Conformational dynamics of the cytoplasmic cap during desensitization of the hP2X3R.

Abstract

The human P2X3 receptor (P2X3R) is a trimeric ligand-gated ion channel activated by extracellular ATP. X-ray crystallography revealed atomic structures of its apo (closed, resting), ATP-bound (open, activated), and ATP-bound (closed, desensitized) states. An intracellular ‘cytoplasmic cap’ was found to stabilize the open pore in the ATP-bound state. However, the cytoplasmic cap was not resolved in the resting or desensitized structures, suggesting a model in which transient formation of the cytoplasmic cap is associated with the opening of the ion pore. To study the functional coupling between (un)folding of the cytoplasmic cap and opening/closing of the P2X3R ion pore, we performed extensive all-atom molecular dynamics (MD) simulations combined with voltage-clamp electrophysiology and mutagenesis. Temperature replica-exchange MD simulations of P2X3R revealed the cytoplasmic cap's unfolding pathway and consequent ion pore closure. Our results show that the N- and C-terminal layers that form the cytoplasmic cap in the open state do not fully unfold during desensitization. By contrast, the partial unfolding of the lower C-terminal layer is sufficient to initiate pore closure/desensitization. In contrast, the N-terminal layers remain folded and keep their spatial proximity to the membrane. Interestingly, not all C-terminal secondary structures unfold, and some structural organization persists, which may allow for rapid transitions between the apo, open, and resting states. The main trigger of pore closure/desensitization is the loss of contact between the C-terminal and the N-terminal part of the cap. We investigated point mutations, aimed at (de)stabilizing the cytoplasmic cap, using free-energy calculations and electrophysiological recordings. These demonstrate that the kinetics of P2X3R desensitization depends on the folding-free energy of the cytoplasmic cap. In summary, our work reveals an allosteric coupling mechanism between the cytoplasmic cap and gating of the transmembrane ion pore in P2X receptors.