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Effects of divalent cations, protons and calmidazolium at the rat P2X7 receptor
C Virginio1, D Church, R A North
1Geneva Biomedical Research Institute, Glaxo Wellcome, Switzerland. CV49987@ggr.co.uk
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; Mw 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 microM) were inhibited by extracellular calcium, magnesium, zinc, copper and protons with half-maximal inhibitory concentrations (IC50) of 2.9 mM, 0.5 mM, 11 microM, 0.5 microM and 0.4 microM, 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.
Insights
The P2X7 receptor has two functions: a small ion channel and a large pore. Divalent cations and protons modulate ATP binding, while calmidazolium blocks the ion channel but not pore formation.
Area of Science:
- Pharmacology
- Cell Biology
- Molecular Biology
Background:
- The P2X7 receptor exhibits bifunctional activity, opening a small cationic channel and a large pore permeable to molecules over 600 Da.
- Large pore activation can rapidly induce cell lysis, highlighting its critical role in cellular processes.
Purpose of the Study:
- To investigate pharmacological distinctions between the P2X7 receptor's cationic channel and its large pore.
- To compare the effects of divalent cations and protons on both channel and pore functions.
Main Methods:
- Whole-cell patch-clamp electrophysiology to measure ion currents.
- Propidium iodide (YO-PRO) uptake assay to assess large pore formation.
- HEK293 cells stably expressing the rat P2X7 receptor were utilized.
Main Results:
- Extracellular divalent cations (Cu2+, Cd2+, Zn2+, Ni2+, Mg2+, Co2+, Mn2+, Ca2+, Ba2+, Sr2+) and protons inhibited P2X7 receptor currents and YO-PRO uptake with varying potencies.
- Divalent cations and protons acted as allosteric modulators of ATP binding affinity.
- Extracellular calmidazolium selectively inhibited the cationic current without affecting YO-PRO uptake, indicating distinct mechanisms.
Conclusions:
- Divalent cations and protons modulate P2X7 receptor activity through allosteric mechanisms affecting ATP binding.
- Calmidazolium specifically blocks the P2X7 receptor's cationic channel, dissociating channel function from large pore formation.
- These findings reveal differential pharmacological profiles for the P2X7 receptor's ion channel and pore activities.