Gating properties of the P2X2a and P2X2b receptor channels: Experiments and mathematical modeling

Laboratory of Biological Modeling, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
The Journal of General Physiology (Impact Factor: 4.79). 05/2012; 139(5):333-48. DOI: 10.1085/jgp.201110716
Source: PubMed


Adenosine triphosphate (ATP)-gated P2X2 receptors exhibit two opposite activation-dependent changes, pore dilation and pore closing (desensitization), through a process that is incompletely understood. To address this issue and to clarify the roles of calcium and the C-terminal domain in gating, we combined biophysical and mathematical approaches using two splice forms of receptors: the full-size form (P2X2aR) and the shorter form missing 69 residues in the C-terminal domain (P2X2bR). Both receptors developed conductivity for N-methyl-D-glucamine within 2-6 s of ATP application. However, pore dilation was accompanied with a decrease rather than an increase in the total conductance, which temporally coincided with rapid and partial desensitization. During sustained agonist application, receptors continued to desensitize in calcium-independent and calcium-dependent modes. Calcium-independent desensitization was more pronounced in P2X2bR, and calcium-dependent desensitization was more pronounced in P2X2aR. In whole cell recording, we also observed use-dependent facilitation of desensitization of both receptors. Such behavior was accounted for by a 16-state Markov kinetic model describing ATP binding/unbinding and activation/desensitization. The model assumes that naive receptors open when two to three ATP molecules bind and undergo calcium-independent desensitization, causing a decrease in the total conductance, or pore dilation, causing a shift in the reversal potential. In calcium-containing media, receptor desensitization is facilitated and the use-dependent desensitization can be modeled by a calcium-dependent toggle switch. The experiments and the model together provide a rationale for the lack of sustained current growth in dilating P2X2Rs and show that receptors in the dilated state can also desensitize in the presence of calcium.

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    • "Channel clustering is not obligatory for P2X2R pore dilation, which most likely reflects permissive motions at the interface between the first and second transmembrane domains of neighboring subunits (Khakh and Egan, 2005). Finally, an experimentally supported mathematical model provides a rationale for the lack of sustained current growth in dilating rP2X2Rs by showing that dilated receptors also desensitize in the presence of Ca 2+ (Khadra et al., 2012). The Ca 2+ -dependent transition from dilated to desensitized states does not occur in rP2X7R-expressing cells. "
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    ABSTRACT: The occupancy of the orthosteric ligand binding sites of P2X receptor (P2XR) channels causes the rapid opening of a small cation-permeable pore, followed by a gradual dilation that renders the pore permeable to large organic cations. Electrophysiologically, this phenomenon was shown using whole-cell current recording on P2X2R-, P2X2/X5R-, P2X4R- and P2X7R-expressing cells that were bathed in N-methyl-D-glucamine (NMDG(+))-containing buffers in the presence and/or absence of small monovalent and divalent cations. The pore dilation of P2X4R and P2X7R caused a secondary current growth, whereas that of P2X2R showed a sustained kinetic coupling of dilation and desensitization, leading to receptor channel closure. The pore size of the P2X7R open and dilated states was estimated to be approximately 0.85 nm and greater than 1 nm, respectively. The P2XR pore dilation was also observed in intact cells by measurement of fluorescent dye uptake/release, application of polyethylene glycols of different sizes, and atomic force microscopy. However, pore dilation was not observed at the single channel level. Structural data describing the dilated state are not available, and the relevance of orthosteric and allosteric ligand interactions to pore dilation was not studied.
    Full-text · Article · Nov 2013 · Frontiers in Cellular Neuroscience
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    • "Two mechanisms have been proposed for pore dilation (Figure 4). For P2X2, P2X4, and P2X7 receptors, pore dilation appears to involve an intrinsic conformational change in the protein itself (Chaumont and Khakh, 2008; Khadra et al., 2012; Yan et al., 2008, 2010, 2011). However, for natively expressed P2X7 channels, an accessory protein may also be required, and pannexin-1 channels may be involved in receptor pore dilation (Jiang et al., 2005; Pelegrin and Surprenant, 2006; Pelegrin and Surprenant, 2007; Surprenant et al., 1996) in a manner that varies with the particular splice variant being studied (Xu et al., 2012). "
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    ABSTRACT: Extracellular adenosine 5' triphosphate (ATP) is a widespread cell-to-cell signaling molecule in the brain, where it activates cell surface P2X and P2Y receptors. P2X receptors define a protein family unlike other neurotransmitter-gated ion channels in terms of sequence, subunit topology, assembly, and architecture. Within milliseconds of binding ATP, they catalyze the opening of a cation-selective pore. However, recent data show that P2X receptors often underlie neuromodulatory responses on slower time scales of seconds or longer. Herein, we review these findings at molecular, cellular and systems levels. We propose that, while P2X receptors are fast ligand-gated cation channels, they are most adept at mediating slow neuromodulatory functions that are more widespread and more physiologically utilized than fast ATP synaptic transmission in the CNS.
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    ABSTRACT: Transcripts and/or proteins of P2X receptor (P2XR) subunits have been found in virtually all mammalian tissues. Generally more than one of the seven known P2X subunits have been identified in a given cell type. Six of the seven cloned P2X subunits can efficiently form functional homotrimeric ion channels in recombinant expression systems. This is in contrast to other ligand-gated ion channel families, such as the Cys-loop or glutamate receptors, where homomeric assemblies seem to represent the exception rather than the rule. P2XR mediated responses recorded from native tissues rarely match exactly the biophysical and pharmacological properties of heterologously expressed homomeric P2XRs. Heterotrimerization of P2X subunits is likely to account for this observed diversity. While the existence of heterotrimeric P2X2/3Rs and their role in physiological processes is well established, the composition of most other P2XR heteromers and/or the interplay between distinct trimeric receptor complexes in native tissues is not clear. After a description of P2XR assembly and the structure of the intersubunit ATP-binding site, this review summarizes the distribution of P2XR subunits in selected mammalian cell types and the biochemically and/or functionally characterized heteromeric P2XRs that have been observed upon heterologous co-expression of P2XR subunits. We further provide examples where the postulated heteromeric P2XRs have been suggested to occur in native tissues and an overview of the currently available pharmacological tools that have been used to discriminate between homo- and heteromeric P2XRs.
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