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

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 -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.

Deciphering the regulation of P2X4 receptor channel gating by ivermectin using Markov models

“…This is consistent with our earlier work [20] and the finding of others that IVM potentiates ATP-induced responses and increases permeability for NMDG + , but cannot activate P2X4R channels on its own [16]. Because strategies that rely on changes in E rev to provide evidence for large pore formation during sustained stimulation with agonist were questioned [13, 15], we examined currents induced by ATP in Ca 2+ -free/NMDG + -containing medium. S2 Fig shows that in the absence of Ca 2+ , a 40-s application of ATP (100 μM) at −60 mV in bi-ionic NMDG + out/Na + in solution (where the reversal potential of ATP-induced current is about -70 mV [27]) evoked only outward Na + current, whereas in the presence of IVM, outward Na + current was followed by inward NMDG + current.…”

A Mechanism-Based Approach to P2X7 Receptor Action

“…Nevertheless, the P2X7R channel is somewhat more permeable to large cations than many other ion channels are. This is the case from the beginning of its activation; its permeability characteristics do not change over time (Riedel et al, 2007a,b, see also Harkat et al (2017) for a similar finding in P2X2R). Furthermore, data obtained by cysteine accessibility mutations suggest that P2X7R conduction pore does not show any sign of dilation during receptor activation (Pippel et al, 2017).…”

Defined extracellular ionic solutions to study and manipulate the cellular resting membrane potential

“…This implies that the final RMP value is not just a function of the substituted ion(s), but also of the voltagedependent change of every permeability variable. Furthermore, since it is possible that the cell is permeable to either NMDG (Wang et al, 2009;Harkat et al, 2017) or gluconate (Chen et al, 2004), the use of more than one osmolarity non-permeable cationic [choline (Knowles et al, 1983)] or anionic ion (i.e. SO 2À 4 ) is strongly recommended to confirm findings.…”