P2X receptors in mouse Leydig cells

ATP-activated currents were studied in Leydig cells of mice with the patch-clamp technique. Whole cell currents were rapidly activating and slowly desensitizing (55% decrement from the peak value on exposure to 100 microM ATP for 60 s), requiring 3 min of washout to recover 100% of the response. The concentration-response relationships for ATP, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), and 2-methylthio-ATP (2-MeS-ATP) were described by the Hill equation with a concentration evoking 50% of maximal ATP response (K(d)) of 44, 110, and 637 microM, respectively, and a Hill coefficient of 2. The order of efficacy of agonists was ATP >or= ATPgammaS > 2-MeS-ATP > 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP). alphabeta-Methylene-ATP (alphabeta-MeATP), GTP, UTP, cAMP, and adenosine were ineffective. Suramin and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) blocked the responses in a concentration-dependent manner. The ATP-activated currents were dependent on extracellular pH, being maximal at pH 6.5 and decreasing with both acidification and alkalinization (apparent dissociation constant (pK(a)) of 5.9 and 7.4, respectively). The whole cell current-voltage relationship showed inward rectification and reversed near 0 mV. Experiments performed in bi-ionic conditions for measurement of reversal potentials showed that this channel is highly permeable to calcium [permeability (P)(Ca)/P(Na) = 5.32], but not to chloride (P(Cl)/P(Na) = 0.03) or N-methyl-D-glucamine (NMDG) (P(NMDG)/P(Na) = 0.09). Unitary currents recorded in outside-out patches had a chord conductance of 27 pS (between -90 and -50 mV) and were inward rectifying. The average current passing through the excised patch decreased with time [time constant (tau) = 13 s], resembling desensitization of the macroscopic current. These findings indicate that the ATP receptor present in Leydig cells shows properties most similar to those of cloned homomeric P2X(2).