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 µM 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) (ATPS), 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 µM, respectively, and a Hill coefficient of 2. The order of efficacy of agonists was ATP ATPS > 2-MeS-ATP > 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP). -Methylene-ATP (-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 () = 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 P2X₂. patch clamp; single channels; ATP; desensitization