Extracellular ATP-induced inward current in isolated epithelial cells of the endolymphatic sac

Using whole-cell patch-clamp technique and Fura-2 fluorescence measurement, the presence of ATP-activated ion channels and its dependence on intracellular Ca2+ concentration ([Ca2+](i)) in the epithelial cells of the endolymphatic sac were investigated. In zero current-clamp configuration, the average resting membrane potential was -66.8 +/- 1.3 mV (n = 18). Application of 30 mu M ATP to the bath induced a rapid membrane depolarization by 43.1 +/- 2.4 mV (n = 18). In voltage-clamp configuration, ATP-induced inward current at holding potential (V-H) of -60 mV was 169.7 +/- 6.3 pA (n = 18). The amplitude of ATP-induced currents increased in sigmoidal fashion over the concentration range between 0.3 and 300 mu M with a Hill coefficient (n) of 1.2 and a dissociation constant (K-d) of 11.7 mu M. The potency order of purinergic analogues in ATP-induced current, which was 2MeSATP > ATP gamma s greater than or equal to ATP > alpha,beta-ATP > ADP = AMP greater than or equal to adenosine = UTP, was consistent with the properties of the Ply receptor. The independence of the reversal potential of the ATP-induced current from Cl- concentration suggests that the current is carried by a cation channel. The relative ionic permeability ratio of the channel modulated by ATP for cations was Ca2+ > Na+ > Li+ > Ba2+ > Cs+ = K+. ATP (10 mu M) increased [Ca2+](i) in an external Ca2+-free solution to a lesser degree than that in the external solution containing 1.13 mM CaCl2. ATP-induced increase in [Ca2+](i) can be mimicked by application of ionomycin in a Ca2+-free solution. These results indicate that ATP increases [Ca2+](i) through the P-2Y. receptor with a subsequent activation of the non-selective cation channel, and that these effects of ATP are dependent on [Ca2+](i) and extracellular Ca2+. (C) 1999 Elsevier Science B.V. All rights reserved.