Activation and Desensitization of the Recombinant P2X₁ Receptor at Nanomolar ATP Concentrations

Activation and desensitization kinetics of the rat P2X₁ receptor at nanomolar ATP concentrations were studied in Xenopus oocytes using two-electrode voltage-clamp recording. The solution exchange system used allowed complete and reproducible solution exchange in <0.5 s. Sustained exposure to 1–100 nM ATP led to a profound desensitization of P2X₁ receptors. At steady-state, desensitization could be described by the Hill equation with a K_1/2 value of 3.2 ± 0.1 nM. Also, the ATP dependence of peak currents could be described by a Hill equation with an EC₅₀ value of 0.7 μM. Accordingly, ATP dose-effect relationships of activation and desensitization practically do not overlap. Recovery from desensitization could be described by a monoexponential function with the time-constant τ = 11.6 ±1.0 min. Current transients at 10–100 nM ATP, which elicited 0.1–8.5% of the maximum response, were compatible with a linear three-state model, C-O-D (closed-open-desensitized), with an ATP concentration-dependent activation rate and an ATP concentration-independent (constant) desensitization rate. In the range of 18–300 nM ATP, the total areas under the elicited current transients were equal, suggesting that P2X₁ receptor desensitization occurs exclusively via the open conformation. Hence, our results are compatible with a model, according to which P2X₁ receptor activation and desensitization follow the same reaction pathway, i.e., without significant C to D transition. We assume that the K_1/2 of 3.2 nM for receptor desensitization reflects the nanomolar ATP affinity of the receptor found by others in agonist binding experiments. The high EC₅₀ value of 0.7 μM for receptor activation is a consequence of fast desensitization combined with nonsteady-state conditions during recording of peak currents, which are the basis of the dose-response curve. Our results imply that nanomolar extracellular ATP concentrations can obscure P2X₁ receptor responses by driving a significant fraction of the receptor pool into a long-lasting refractory closed state.