Analysis of voltage and [ATP] dependent gating of P2X₂ receptor channel by mutagenesis of the ATP binding region

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P2X receptor channels are extracellular ATP gated cation channels. We previously observed in Xenopus oocytes expression system that the P2X₂ channel current at the steady state after ATP application shows gradual increase upon hyperpolarization, showing presence of voltage-dependent gating in spite of the lack of a transmembrane voltage sensor domain. The analyses of tail current amplitude and voltage-dependent activation time constant revealed that the G-V relationship was shifted towards depolarized potentials in accordance with the increase in [ATP] and the activation kinetics was accelerated; implying that the gating mechanism at the steady state is dependent on both voltage and [ATP]. We hypothesized that the binding itself of negatively charged ATP to the binding pocket of P2X₂ could be a source of this mechanism and analyzed K71A/R, K69A/R, R290A/K, K308A/R mutants of the ATP binding region. Having lowered sensitivity to ATP (10 to 400 fold), none showed any [ATP] dependent acceleration of kinetics but all were differed in activation time constant corresponding to WT responding to various [ATP]. Other than K69R no remarkable [ATP] dependent shift of G-V relationship was observed. These findings suggest that residues in the ATP binding site are involved in voltage and [ATP] dependent gating mechanism and the ATP binding is a critical step for voltage dependency. [J Physiol Sci. 2008;58 Suppl:S84]