Properties of the pore-forming P2X7 purinoceptor in mouse NTW8 microglial cells

1. We have used whole-cell patch clamping methods to study and characterize the cytolytic P2X7 (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 microM), 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 microM 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 microM, respectively. These affects of ATP were antagonized by pyridoxal-phosphate-6-azophenyl- 2', 4'-disulphonic acid (PPADS; 30 microM) but not suramin (100 microM). 4. In low divalent solution, repeated application of 1 mM ATP for 1 s produced successively larger currents which reached a plateau, after 8 applications, of 466% of the first application current. PPADS (30 microM) prevented this augmentation, while 5-(N,N-hexamethylene)-amiloride (HMA) (100 microM) 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 P2X7 receptor. We suggest that the current augmentation phenomenon observed reflects formation of the large 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.