Different localization of P2X4 and P2X7 receptors in native mouse lung - lack of evidence for a direct P2X4-P2X7 receptor interaction.

Introduction: P2X receptors are a family of homo- and heterotrimeric cation channels gated by extracellular ATP. The P2X4 and P2X7 subunits show overlapping expression patterns and have been involved in similar physiological processes, such as pain and inflammation as well as various immune cell functions. While formation of P2X2/P2X3 heterotrimers produces a distinct pharmacological phenotype and has been well established, functional identification of a P2X4/P2X7 heteromer has been difficult and evidence for and against a physical association has been found. Most of this evidence stems, however, from in vitro model systems. Methods: Here, we used a P2X7-EGFP BAC transgenic mouse model as well as P2X4 and P2X7 knock-out mice to re-investigate a P2X4-P2X7 interaction in mouse lung by biochemical and immunohistochemical experiments as well as quantitative expression analysis. Results: No detectable amounts of P2X4 could be co-purified from mouse lung via P2X7-EGFP. In agreement with these findings, immuno-histochemical analysis using a P2X7-specific nanobody revealed only limited overlap in the cellular and subcellular localizations of P2X4 and P2X7 in both the native lung tissue and primary cells. Comparison of P2X4 and P2X7 transcript and protein levels in the respective gene-deficient and wild type mice showed no mutual interrelation between their expression levels in whole lungs. However, a significantly reduced P2rx7 expression was found in alveolar macrophages of P2rx4 -/- mice. Discussion: In summary, our detailed analysis of the cellular and subcellular P2X4 and P2X7 localization and expression does not support a physiologically relevant direct association of P2X4 and P2X7 subunits or receptors in vivo.

Persistent motion of a Brownian particle subject to repulsive feedback with time delay.

Based on analytical and numerical calculations we study the dynamics of an overdamped colloidal particle moving in two dimensions under time-delayed, nonlinear feedback control. Specifically, the particle is subject to a force derived from a repulsive Gaussian potential depending on the difference between its instantaneous position, r(t), and its earlier position r(t-τ), where τ is the delay time. Considering first the deterministic case, we provide analytical results for both the case of small displacements and the dynamics at long times. In particular, at appropriate values of the feedback parameters, the particle approaches a steady state with a constant, nonzero velocity whose direction is constant as well. In the presence of noise, the direction of motion becomes randomized at long times, but the (numerically obtained) velocity autocorrelation still reveals some persistence of motion. Moreover, the mean-squared displacement (MSD) reveals a mixed regime at intermediate times with contributions of both ballistic motion and diffusive translational motion, allowing us to extract an estimate for the effective propulsion velocity in presence of noise. We then analyze the data in terms of exact, known results for the MSD of active Brownian particles. The comparison indeed indicates a strong similarity between the dynamics of the particle under repulsive delayed feedback and active motion. This relation carries over to the behavior of the long-time diffusion coefficient D_{eff} which, similarly to active motion, is strongly enhanced compared to the free case. Finally, we show that, for small delays, D_{eff} can be estimated analytically.

Functional P2X7 Receptors in the Auditory Nerve of Hearing Rodents Localize Exclusively to Peripheral Glia.

P2X7 receptors (P2X7Rs) are associated with numerous pathophysiological mechanisms, and this promotes them as therapeutic targets for certain neurodegenerative conditions. However, the identity of P2X7R-expressing cells in the nervous system remains contentious. Here, we examined P2X7R functionality in auditory nerve cells from rodents of either sex, and determined their functional and anatomic expression pattern. In whole-cell recordings from rat spiral ganglion cultures, the purinergic agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) activated desensitizing currents in spiral ganglion neurons (SGNs) but non-desensitizing currents in glia that were blocked by P2X7R-specific antagonists. In imaging experiments, BzATP gated sustained Ca2+ entry into glial cells. BzATP-gated uptake of the fluorescent dye YO-PRO-1 was reduced and slowed by P2X7R-specific antagonists. In rats, P2X7Rs were immuno-localized predominantly within satellite glial cells (SGCs) and Schwann cells (SCs). P2X7R expression was not detected in the portion of the auditory nerve within the central nervous system. Mouse models allowed further exploration of the distribution of cochlear P2X7Rs. In GENSAT reporter mice, EGFP expression driven via the P2rx7 promoter was evident in SGCs and SCs but was undetectable in SGNs. A second transgenic model showed a comparable cellular distribution of EGFP-tagged P2X7Rs. In wild-type mice the discrete glial expression was confirmed using a P2X7-specific nanobody construct. Our study shows that P2X7Rs are expressed by peripheral glial cells, rather than by afferent neurons. Description of functional signatures and cellular distributions of these enigmatic proteins in the peripheral nervous system (PNS) will help our understanding of ATP-dependent effects contributing to hearing loss and other sensory neuropathies.SIGNIFICANCE STATEMENT P2X7 receptors (P2X7Rs) have been the subject of much scrutiny in recent years. They have been promoted as therapeutic targets in a number of diseases of the nervous system, yet the specific cellular location of these receptors remains the subject of intense debate. In the auditory nerve, connecting the inner ear to the brainstem, we show these multimodal ATP-gated channels localize exclusively to peripheral glial cells rather than the sensory neurons, and are not evident in central glia. Physiologic responses in the peripheral glia display classical hallmarks of P2X7R activation, including the formation of ion-permeable and also macromolecule-permeable pores. These qualities suggest these proteins could contribute to glial-mediated inflammatory processes in the auditory periphery under pathologic disease states.

