Identification of a distinct desensitisation gate in the ATP-gated P2X2 receptor

Abstract

P2X receptors are trimeric ATP-gated ion channels. In response to ATP binding, conformational changes lead to opening of the channel and ion flow. Current flow can decline during continued ATP binding in a process called desensitisation. The rate and extent of desensitisation is affected by multiple factors, for instance the T18A mutation in P2X2 makes the ion channel fast desensitising. We have used this mutation to investigate whether the gate restricting ion flow is different in the desensitised and the closed state, by combining molecular modelling and cysteine modification using MTSET (2-(Trimethylammonium)ethyl methanethiosulfonate). Homology modelling of the P2X2 receptor and negative space imaging of the channel suggested a movement of the restriction gate with residue T335 being solvent accessible in the desensitised, but not the closed state. This was confirmed experimentally by probing the accessibility of T335C in the P2X2 T18A/T335C (fast desensitisation) and T335C (slow desensitisation) mutants with MTSET which demonstrates that the barrier to ion flow is different in the closed and the desensitised states. To investigate the T18A induced switch in desensitisation we compared molecular dynamics simulations of the wild type and T18A P2X2 receptor which suggest that the differences in time course of desensitisation are due to structural destabilization of a hydrogen bond network of conserved residues in the proximity of T18.

Keywords: Desensitisation; Electrophysiology; Molecular modelling; P2X receptors; P2X2; Purinergic signalling.

Graphical abstract

Fig. 1

P2X2R Ionic gate in different conformation states. (A) hP2X2R homology model in the apo state (closed). The black box in (A) corresponds to the area displayed in other panels indicating the location of the channel gate. In (B), (C), (D) the voids are shown as a grey-dot tunnel. MTSET access through the transmembrane region is blocked by the activation gate near residue T335 (yellow, B). In the open state (C) and the desensitised state (D) residue T335 is accessible.

Fig. 2

Effect of MTSET at different stages of ATP-response of P2X2 cysteine mutants. (A) MTSET (1 mM, 30s) in the absence of ATP had no effect on subsequent ATP currents at WT, T335C or T18A/T335C (fully recovered from desensitisation) showing inaccessibility of T335C in the closed state. Histogram of mean % inhibition of ATP response by MTSET. For all figures black bar indicates ATP application (100 μM, 10 s). (B) MTSET effects when co-applied with ATP showing gated access to residue T335C. There was no effect on the control WT even after the 3rd co-application. Co-application (30s) abolished responses in both the slowly desensitising mutant T335C and the fast desensitising T18A/T335 by more than 90% (C). To determine whether block at T18A/T335C was at the open or desensitised state, MTSET (1 mM, 30s) was applied to the desensitised channel (lower panel). Following washout and the normal period required for recovery from desensitisation (indicated by hashed line, full recovery in control upper panel) subsequent ATP responses were reduced by >90%. Data are shown as mean ± SEM, n = 3–6, ***p < 0.001. The corresponding states of the receptor are indicated on the right-hand side.

Fig. 3

MD Simulation of P2X2R wildtype and T18A mutant (A) P2X2R TM and intracellular cap regions in cartoon representation with different subunits in light pink, grey and light blue – colour scheme as in Fig. 1. One instance of residues Y16, T18, D348 and K356 is shown as spheres. (B) Enlarged version of (A) with key residues shown as sticks and dotted lines indicating distance measurements shown in (C) and (D). (C) Representative example for Y16/D348 interaction monitored over a 200 ns molecular dynamics simulations for the P2X2R wildtype. Raw data shown in lightblue and rolling average over 10 data points in black. (D) As in (C) for the P2X2R T18A mutant with raw data shown in firebrick and rolling average over 10 data points in black. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)