The Gating Mechanism of a P2X4 Receptor: Normal Mode Analysis and Molecular Dynamics Simulations

Abstract

P2X4 receptors are trimeric ATP-gated non-selective cation channels which play crucial roles in various physiological processes. It remains unclear how ATP binding triggers channel opening. Here, we propose a gating mechanism of a P2X4 receptor based on normal mode analysis and molecular dynamics (MD) simulations. Starting with the first available P2X4 crystal structure in the resting state, a normal mode that couples the motions of three β strands (β1, β13 and β14) at the trimeric interface of the ligand binding domain (LBD) and the motion of the pore-forming helix (TM2) of the transmembrane domain (TMD) was identified. The resulting widening of the fenestrations above the TMD and the opening of the TMD pore are in close agreement with observed signatures of channel activation. Four charged residues implicated in ATP binding are located in β1, β13 and β14. P2X4 activation was further investigated by MD simulations in explicit water. ATP was placed near the putative binding site in two opposite orientations, with the adenine either promixal or distal to the TMD. In simulations with proximal adenine, the adenine ring inserted between β1 and β13 and the phosphate group moved downward. At the same time β1 and β14 approached each other to close in on the ATP, allowing close interactions with the four charged residues. The motions of these β strands are similar to those in the normal mode putatively representing channel activation. In simulations with distal adenine, the ATP hindered the closure between β1 and β14, perhaps representing a desensitized state. Our computational studies produced the first complete model, supported by electrophysiological data, for how ATP binding leads to P2X4 channel activation. The detailed gating mechanism will be essential for the rational drug design.