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Cibacron blue allosterically modulates the rat P2X4 receptor
K J Miller1, A D Michel, I P Chessell
1Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, UK. ADM7393@glaxowellcome.co.uk
Abstract:
We have used whole-cell patch clamp electrophysiology to characterise the actions of the P2 antagonist, cibacron blue, on the rat recombinant P2X4 receptor, stably expressed in human embryonic kidney 293 (HEK293) cells. In single cells, adenosine triphosphate (ATP) evoked inward currents, but the response was subject to considerable run down which precluded obtaining quantitative data. However, when recordings were made from cells that were part of a group of 20-40 electrically coupled cells (cell rafts), run-down of current was not observed and reproducible responses could be obtained. When studied using cell rafts, cibacron blue was a weak antagonist of the rat P2X4 receptor (IC50 > 300 microM) when co-applied with ATP. However, when cell rafts were preincubated with low concentrations of cibacron blue (3-30 microM) for 5 min prior to ATP addition, cibacron blue increased responses to ATP by increasing its potency (up to 4-fold) without affecting the maximum current. Potentiation of ATP-evoked currents was also observed following washout of high, inhibitory concentrations of cibacron blue (300 microM). In contrast to these effects on P2X4 receptors, cibacron blue inhibited the ATP-induced response in both single cells and rafts of HEK293 cells expressing the P2X2 receptor (IC50 approximately 600-800 nM). The effects of cibacron blue on the P2X4 receptor were quantitatively similar to those of Zn2+ which also increased ATP-evoked currents by decreasing the EC50 of ATP (up to 3.5-fold). These data are consistent with the concept that cibacron blue, like zinc, allosterically regulates the function of the P2X4 receptor.
Insights
Cibacron blue acts as a weak antagonist or potentiator of P2X4 receptors, unlike its inhibitory effect on P2X2 receptors. This suggests cibacron blue allosterically regulates P2X4 receptor function, similar to zinc.
Area of Science:
- Pharmacology
- Neuroscience
- Molecular Biology
Background:
- Purinergic signaling via P2 receptors is crucial in cellular communication.
- P2X4 receptors are ligand-gated ion channels activated by adenosine triphosphate (ATP).
- Understanding P2X receptor modulation is key for developing targeted therapeutics.
Purpose of the Study:
- To characterize the effects of cibacron blue on rat recombinant P2X4 and P2X2 receptors.
- To investigate the potential allosteric modulation of P2X4 receptors by cibacron blue.
- To compare cibacron blue's actions with known modulators like zinc.
Main Methods:
- Whole-cell patch clamp electrophysiology was employed.
- Experiments were conducted on human embryonic kidney 293 (HEK293) cells stably expressing P2X4 or P2X2 receptors.
- Cell rafts (20-40 electrically coupled cells) were used to overcome current rundown.
Main Results:
- Cibacron blue weakly antagonized P2X4 receptors (IC50 > 300 µM) but potentiated ATP-evoked currents at lower concentrations (3-30 µM).
- Potentiation involved increased ATP potency (up to 4-fold) without altering maximum current.
- Cibacron blue inhibited P2X2 receptor activity (IC50 ≈ 600-800 nM).
- Effects on P2X4 receptors mirrored those of Zn2+, suggesting allosteric regulation.
Conclusions:
- Cibacron blue exhibits dual activity on P2X4 receptors: weak antagonism and potentiation.
- The potentiating effect of cibacron blue on P2X4 receptors is concentration-dependent and suggests allosteric modulation.
- Cibacron blue's differential effects on P2X4 and P2X2 receptors highlight subtype-specific interactions.