Brain Research 904 (2001) 307–317 www.elsevier.com / locate / bres Research report 1 ATP modulates Na channel gating and induces a non-selective cation current in a neuronal hippocampal cell line a,b,e a,c a,d e, * Yasir El-Sherif , Andrzej Wieraszko , Probal Banerjee , Nicholas J. Penington a CSI / IBR Center for Developmental Neuroscience and Developmental Disabilities, The College of Staten Island / CUNY, New York, USA b CUNY Graduate Center, New York, USA c Department of Biology ( CSI), New York, USA d Department of Chemistry ( CSI), New York, USA e Department of Physiology and Pharmacology, State University of New York, Health Science Center at Brooklyn, Box 29, 450 Clarkson Ave., Brooklyn, NY 11203-2098, USA Accepted 19 April 2001 Abstract Extracellular ATP evoked two excitatory responses in hippocampal neuroblastoma cells (HN2). The first, an opening of a 1 receptor-operated non-selective cation channel and the second was a leftward shift in Na channel activation. Both ATP (5–1000 mM) 1 and 29,39-(4-benzoyl)-benzoyl-ATP (Bb-ATP, 50 mM) activated a non-selective cation current reversing near 0 mV and shifted the Na activation and inactivation curves to the left. Based on a comparison of a series of agonists and antagonists, the inward current appeared to 1 be partially mediated by activation of a P2X receptor, although hybrid channels cannot be ruled out. The shift in Na channel gating 7 1 could be separated from the opening of the cation channel, as application of the P2Y antagonist Reactive Blue-2 and GTP shifted the Na current activation to the left but failed to elicit the inward cation current. Both responses to ATP and Bb-ATP were insensitive to block by the P2X antagonist suramin (300 mM) but were prevented by incubation in oxidized ATP (200 mM); a putative P2X receptor antagonist. 7 Prior screening of the surface negative charge of the membrane with a high concentration of divalent cations prevented both responses. 42 1 We suggest that ATP activates a P2X receptor and becomes trapped on a site, on or near the Na channel. Activation of the P2X 42 1 receptor leads to the opening of a non-specific cation channel, while the binding of ATP leads to a modified charge sensed by the Na channel, similar to what occurs in the presence of charged amphiphiles as well as a number of b-scorpion toxins.  2001 Elsevier Science B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Sodium channels 1 Keywords: ATP; Charge screening; HN2 cell; Na channel; Reactive Blue-2; P2X receptor 7 1. Introduction mechanisms. The first of these involves the opening of receptor-operated non-selective cation channels [6] and a ATP, a source of metabolic energy and a neurotrans- second, infrequently studied mechanism, requires regula- mitter substance that can act on neuronal receptors, is tion of the voltage dependence of ion channel gating [39]. released in high concentrations both from nerve terminals The discovery of the role of extracellular ATP as a in a calcium-dependent manner [8,43,44], and from dam- neurotransmitter and the classification of purinergic re- aged cells [17]. The effects of ATP, in the extracellular ceptors has been reviewed recently by Burnstock [9] and space, during hypoxic trauma are likely to be important in North and Barnard [35]. Essentially if ATP, rather than one producing further neuronal depolarization by at least two of its metabolites, mediates an observed effect then the effect is probably mediated by P2 receptors. The P2 receptors have recently been reclassified into P2Y and P2X *Corresponding author. Tel.: 11-718-270-3399; fax: 11-718-270- subtypes based on whether they couple to a G-protein, or if 2241. E-mail address: npenington@downstate.edu (N.J. Penington). the receptor is a ligand-gated ion channel, respectively. 0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993(01)02487-8