Intermediate closed channel state(s) precede(s) activation in the ATP-gated P2X2 receptor

Figures & data

Figure 1. (A) Structural model of the binding jaw of the rP2X2 receptor built from the X-ray structures of zfP2X4 resolved in the absence (left, open jaw) and presence (right, tightened jaw) of ATP. The distance separating the α-carbons of the two histidines forming the Zn²⁺-binding site, and the distance between the α-carbon of the Leu186 and position 8 of adenine ring of ATP are indicated. (B) Chemical structures of NCS-ATP and MTSEA covalently tethered to Cys186 in the rP2X2 L186C mutant. R = P₃O₉^4-. (C) Representative traces (left) and corresponding pooled data (right, n = 3–5 for each condition) showing the potentiating effect of NCS-ATP (10 µM, 30 sec) or MTSEA (1 mM, 30 sec) application on ATP- (100 µM) or Zn²⁺- (100 µM) evoked currents on cells expressing the L186C/T339S mutant. Potentiation was defined as the ratio of Zn²⁺- or ATP-gated currents recorded after exposure to either NCS-ATP or MTSEA to those recorded before exposure. (D) Dose-response curves of Zn²⁺-evoked currents for the double mutant L186C/T339S before (EC₅₀ = 102 ± 24 µM, n_H = 1.2 ± 0.1, n = 4) and after (EC₅₀ = 42 ± 5 µM, n_H = 1.7 ± 0.2, n = 4) NCS-ATP (10 µM, 30 sec) treatment. (E) Single channel currents (upperleft) from outside-out patches expressing the double mutant L186C/T339S showing that channels open spontaneously before and after NCS-ATP treatment (10 µM, 12 sec). Corresponding dwell-time distributions (right) of open- and shut-times of channel activities (pooled data from 6 patches containing 3077 events for the control, and 7 patches containing 6872 events for the condition after NCS-ATP treatment). The open-time distribution can be best fit with two components [τ₁ = 0.43 ms (87.9%), τ₂ = 2.34 ms (12.1%) for control; τ₁ = 0.41 ms (88.4%), τ₂ = 1.88 ms (11.6%) after NCS-ATP treatment]. The shut-time distribution can be best fit with three components [τ₁ = 8.48 ms (42.2%), τ₂ = 39.09 ms (37.0%), τ₃ = 195.9 ms (20.8%) for control; τ₁ = 8.16 ms (58.3%), τ₂ = 46.91 ms (31.8%), τ₃ = 263.8 ms (9.9%) after NCS-ATP treatment]. Histograms (bottom left) showing the NP_o and the single-channel conductance remain unaltered after NCS-ATP treatment.