Deviant reporter expression and P2X4 passenger gene overexpression in the soluble EGFP BAC transgenic P2X7 reporter mouse model.

The ATP-gated P2X7 receptor is highly expressed in microglia and has been involved in diverse brain diseases. P2X7 effects were also described in neurons and astrocytes but its localisation and function in these cell types has been challenging to demonstrate in situ. BAC transgenic mouse lines have greatly advanced neuroscience research and two BAC-transgenic P2X7 reporter mouse models exist in which either a soluble EGFP (sEGFP) or an EGFP-tagged P2X7 receptor (P2X7-EGFP) is expressed under the control of a BAC-derived P2rx7 promoter. Here we evaluate both mouse models and find striking differences in both P2X expression levels and EGFP reporter expression patterns. Most remarkably, the sEGFP model overexpresses a P2X4 passenger gene and sEGFP shows clear neuronal localisation but appears to be absent in microglia. Preliminary functional analysis in a status epilepticus model suggests functional consequences of the observed P2X receptor overexpression. In summary, an aberrant EGFP reporter pattern and possible effects of P2X4 and/or P2X7 protein overexpression need to be considered when working with this model. We further discuss reasons for the observed differences and possible caveats in BAC transgenic approaches.

The antiarrhythmic compound efsevin directly modulates voltage-dependent anion channel 2 by binding to its inner wall and enhancing mitochondrial Ca2+ uptake.

BACKGROUND AND PURPOSE: The synthetic compound efsevin was recently identified to suppress arrhythmogenesis in models of cardiac arrhythmia, making it a promising candidate for antiarrhythmic therapy. Its activity was shown to be dependent on the voltage-dependent anion channel 2 (VDAC2) in the outer mitochondrial membrane. Here, we investigated the molecular mechanism of the efsevin-VDAC2 interaction. EXPERIMENTAL APPROACH: To evaluate the functional interaction of efsevin and VDAC2, we measured currents through recombinant VDAC2 in planar lipid bilayers. Using molecular ligand-protein docking and mutational analysis, we identified the efsevin binding site on VDAC2. Finally, physiological consequences of the efsevin-induced modulation of VDAC2 were analysed in HL-1 cardiomyocytes. KEY RESULTS: In lipid bilayers, efsevin reduced VDAC2 conductance and shifted the channel's open probability towards less anion-selective closed states. Efsevin binds to a binding pocket formed by the inner channel wall and the pore-lining N-terminal α-helix. Exchange of amino acids N207, K236 and N238 within this pocket for alanines abolished the channel's efsevin-responsiveness. Upon heterologous expression in HL-1 cardiomyocytes, both channels, wild-type VDAC2 and the efsevin-insensitive VDAC2AAA restored mitochondrial Ca2+ uptake, but only wild-type VDAC2 was sensitive to efsevin. CONCLUSION AND IMPLICATIONS: In summary, our data indicate a direct interaction of efsevin with VDAC2 inside the channel pore that leads to modified gating and results in enhanced SR-mitochondria Ca2+ transfer. This study sheds new light on the function of VDAC2 and provides a basis for structure-aided chemical optimization of efsevin.

P2X7 Interactions and Signaling - Making Head or Tail of It.

Extracellular adenine nucleotides play important roles in cell-cell communication and tissue homeostasis. High concentrations of extracellular ATP released by dying cells are sensed as a danger signal by the P2X7 receptor, a non-specific cation channel. Studies in P2X7 knockout mice and numerous disease models have demonstrated an important role of this receptor in inflammatory processes. P2X7 activation has been shown to induce a variety of cellular responses that are not usually associated with ion channel function, for example changes in the plasma membrane composition and morphology, ectodomain shedding, activation of lipases, kinases, and transcription factors, as well as cytokine release and apoptosis. In contrast to all other P2X family members, the P2X7 receptor contains a long intracellular C-terminus that constitutes 40% of the whole protein and is considered essential for most of these effects. So far, over 50 different proteins have been identified to physically interact with the P2X7 receptor. However, few of these interactions have been confirmed in independent studies and for the majority of these proteins, the interaction domains and the physiological consequences of the interactions are only poorly described. Also, while the structure of the P2X7 extracellular domain has recently been resolved, information about the organization and structure of its C-terminal tail remains elusive. After shortly describing the structure and assembly of the P2X7 receptor, this review gives an update of the identified or proposed interaction domains within the P2X7 C-terminus, describes signaling pathways in which this receptor has been involved, and provides an overlook of the identified interaction partners.

Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody.

The P2X7 channel is involved in the pathogenesis of various CNS diseases. An increasing number of studies suggest its presence in neurons where its putative functions remain controversial for more than a decade. To resolve this issue and to provide a model for analysis of P2X7 functions, we generated P2X7 BAC transgenic mice that allow visualization of functional EGFP-tagged P2X7 receptors in vivo. Extensive characterization of these mice revealed dominant P2X7-EGFP protein expression in microglia, Bergmann glia, and oligodendrocytes, but not in neurons. These findings were further validated by microglia- and oligodendrocyte-specific P2X7 deletion and a novel P2X7-specific nanobody. In addition to the first quantitative analysis of P2X7 protein expression in the CNS, we show potential consequences of its overexpression in ischemic retina and post-traumatic cerebral cortex grey matter. This novel mouse model overcomes previous limitations in P2X7 research and will help to determine its physiological roles and contribution to diseases.