Cibacron blue allosterically modulates the rat P2X(4) receptor

We have used whole-cell patch clamp electrophysiology to characterise the a ctions of the PZ antagonist, cibacron blue, on the rat recombinant P2X(4) r eceptor, 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 qua ntitative data. However, when recordings were made from cells that were par t 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 P 2X(4) receptor (IC50 > 300 mu M) when co-applied with ATP. However, when ce ll rafts were preincubated with low concentrations of cibacron blue (3-30 m u M) 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 wa shout of high, inhibitory concentrations of cibacron blue (300 mu M). In co ntrast to these effects on P2X(4) receptors, cibacron blue inhibited the AT P-induced response in both single cells and rafts of HEK293 cells expressin g the P2X(2) receptor (IC50 similar to 600-800 nM). The effects of cibacron blue on the P2X(4) receptor were quantitatively similar to those of Zn2+ w hich also increased ATP-evoked currents by decreasing the EC50 of ATP (up t o 3.5-fold). These data are consistent with the concept that cibacron blue, like zinc, allosterically regulates the function of the P2X(4) receptor. ( C) 1998 Elsevier Science Ltd, hll rights reserved.