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

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.

P2X-GCaMPs as Versatile Tools for Imaging Extracellular ATP Signaling.

ATP is an extracellular signaling molecule involved in numerous physiological and pathologic processes. However, in situ characterization of the spatiotemporal dynamic of extracellular ATP is still challenging because of the lack of sensor with appropriate specificity, sensitivity, and kinetics. Here, we report the development of biosensors based on the fusion of cation permeable ATP receptors (P2X) to genetically encoded calcium sensors [genetically encoded calcium indicator (GECI)]. By combining the features of P2X receptors with the high signal-to-noise ratio of GECIs, we generated ultrasensitive green and red fluorescent sniffers that detect nanomolar ATP concentrations in situ and also enable the tracking of P2X receptor activity. We provide the proof of concept that these sensors can dynamically track ATP release evoked by depolarization in mouse neurons or by extracellular hypotonicity. Targeting these P2X-based biosensors to diverse cell types should advance our knowledge of extracellular ATP dynamics in vivo.

New Insights Into Permeation of Large Cations Through ATP-Gated P2X Receptors.

The permeability of large cations through the P2X pore has remained arguably the most controversial and complicated topic in P2X-related research, with the emergence of conflicting studies on the existence, mechanism and physiological relevance of a so-called "dilated" state. Due to the important role of several "dilating" P2X subtypes in numerous diseases, a clear and detailed understanding of this phenomenon represents a research priority. Recent advances, however, have challenged the existence of a progressive, ATP-induced pore dilation, by demonstrating that this phenomenon is an artifact of the method employed. Here, we discuss briefly the history of this controversial and enigmatic dilated state, from its initial discovery to its recent reconsideration. We will discuss the literature in which mechanistic pathways to a large cation-permeable state are proposed, as well as important advances in the methodology employed to study this elusive state. Considering recent literature, we will also open the discussion as to whether an intrinsically dilating P2X pore exists, as well as the physiological relevance of such a large cation-permeable pore and its potential use as therapeutic pathway.

P2X2 Dominant Deafness Mutations Have No Negative Effect on Wild-Type Isoform: Implications for Functional Rescue and in Deafness Mechanism.

The P2X2 receptor is an ATP-gated ion channel, assembled by three subunits. Recently, it has been found that heterozygous mutations of P2X2 V60L and G353R can cause autosomal dominant nonsyndromic hearing loss. However, the underlying mechanism remains unclear. The fact that heterozygous mutations cause deafness suggests that the mutations may have dominant-negative effect (DNE) on wild-type (WT) P2X2 isoforms and/or other partners leading to hearing loss. In this study, the effect of these dominant deafness P2X2 mutations on WT P2X2 was investigated. We found that sole transfection of both V60L and G353R deafness mutants could efficiently target to the plasma membrane, like WT P2X2, but exhibit a significantly reduced response to ATP stimulation. Both mutants reduced the channel conductance, but G353R mutation also altered the voltage dependency. Co-expression with WT P2X2 could restore the response to ATP. As the ratio of WT P2X2 vs. mutants increased, the response to ATP was also increased. Computer modeling confirmed that both V60L and G353R dominant-deafness mutant subunits do not have any negative effect on WT P2X2 subunit, when assembled as a heterotrimer. Improper docking or defective gating is the more likely mechanism for impaired channel function by these P2X2 deafness mutations. These results suggest that P2X2 dominant deafness mutations do not have negative effects on WT P2X2 isoforms, and that adding additional WT P2X2 could rescue the lost channel function caused by the deafness mutations. These P2X2 dominant deafness mutations may have negative-effects on other partners leading to hearing loss.