Untangling Macropore Formation and Current Facilitation in P2X7

Abstract

Macropore formation and current facilitation are intriguing phenomena associated with ATP-gated P2X7 receptors (P2X7). Macropores are large pores formed in the cell membrane that allow the passage of large molecules. The precise mechanisms underlying macropore formation remain poorly understood, but recent evidence suggests two alternative pathways: a direct entry through the P2X7 pore itself, and an indirect pathway triggered by P2X7 activation involving additional proteins, such as TMEM16F channel/scramblase. On the other hand, current facilitation refers to the progressive increase in current amplitude and activation kinetics observed with prolonged or repetitive exposure to ATP. Various mechanisms, including the activation of chloride channels and intrinsic properties of P2X7, have been proposed to explain this phenomenon. In this comprehensive review, we present an in-depth overview of P2X7 current facilitation and macropore formation, highlighting new findings and proposing mechanistic models that may offer fresh insights into these untangled processes.

Keywords: ATP sensitization; P2X7; current facilitation; macropore formation.

Figure 1

(A). Cryo-EM structure of rat P2X7 (pdb code: 6u9w) [40] embedded in a lipid bilayer, represented as spheres. The two nucleotide-binding sites, ATP and GDP, are indicated. (B). Ribbon representation of the same view, highlighting relevant structural features (ATP and GDP in green, Zn²⁺ in violet, palmitoyl groups in orange and F11 residue in red). Dotted lines depict the approximate position of the membrane boundaries. Structures were displayed using Protein Imager (https://3dproteinimaging.com/protein-imager/, accessed on 9 May 2023).

Figure 2

(A). Examples of current facilitation resulting from either the prolonged (30 s, left) or the repeated (2 s, right) application of 10 μM (2′(3′)-O-(4-benzoylbenzoyl)ATP) (BzATP), which is an ATP analogue that is more potent than ATP in activating P2X7. Notice the increase in inward current during the late stage compared to the early stage. Traces were adapted from [47]. (B,C). Proposed mechanistic models underlying current facilitation. (B). Model #1 suggests that current facilitation occurs through the secondary activation of Cl⁻ channels. This secondary activation can be mediated by Ca²⁺, functioning as a second messenger, as observed in oocytes [51] or through PLA2 (Q9NP80 predicted from AlphaFold [52]), in a Ca²⁺-independent manner, as demonstrated in macrophages [49]. The specific identity of the Cl⁻ channels remains unknown, but they could be CaCCs (here exemplified by Q4KMQ2 predicted from AlphaFold). (C). Model #2 suggests that current facilitation is an intrinsic property of P2X7 itself (pdb code: 6u9w [40]), as evidenced by an increase in open channel probability following a 30 s perfusion of 10 μM BzATP (recorded through single-channel currents [47]) or an acceleration of the ANAP fluorescent change upon a second 300 μM ATP application (recorded through voltage-clamp experiments [53], note the different time scales). It has been suggested that cholesterol exerts inhibitory effects on P2X7 activity by directly interacting with the transmembrane domain [45,46,47]. This implies that the dissociation of cholesterol from the receptor may alleviate its inhibition. Traces were adapted from [47,53]. Structures were displayed using Protein Imager (https://3dproteinimaging.com/protein-imager/, accessed on 9 May 2023).

Figure 3

(A). Example of YO-PRO-1 dye uptake used to monitor macropore formation in HEK cells upon 10 μM BzATP application at time 0 (indicated by the arrow). Data were taken from [47]. (B,C). Proposed pathways involved in macropore formation. (B). Pathway #1: At low eATP concentrations, the low channel activity permits the passage of Na⁺ or Ca²⁺ ions, as well as small cations such as spermidine (145 Da), NMDG⁺ (195 Da), ethidium (314 Da) or YO-PRO-1 (376 Da) directly through the P2X7 pore itself, albeit at considerably slower rates. This slow channel activity, induced by low eATP concentration or during the initial stage of large ATP application (refer to Figure 2A), is suggested to favor cell survival. (C). Pathway #2: With higher eATP concentrations or prolonged activation, enhanced P2X7 pore activity triggers secondary scramblases, such as TMEM16F (Q4KMQ2 predicted from AlphaFold) or XK (P51811 predicted from AlphaFold), facilitating the passage of larger molecules, such as YOYO-1 (763 Da) or TOTO-1 (795 Da), for which their size may exceed the open diameter of P2X7 (≥5 Å). This mechanism responsible for the activation of the secondary pathway remains unknown, but it is speculated that the involvement of the ballast domain may play a role in this process. It should be noted that increased channel activity resulting from prolonged ATP application or high ATP concentrations further amplifies these pathways, which can contribute to cell death. Pathway #1 may coexist with pathway #2. Structures were displayed using Protein Imager (https://3dproteinimaging.com/protein-imager/, accessed on 9 May 2023).