Electrophysiological evidence for an ATP-gated ion channel in the principal cells of the frog skin epithelium

In the present study we investigated the effects of adenosine 5'-triphosphate (ATP) on Na+ transport in frog skin epithelium. An experimental set-up was constructed to allow simultaneous measurement of Na+ transport, measured as the amiloride-sensitive short circuit current (I-sc), and free cytosolic Ca2+ concentration ([Ca2+](i)) measured with the Ca2+-sensitive dye fura-2. The cell potential (V-sc) was measured with microelectrodes. Addition of ATP (100 mu M) to the basolateral solution resulted in a fast transient decrease in I-sc followed by a slower increase and a transient increase in [Ca2+](i). Microelectrode measurements showed that the primary response, i.e. the decline in I-sc, was accompanied by transient depolarisation, followed by a return to the control value. The decrease in current was Ca2+ independent; i.e. treatment with thapsigargin in Ca2+-free solutions abolished the Ca2+ transient but did not influence the current transient. The secondary response, i.e. the slow increase in current, was accompanied by slow depolarisation of the cell. Measurements of apical Na+ permeability showed that this was due to an opening or activation of apical Na channels. These data show that ATP causes a fast initial drop and a secondary, long-lasting increase in Na+ absorption. The ability of ATP to cause the initial decline in current is independent of Ca2+, i.e. it is not caused by secondary effects of the P2Y-type receptors present in the tissue. Measurements of intracellular potential indicate that the initial depolarisation is caused by opening of non-selective cation channels, suggesting that this decrease is due to a transient activation of P2X-type ATP receptors.