Conductive cation transport in apical membrane vesicles prepared from fetal lung

Abstract

In order to characterise the apically-located conductive cation pathway of the type II pneumocyte, apical plasma membranes were prepared from mature fetal guinea pig lung. The protocol yielded purified apical membranes that enriched 19-fold with the brush border enzyme marker alkaline phosphatase; there was no significant contamination with other cellular membranes. A technique for imposing an outwardly-directed electrochemical Na + gradient was used to amplify conductive 22Na + uptake into vesicles. Uptake of 22Na + was time-dependent, proportional to the magnitude of the Na + gradient, specific and sensitive to the amiloride analogues phenamil and EIPA (apparent minimum K i values of 50 nM and 10 μM, respectively, with maximum uptake inhibition of 42% and 39% at 100 μM). Uptake experiments in which the outwardly-directed Na + gradient was replaced by outwardly-directed gradients of small monovalent cations and molecular cations were performed. The Na +/K + permeability ratio was 1.2:1, and over the extended range of small monovalent cations, a permeability sequence of Na +>K +>Li +>Rb +>Cs + was observed, indicating the presence of fixed negative charge in or spatially close to the pore. The molecular cation permeability sequence of NH 4 +> methylamine +> dimethylamine +> choline +>N- methyl- d-glucamine +> tetraethylammonium +> tetramethylammonium + , after transformation, gives an estimate of 8 Å for the conducting pore diameter. These data are consistent with the presence in the apical membrane of fetal type II pneumocytes of a cation specific channel with low Na + selectivity and amiloride sensitivity.