Contribution of the region Glu181 to Val200 of the extracellular loop of the human P2X1 receptor to agonist binding and gating revealed using cysteine scanning mutagenesis¹

Abstract

At the majority of mutants in the region Glu181‐Val200 incorporating a conserved AsnPheThrΦΦxLys motif cysteine substitution had no effect on sensitivity to ATP, partial agonists, or methanethiosulfonate (MTS) compounds. For the F185C mutant the efficacy of partial agonists was reduced by ∼ 90% but there was no effect on ATP potency or the actions of MTS reagents. At T186C, F188C and K190C mutants ATP potency and partial agonists responses were reduced. The ATP sensitivity of the K190C mutant was rescued towards WT levels by positively charged (2‐aminoethyl)methanethiosulfonate hydrobromide and reduced by negatively charged sodium (2‐sulfonatoethyl) methanethiosulfonate. Both MTS reagents decreased ATP potency at the T186C mutant, and abolished responses at the F195C mutant. 32P‐2‐azido ATP binding to the mutants T186C and K190C was sensitive to MTS reagents consistent with an effect on binding, however binding at F195C was unaffected indicating an effect on gating. The accessibility of the introduced cysteines was probed with (2‐aminoethyl)methanethiosulfonate hydrobromide‐biotin, this showed that the region Thr186‐Ser192 is likely to form a beta sheet and that accessibility is blocked by ATP. Taken together these results suggest that Thr186, Phe188 and Lys190 are involved in ATP binding to the receptor and Phe185 and Phe195 contribute to agonist evoked conformational changes.

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.…”

“…4E) from two adjacent subunits. Of note, the residues previously found to be important for ATP function (12)(13)(14)(15)(16)(17)(18)(19) are in close proximity to docked NCS-ATP. Our results thus demonstrate that the intersubunit cavities found in the X-ray structure (9) are the ATP-binding sites.…”

“…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).…”

A Highly Conserved Salt Bridge Stabilizes the Kinked Conformation of β2,3-Sheet Essential for Channel Function of P2X4 Receptors

“…Knowledge of the gating mechanism of those channels will advance our understanding of the structure-function relationship of those trimeric receptors, which in turn will contribute to the development of new drugs targeting P2X receptors (6). In the past 2 decades, the functional roles of the essential structural elements, such as conserved residues/motifs in P2X receptors activation, have been identified through sequence alignment, mutagenesis, and electrophysiological recording before the crystal structure was determined (3,(11)(12)(13)(14)(15). The recent structural determination of zebrafish P2X4 receptors (zfP2X4) at the resting and open states allowed us to study both conserved and non-conserved key residues/motifs and their allosteric transitions during channel activation at the atomic level (16,17).…”