Voltage‐ and ATP‐dependent structural rearrangements of the P2X2 receptor associated with the gating of the pore
Authors: Keceli Batu , Kubo Yoshihiro 
Published: August 2014

Key points
We previously reported that the ATP receptor channel P2X2 shows gating which depends not only on ATP but also on membrane voltage, in spite of the absence of a canonical voltage sensor.
The purpose of the present study was to capture the structural rearrangements of P2X2 associated with voltage‐ and ATP‐dependent gating.
Cysteine residues were introduced by mutation at Asp315 and Ile67 in the linker region located between the ATP binding site and the channel pore. We analysed the modification rate of cysteine residues by Cd2+ by recording current under two‐electrode voltage clamp using Xenopus oocytes.
Two cysteine residues at 315 and 67 were bridged by Cd2+ resulting in current decline, and the speed of modification was faster at hyperpolarized than at depolarized potential, and also faster in the presence than in the absence of ATP.
The bridging by Cd2+ between 315 and 67 was not intra‐ but inter‐subunit.
The results demonstrate the structural rearrangements in the linker region of P2X2 associated with voltage‐ and ATP‐dependent gating of the pore.
AbstractP2X2 is an extracellular ATP‐gated cation channel which has a voltage‐dependent gating property even though it lacks a canonical voltage sensor. It is a trimer in which each subunit has two transmembrane helices and a large extracellular domain. The three inter‐subunit ATP binding sites are linked to the pore forming transmembrane (TM) domains by β‐strands. We analysed structural rearrangements of the linker strands between the ATP binding site and TM domains upon ligand binding and voltage change, electrophysiologically in Xenopus oocytes, using mutants carrying engineered thiol‐modifiable cysteine residues. (1) We demonstrated that the double mutant D315C&I67C (at β‐14 and β‐1, respectively) shows a 2‐ to 4‐fold increase in current amplitude after treatment with a reducing reagent, dithiothreitol (DTT). Application of the thiol‐reactive metal Cd2+ induced current decline due to bond formation between D315C and I67C. This effect was not observed in wild type (WT) or in single point mutants. (2) Cd2+‐induced current decline was analysed in hyperpolarized and depolarized conditions with different pulse protocols, and also in the presence and absence of ATP. (3) Current decline induced by Cd2+ could be clearly observed in the presence of ATP, but was not clear in the absence of ATP, showing a state‐dependent modification. (4) In the presence of ATP, Cd2+ modification was significantly faster in hyperpolarized than in depolarized conditions, showing voltage‐dependent structural rearrangements of the linker strands. (5) Experiments using tandem trimeric constructs (TTCs) with controlled number and position of mutations in the trimer showed that the bridging by Cd2+ between 315 and 67 was not intra‐ but inter‐subunit. (6) Finally, we performed similar analyses of a pore mutant T339S, which makes the channel activation voltage insensitive. Cd2+ modification rates of T339S were similar in hyperpolarized and depolarized conditions. Taking these results together, we demonstrated that structural rearrangements of the linker region of the P2X2 receptor channel are induced not only by ligand binding but also by membrane potential change.