Na+ dependence of single-channel current and channel density generate saturation of Na+ uptake in A6 cells

Abstract

In high-resistance, salt-absorbing epithelia the apical amiloride-sensitive Na+ channel is the key site for regulation of salt and water balance. The saturation of macroscopic Na+ transport through these channels was investigated using A6 epithelial monolayers. The relation between transepithelial Na+ transport (I-Na) and apical Na+ concentration ([Na+](ap)) under short-circuit conditions was studied. Michaelis-Menten analysis of the saturable short-circuit current (I-sc) yielded an apparent Michaelis-Menten constant (K-m(I)) of 5 mmol/l and a maximal current (I-max) of 8 mu A/cm(2). The microscopic parameters underlying I-Na, namely the single-channel current (i) and the open channel density (N-o,), were investigated by the analysis of current fluctuations induced by the electroneutral amiloride analogue CDPC (6-chloro-3,5-diaminopyrazine-2-carboxamide). A two-state model analysis yielded the absolute values of i (0.18 +/- 0.01 pA) and N-o, (65.38 +/- 9.57 million channels/cm(2) of epithelium) at [Na+](ap),, 110 mmol/l containing 50 mu mol/l CDPC. Our data indicate that in A6 cells both i and N-o, depend on [Na+](ap). Between 3 and approximate to 20 mmol/l the density of conducting pores, N-o,, decreases sharply and behaves again as an almost [Na+](ap)-independent parameter at higher [Na+](ap). The single-channel current clearly saturates with an apparent Michaelis-Menten constant, K-m(i),, of approximate to 17 mmol/l. Thus, the [Na+](ap),, dependence of N-o, as well as the limited transport capacity of the amiloride-sensitive Na+ channel are both responsible for the saturation of I-Na.