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1.
Methods Mol Biol ; 2510: 239-252, 2022.
Article in English | MEDLINE | ID: mdl-35776328

ABSTRACT

P2X7 receptors are ATP-gated ion channels permeable to metal cations, such as Na+, K+, and Ca2+. They also exhibit permeability to various large molecular weight species, reaching up to 900 Da, in a process known as macropore formation, which is a unique functional hallmark across the P2X family. While well-documented in a range of different cell types, the molecular mechanism underlying this phenomenon is poorly understood, and has been clouded through the use of electrophysiological methodology prone to artifacts as a result of significant changes in ionic concentrations in asymmetric conditions. In this chapter, we discuss the permeation properties of P2X7, the related methodological challenges and the use of symmetrical organic cation solutions as a useful technique for probing P2X7 permeation.


Subject(s)
Sodium , Cations
2.
Curr Opin Pharmacol ; 62: 109-116, 2022 02.
Article in English | MEDLINE | ID: mdl-34965483

ABSTRACT

Ligand-gated ion channels (LGIC, also referred to as ionotropic receptors) are important transmembrane proteins that open to allow ions to flow across the membrane and locally modify the membrane potential in response to the binding of a ligand. For more than a decade, a tremendous effort has been carried out in the determination of many LGIC structures in high resolution, leading to an unprecedented molecular description of channel gating. However, it is sometimes difficult to classify experimentally derived structures to their corresponding functional states, and alternative methods may help resolve or refine this issue. In this review, we focus on the application of photo-isomerizable tweezers (PIT) as a powerful strategy to interrogate molecular mechanisms of LGIC while assessing their functionality by electrophysiology.


Subject(s)
Membrane Potentials , Humans , Ligands
3.
Int J Mol Sci ; 22(12)2021 Jun 18.
Article in English | MEDLINE | ID: mdl-34207150

ABSTRACT

P2X7 receptors (P2X7) are cationic channels involved in many diseases. Following their activation by extracellular ATP, distinct signaling pathways are triggered, which lead to various physiological responses such as the secretion of pro-inflammatory cytokines or the modulation of cell death. P2X7 also exhibit unique behaviors, such as "macropore" formation, which corresponds to enhanced large molecule cell membrane permeability and current facilitation, which is caused by prolonged activation. These two phenomena have often been confounded but, thus far, no clear mechanisms have been resolved. Here, by combining different approaches including whole-cell and single-channel recordings, pharmacological and biochemical assays, CRISPR/Cas9 technology and cell imaging, we provide evidence that current facilitation and macropore formation involve functional complexes comprised of P2X7 and TMEM16, a family of Ca2+-activated ion channel/scramblases. We found that current facilitation results in an increase of functional complex-embedded P2X7 open probability, a result that is recapitulated by plasma membrane cholesterol depletion. We further show that macropore formation entails two distinct large molecule permeation components, one of which requires functional complexes featuring TMEM16F subtype, the other likely being direct permeation through the P2X7 pore itself. Such functional complexes can be considered to represent a regulatory hub that may orchestrate distinct P2X7 functionalities.


Subject(s)
Anoctamins/metabolism , Receptors, Purinergic P2X7/metabolism , Adenosine Triphosphate/metabolism , Algorithms , Animals , Anoctamins/chemistry , CRISPR-Cas Systems , Cell Membrane/metabolism , Cell Membrane Permeability , Cholesterol/metabolism , HEK293 Cells , Humans , Immunohistochemistry , Models, Biological , Oocytes , Receptors, Purinergic P2X7/chemistry
4.
Methods Enzymol ; 653: 349-376, 2021.
Article in English | MEDLINE | ID: mdl-34099179

ABSTRACT

The modulation of ion channel activity is of central importance within the nervous system, and an in-depth understanding of how such activity occurs on the molecular level is of prime importance for enhancing our understanding of neuronal systems in physiological and pathological states. The use of light as a stimulus has presented the unique opportunity to study these dynamic processes with exquisite spatiotemporal control. We have developed the photoswitchable tweezers method, an optogenetic pharmacology-based technique which relies on the use of a photoswitchable crosslinker as "tweezers" to manipulate the molecular movements involved in ion channel functionalities. Not only does this allow optical control of ion channel activity, but also investigation into the molecular motions and inter-residue distances implicated in such activity. In this chapter we discuss the principles behind the photoswitchable tweezers method, its strategic design and the key experimental steps involved in this technique, using purinergic P2X2 receptor as a case study system.


Subject(s)
Ion Channel Gating , Ion Channels , Adenosine Triphosphate , Ion Channels/genetics , Ion Channels/metabolism , Neurons/metabolism , Optogenetics
5.
Front Mol Neurosci ; 11: 265, 2018.
Article in English | MEDLINE | ID: mdl-30108481

ABSTRACT

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.

6.
Proc Natl Acad Sci U S A ; 114(19): E3786-E3795, 2017 05 09.
Article in English | MEDLINE | ID: mdl-28442564

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.


Subject(s)
Cell Membrane Permeability , Glutamates/metabolism , Models, Biological , Receptors, Purinergic P2X/metabolism , Spermidine/metabolism , HEK293 Cells , Humans
7.
Neuropharmacology ; 104: 18-30, 2016 05.
Article in English | MEDLINE | ID: mdl-26231831

ABSTRACT

ATP-gated P2X receptors are trimeric ion channels selective to cations. Recent progress in the molecular biophysics of these channels enables a better understanding of their function. In particular, data obtained from biochemical, electrophysiogical and molecular engineering in the light of recent X-ray structures now allow delineation of the principles of ligand binding, channel opening and allosteric modulation. However, although a picture emerges as to how ATP triggers channel opening, there are a number of intriguing questions that remain to be answered, in particular how the pore itself opens in response to ATP and how the intracellular domain, for which structural information is limited, moves during activation. In this review, we provide a summary of functional studies in the context of the post-structure era, aiming to clarify our understanding of the way in which P2X receptors function in response to ATP binding, as well as the mechanism by which allosteric modulators are able to regulate receptor function. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.


Subject(s)
Adenosine Triphosphate/metabolism , Ion Channel Gating , Receptors, Purinergic P2X/metabolism , Allosteric Regulation , Animals , Binding Sites , Humans , Optogenetics , Protein Binding , Protein Conformation , Protein Domains , Protein Structure, Tertiary , Receptors, Purinergic P2X/chemistry
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