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

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.