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.
Highlights
IntroductionPurinergic P2X7 receptors (P2X7) are members of the P2X receptor family, a class of membrane proteins forming trimeric channels activated by extracellular ATP [1]
To avoid non-specific channel activation evoked by millimolar concentrations of ATP [35], we used the more potent P2X7 agonist 20 (30 )-O-(4-benzoylbenzoyl)ATP (BzATP), which activates the channel in the low μM range
The dead time was equal to 1.8 ms, and transitions shorter than td were ignored. These unitary currents can be attributed to rat P2X7 (rP2X7) as no currents were recorded in the absence of BzATP, in the presence of the highly selective P2X7 antagonist AZ10606120 co-applied with BzATP, nor in non-transfected cells stimulated with BzATP (Figure 1A, right, and Figure S1C,D)
Summary
Purinergic P2X7 receptors (P2X7) are members of the P2X receptor family, a class of membrane proteins forming trimeric channels activated by extracellular ATP [1]. They are expressed in different cell types, mainly in immune and glial cells. Following ATP gating, P2X7 initiates nonselective metal cation flux (Na+ , K+ and Ca2+ ), which in turn triggers distinct activation pathways such as the secretion of pro-inflammatory cytokines or modulation of cell death. Owing to its key role in multiple pathologies, including chronic inflammation, neurodegeneration, neuropathic pain, metabolic diseases, rheumatoid arthritis, Crohn’s disease and cancer, P2X7 has become a relevant therapeutic target, sparking intense interest for drug development [2]
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