Voltage-clamp fluorometry analysis of structural rearrangements of ATP-gated channel P2X2 upon hyperpolarization

Abstract

The gating of the ATP-activated channel P2X2 has been shown to be dependent not only on [ATP] but also on membrane voltage, despite the absence of a canonical voltage-sensor domain. We aimed to investigate the structural rearrangements of the rat P2X2 during ATP- and voltage-dependent gating by voltage-clamp fluorometry technique. We observed fast and linearly voltage-dependent fluorescence intensity (F) changes at Ala337 and Ile341 in the TM2 domain, which could be due to the electrochromic effect, reflecting the presence of a converged electric field here. We also observed slow and voltage-dependent F changes at Ala337, which reflect the structural rearrangements. Furthermore, we identified that the interaction between Ala337 in TM2 and Phe44 in TM1, located in close proximity in the ATP-bound open state, is critical for activation. Taken together, we propose that the voltage dependence of the interaction in the converged electric field underlies the voltage-dependent gating.