations in the P2X2 receptor produced modest increases in antagonist sensitivity. In silico docking models highlighted possible sites of action for NF449 and suramin on the P2X1 receptor showing that the base of the cysteine rich head region may be important in the binding of antagonists. In summary, this research furthered understanding of ligand action at the P2X1 receptor.