Pore dilation is thought to be a hallmark of purinergic P2X receptors. The most commonly held view of this unusual process posits that under prolonged ATP exposure the ion pore expands in a striking manner from an initial small-cation conductive state to a dilated state, which allows the passage of larger synthetic cations, such as -methyl-d-glucamine (NMDG). However, this mechanism is controversial, and the identity of the natural large permeating cations remains elusive. Here, we provide evidence that, contrary to the time-dependent pore dilation model, ATP binding opens an NMDG-permeable channel within milliseconds, with a conductance that remains stable over time. We show that the time course of NMDG permeability superimposes that of Na and demonstrate that the molecular motions leading to the permeation of NMDG are very similar to those that drive Na flow. We found, however, that NMDG "percolates" 10 times slower than Na in the open state, likely due to a conformational and orientational selection of permeating molecules. We further uncover that several P2X receptors, including those able to desensitize, are permeable not only to NMDG but also to spermidine, a large natural cation involved in ion channel modulation, revealing a previously unrecognized P2X-mediated signaling. Altogether, our data do not support a time-dependent dilation of the pore on its own but rather reveal that the open pore of P2X receptors is wide enough to allow the permeation of large organic cations, including natural ones. This permeation mechanism has considerable physiological significance.

Glycine receptors (GlyR) mediate fast inhibitory neurotransmission by switching between discrete states in response to ligand-binding events. Recent high-resolution structures from cryoelectron microscopy (cryo-EM) and X-ray crystallography have provided atomistic models for the open and closed states. Notably, the cryo-EM structure in complex with glycine illuminated a previously unreported wide-open state, whose physiological significance is debated. Here, we present the structure of an ion-conducting state of GlyR α1 captured by molecular dynamics and validate its physiological relevance with computational electrophysiology and polyatomic anion permeation simulations. Our analysis suggests that none of the experimental structures is a true representation of the physiologically active state, although previously characterized open channels in GLIC at pH 4, or GluCl/GlyR with ivermectin bound, provide reasonable models. These results open the door to an original functional annotation and support the conclusion that pore closing by desensitization versus deactivation involves the reorientation of the pore-lining helices in opposite directions.