On the permeation of large organic cations through the pore of ATP-gated P2X receptors

Significance

Unlike many ion channels whose pore conductances remain relatively stable over time, it is thought that prolonged ATP applications to P2X receptors cause a striking increase over time in the permeability of large molecules, a process dubbed pore dilation. However, this mechanism remains poorly understood and highly controversial. Here, we use different methods spanning single-channel recordings, photochemistry, molecular biology, and computations to show that contrary to longstanding view, rapid activation by ATP allows the stable passage of large cations through the P2X pore. We further discover that spermidine, a large natural cation known to modulate other ion channels, is able to transit through many P2X receptors, including those thought to be nondilating. Our data thus reveal an unacknowledged P2X-mediated signaling.

Abstract

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 N-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.