Properties of the pore-forming P2X(7) purinoceptor in mouse NTW8 microglial cells

1 We have used whole-cell patch clamping methods to study and characterize the cytolytic P2X, (P2Z) receptor in the NTW8 mouse microglial cell line. 2 At room temperature, in an extracellular solution containing 2 mM Ca2+ and 1 mM Mg2+, 2'- and 3'-O-(4-benzoylbenzoyl)-adenosine-5'-triphosphate (Bz-ATP; 300 mu M), or ATP (3 mM), evoked peak whole cell inward currents, at a holding potential of -90 mV, of 549 +/- 191 and 644 +/- 198 pA, respectively. Current-voltage relationships generated with 3 mM ATP reversed at 4.6 mV and did not display strong rectification. 3 In an extracellular solution containing zero Mg2+ and 500 mu M Ca2+ (low divalent solution), brief (0.5 s) application of these agonists elicited larger maximal currents (909 +/- 138 and 1818 +/- 218 pA, Bz-ATP and ATP, respectively). Longer application of ATP (1 mM for 30 s) produced larger, slowly developing, currents which reached a plateau after approximately 15-20 s and were reversible on washing. Under these conditions, in the presence of ATP, ethidium bromide uptake could be demonstrated. Further applictions of 1 mM ATP produced rapid currents of the same magnitude as those observed during the 30 s application. Subsequent determination of concentration-effect curves to Bz-ATP, ATP and 2-methylthio-ATP yielded EC50 values of 58.3, 298 and 505 mu M, respectively. These affects of ATP were antagonized by pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 30 mu M) but not suramin (100 mu M). 4 In low divalent solution, repeated application of 1 mM ATP for is produced successively larger currents which reached a plateau, after 8 applications, of 466% of the first application current. PPADS (30 mu M) prevented this augmentation, while 5-(N,N-hexamethylene)-amiloride (HMA) (100 mu M) accelerated it such that maximal augmentation was observed after only one application of ATP in the presence of HMA. At a bath temperature of 32 degrees C, current augmentation also occurred in normal divalent cation containing solution. 5 These data demonstrate that mouse microglial NTW8 cells possess a purinoceptor with pharmacological characteristics resembling the P2X receptor. We suggest that the current augmentation phenomenon observed reflects formation of the la:ge cytolytic pore characteristic of this receptor. We have demonstrated that pore formation can occur under normal physiological conditions and can be modulated pharmacologically, both positively and negatively.