Amiloride-sensitive sodium current in evertedAmbystoma initial collecting tubule: short-term insulin effects

Abstract

Whole cell patch-clamp techniques were used to investigate amiloride-sensitive sodium conductance ( G Na) in the everted initial collecting tubule of Ambystoma. Accessibility to both the apical and basolateral membranes made this preparation ideal for studying the regulation of sodium transport by insulin. G Na accounted for 20% of total cell conductance ( G T) under control conditions. A resting membrane potential of −75 ± 2 mV ( n = 7) together with the fact that G T is stable with time suggested that the cells studied were viable. Measurements of capacitance and use of a known uncoupling agent, heptanol, suggested that cells were not electrically coupled. Thus the values of G T and G Na represented individual principal cells. Exposure of the basolateral membrane to insulin (1 mU/ml) for 10–60 min significantly ( P< 0.05) increased the normalized G Na [1.2 ± 0.3 nS ( n = 6) vs. 2.0 ± 0.4 nS ( n = 6)]. Cell-attached patch-clamp techniques were used to further elucidate the mechanism by which insulin increases amiloride-sensitive epithelial sodium channel (ENaC) activity. In the presence of insulin there was no apparent change in either the number of active levels/patch or the conductance of ENaC. The open probability increased significantly ( P < 0.01) from 0.21 ± 0.04 ( n = 6) to 0.46 ± 0.07 ( n = 6). Thus application of insulin enhanced sodium reabsorption by increasing the fraction of time the channel spent in the open state.