Cysteine Substitution Mutagenesis and the Effects of Methanethiosulfonate Reagents at P2X2 and P2X4 Receptors Support a Core Common Mode of ATP Action at P2X Receptors

Abstract

The agonist binding site of ATP-gated P2X receptors is distinct from other ATP-binding proteins. Mutagenesis on P2X 1 receptors of conserved residues in mammalian P2X receptors has established the paradigm that three lysine residues, as well as FT and NFR motifs, play an important role in mediating ATP action. In this study we have determined whether cysteine substitution mutations of equivalent residues in P2X 2 and P2X 4 receptors have similar effects and if these mutant receptors can be regulated by charged methanethiosulfonate (MTS) compounds. All the mutants (except the P2X 2 K69C and K71C that were expressed, but non-functional) showed a significant decrease in ATP potency, with >300-fold decreases for mutants of the conserved asparagine, arginine, and lysine residues close to the end of the extracellular loop. MTS reagents had no effect at the phenylalanine of the FT motif, in contrast, cysteine mutation of the threonine was sensitive to MTS reagents and suggested a role of this residue in ATP action. The lysine-substituted receptors were sensitive to the charge of the MTS reagent consistent with the importance of positive charge at this position for coordination of the negatively charged phosphate of ATP. At the NFR motif the asparagine and arginine residues were sensitive to MTS reagents, whereas the phenylalanine was either unaffected or showed only a small decrease. These results support a common site of ATP action at P2X receptors and suggest that non-conserved residues also play a regulatory role in agonist action.P2X receptors for ATP comprise a distinct family of ligandgated ion channels, with two transmembrane domains, intracellular amino and carboxyl termini and a large extracellular ligand binding loop. Seven P2X receptor genes have been identified (P2X 1-7 ) and the subunits form functional homo-and heterotrimeric ion channels with a variety of phenotypes (1). ATP is released from neurons, in response to shear stress, as well as from damaged cells. P2X receptors have been shown to be involved in a wide range of physiological roles including blood clotting (2), sensory perception (including pain (3-5), bladder voiding (6), and taste (7)), as well as bone formation (8).As the receptors provide novel drug targets for a range of diseases (9) an understanding of the agonist binding site would be useful for rational drug design. However, it is clear that common ATP binding sequences, e.g. the Walker motif (10), are not present in P2X receptors. In the absence of a crystal structure a site-directed mutagenesis approach has been used to gain insight into amino acids important in mediating the actions of ATP at P2X receptors.We have used the human P2X 1 receptor and alanine mutagenesis of conserved residues in the extracellular loop ( Fig. 1) to propose a model of the ATP binding site (reviewed in Refs. 11 and 12). Cysteine substitution mutagenesis and charged methanethiosulfonate (MTS) 3 compounds have been useful in investigating a variety of ion channels including the pore region (13, 14) as well as...

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

“…In addition, certain histidine residues within the protein can regulate P2X function [2,5]. It has been reported that glycine residues enhance flexibility in the extracellular loop, raising the possibility that they are involved in conformational changes in P2X receptors upon agonist binding [2,[76][77][78]. Swapping fragments and point mutations in the extracellular loop between rat and mouse P2X 7 receptors also demonstrated that amino acid residues in the ectodomain of the P2X 7 receptor are involved in the differential sensitivity to agonists between species [79].…”

Agonist trapped in ATP-binding sites of the P2X2 receptor

“…Also indicated is the sequence alignment of the investigated segments (I to VI) of rat (r) and zebrafish (zf) P2X1-P2X4 and P2X7. Conserved residues previously identified as important for ATP function are underscored (12)(13)(14)(15)(16)(17)(18)(19). Pooled data in this and all other figures represent mean AE SEM.…”

“…On the basis of our P2X2 homology model (5), we individually mutated into cysteine 26 positions that protrude in the putative binding cavity, including those previously identified conserved residues (12)(13)(14)(15)(16)(17)(18)(19) (Fig. 1D).…”

“…Early studies based on mutagenesis data have identified highly conserved extracellular residues important for ATP function and have proposed that ATP binding occurs through the extracellular domain (12)(13)(14)(15)(16)(17)(18)(19), presumably at the subunit interface (15,17). When mapped on the crystal structure, most of these residues are observed to line a large and deep intersubunit cavity shaped like an open "jaw" and located approximately 45 Å away from the ion channel domain (9).…”

Amino acid variations resulting in functional and nonfunctional zebrafish P2X1 and P2X5.1 receptors

“…In P2X [1][2][3][4][5][6][7] receptors from most other species, this site is occupied by a highly conserved KXK motif [17]. In human P2X 1 , rat P2X 2 , and rat P2X 4 , mutation of either of these two highly conserved lysines severely impaired ATP potency [20][21][22]. Fig.…”