Effects of divalent cations, protons and calmidazolium at the rat P2X7 receptor

doi:10.1016/S0028-3908(97)00141-X

Neuropharmacology

Volume 36, Issue 9, September 1997, Pages 1285-1294

https://doi.org/10.1016/S0028-3908(97)00141-X Get rights and content

Abstract

The P2X₇ receptor is a uniquely bifunctional molecule through which ATP can open a small cationic channel typical of ionotropic receptors and also induce a large pore permeable to high molecular weight molecules (> 600 Da). Activation of this large pore can lead to cell lysis within 1–2 min. We asked whether pharmacological differences existed between the cationic channel and the cell permeabilizing pore by measuring whole-cell currents and uptake of a propidium dye (YO-PRO; M_w 629) in HEK293 cells stably expressing the rat P2X₇ receptor, and comparing the actions of divalent cations and protons in these two assays. Currents in response to 2′-3′-(O)-(4-benzoyl benzoyl) ATP (BzATP, 30 μM) were inhibited by extracellular calcium, magnesium, zinc, copper and protons with half-maximal inhibitory concentrations (IC₅₀) of 2.9 mM, 0.5 mM, 11 μM, 0.5 μM and 0.4 μM, respectively. The inhibition was voltage independent in each case. YO-PRO uptake induced by BzATP was also inhibited with similar IC₅₀ values. The rank order of potency of a range of divalents was Cu²⁺ > Cd²⁺ = Zn²⁺ > Ni²⁺ ⪢ Mg²⁺ = Co²⁺ > Mn²⁺ > Ca²⁺ = Ba²⁺ ⪢ Sr²⁺. These results suggest that these divalent cations and protons all act primarily as allosteric modulators to alter the affinity of ATP binding to the P2X₇ receptor. In contrast, extracellular (but not intracellular) calmidazolium inhibited the BzATP-evoked current by up to 90% (IC₅₀ = 15 nM) but had no effect on YO-PRO uptake. Thus, calmidazolium can block activation of the ionic channel but this does not prevent the formation of the large permeabilizing pore.

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Activation of ionotropic P2X receptors increases free intracellular Ca²⁺ ([Ca²⁺]_i) by initiating a transmembrane cation flux. We studied the “a” and “k” splice variants of the rat purinergic P2X7 receptor (rP2X7aR and rP2X7kR) to exhibit a significant difference in Ca²⁺ flux through this channel. This difference is surprising because the variants share absolute sequence identity in the area of the pore that defines ionic selectivity. Here, we used patch-clamp fluorometry and chimeric receptors to show that the fraction of the total current carried by Ca²⁺ is a function of the primary sequence of the cytoplasmic N terminus. Using scanning mutagenesis, we identified five sites within the N terminus that respond to mutagenesis with a decrease in fractional calcium current and an increase in permeability to the polyatomic cation, N-methyl-d-glucamine (NMDG⁺), relative to Na⁺ (P_NMDG/P_Na). We tested the hypothesis that these sites line the permeation pathway by measuring the ability of thiol-reactive MTSET⁺ to alter the current of cysteine-substituted variants, but we detected no effect. Finally, we studied the homologous sites of the rat P2X2 receptor (rP2X2R) and observed that substitutions at Glu¹⁷ significantly reduced the fractional calcium current. Taken together, our results suggest that a change in the structure of the N terminus alters the ability of an intra-pore Ca²⁺ selectivity filter to discriminate among permeating cations. These results are noteworthy for two reasons: they identify a previously unknown outcome of mutagenesis of the N-terminal domain, and they suggest caution when assigning structure to function for truncated P2X receptors that lack a part of the N terminus.
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ReviewEffects of divalent cations, protons and calmidazolium at the rat P2X7 receptor

Abstract

Trends in Pharmacological Science

Biophysical Journal

Blood

Progress in Neurobiology

Neuroscience Letters

Molecular Cellular Endocrinology

Biophysical Journal

Current Opinions in Cell Biology

Journal of Biological Chemistry

Journal of Biological Chemistry

Biophysical Journal

Progress in Neurobiology

Journal of Biological Chemistry

Trends in Neuroscience

The permeability of endplate channels to monovalent and divalent metal cations

Journal of General Physiology

Nicotinic receptor function in the mammalian central nervous system

Annals of the New York Academy of Science

Rat mast cells permeabilized with ATP secrete histamine in response to calcium ions buffered in the micromolar range

Journal of Physiology

New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor

Nature

P2X receptors: an emerging channel family

European Journal of Neuroscience

Zn2+ potentiates ATP-activated current in rat sympathetic neurons

Pflugers Archives

ATP induces nucleotide permeability in rat mast cells

Nature

Pituitary Ca2+ channels: blockade by conventional and novel Ca2+ antagonists

American Journal of Physiology

Ionic permeability of, and divalent cation effects on, two ATP-gated cation channels (P2X receptors) expressed in mammalian cells

Journal of Physiology

Review
Effects of divalent cations, protons and calmidazolium at the rat P2X₇ receptor

Zn²⁺ potentiates ATP-activated current in rat sympathetic neurons

Pituitary Ca²⁺ channels: blockade by conventional and novel Ca²⁺ antagonists