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

Abstract

Using whole-cell patch-clamp technique and Fura-2 fluorescence measurement, the presence of ATP-activated ion channels and its dependence on intracellular Ca 2+ concentration ([Ca 2+] 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 μM 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 μM with a Hill coefficient ( n) of 1.2 and a dissociation constant ( K d) of 11.7 μM. The potency order of purinergic analogues in ATP-induced current, which was 2MeSATP>ATPγs≥ATP>α,β-ATP>ADP=AMP≥adenosine=UTP, was consistent with the properties of the P 2Y 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 Ca 2+>Na +>Li +>Ba 2+>Cs +=K +. ATP (10 μM) increased [Ca 2+] i in an external Ca 2+-free solution to a lesser degree than that in the external solution containing 1.13 mM CaCl 2. ATP-induced increase in [Ca 2+] i can be mimicked by application of ionomycin in a Ca 2+-free solution. These results indicate that ATP increases [Ca 2+] 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 [Ca 2+] i and extracellular Ca 2+.