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Pannexin1-Mediated ATP Release Provides Signal Transmission Between Neuro2A Cells

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Abstract

Pannexin1 (Panx1), a protein related to the gap junction proteins of invertebrates, forms nonjunctional channels that open upon depolarization and in response to mechanical stretch and purinergic receptor stimulation. Importantly, ATP can be released through Panx1 channels, providing a possible role for these channels in non-vesicular signal transmission. In this study we expressed exogenous human and mouse Panx1 in the gap junction deficient Neuro2A neuroblastoma cell line and explored the contribution of Panx1 channels to cell–cell communication as sites of ATP release. Electrophysiological (patch clamp) recordings from Panx1 transfected Neuro2A cells revealed membrane conductance that increased beyond 0 mV when applying voltage ramps from −60 to +100 mV; threshold was correlated with extracellular K+, so that at 10 mM K+, channels began to open at −30 mV. Evaluation of cell–cell communication using dual whole cell recordings from cell pairs revealed that activation of Panx1 current in one cell of the pair induced an inward current in the second cell after a latency of 10–20 s. This paracrine response was amplified by an ATPase inhibitor (ARL67156, 100 μM) and was blocked by the ATP-degrading enzyme apyrase (6.7 U/ml), by the P2 receptor antagonist suramin (50 μM) and by the Panx1 channel blocker carbenoxolone. These results provide additional evidence that ATP release through Panx1 channels can mediate nonsynaptic bidirectional intercellular communication. Furthermore, current potentiation by elevated K+ provides a mechanism for enhancement of ATP release under pathological conditions.

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Acknowledgments

We appreciate the technical support of Ms. Marcia Urban-Maldonado with Panx1 constructs, and the original gifts of hPanx1 and mPnx1 constructs from Dr. Gerhard Dahl (Miami) and Dr. George Zoidl (formerly Bochum, now Toronto). Supported in part by the National Institute of Neurological Disorders (NINDS) of the National Institutes of Health (NIH), NS041282 (to DCS). Studies described here were performed on the Neuro2A cell line to which DCS was introduced by Dr. Robert Ledeen and has been widely used as an exogenous expression system to characterize gap junction channels formed of various connexins (16).

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Authors and Affiliations

  1. Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, K 840, Bronx, NY, 10461, USA

    Rodolfo M. Iglesias & David C. Spray

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  1. Rodolfo M. Iglesias
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  2. David C. Spray
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Correspondence to David C. Spray.

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Special Issue: In Honor of Bob Leeden.

Electronic supplementary material

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11064_2012_720_MOESM1_ESM.tif

Supplemental Figure 1. Lack of response in directly opposed Cell2 to triple voltage ramps delivered to cells in Neuro2A cells not transfected with Panx1. (TIFF 119 kb)

11064_2012_720_MOESM2_ESM.tif

Supplemental Figure 2. Correlation between current in Cell1 at 60 mV (to which Pannexin1 channels largely contribute) and amplitude of response in Cell2 in terms of experiments performed with various K concentrations shown in Fig. 5. Note that cell current amplitudes tend to be higher in higher K concentrations and that the two parameters are highly correlated (r2 = 0.73). (TIFF 6862 kb)

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Iglesias, R.M., Spray, D.C. Pannexin1-Mediated ATP Release Provides Signal Transmission Between Neuro2A Cells. Neurochem Res 37, 1355–1363 (2012). https://doi.org/10.1007/s11064-012-0720-6

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  • DOI: https://doi.org/10.1007/s11064-012-0720-6

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