Low-amiloride-affinity Na+ channel in the epithelial cells isolated from the endolymphatic sac of guinea-pigs.

Abstract

By using the whole-cell patch-clamp technique, an amiloride-sensitive Na+-selective conductance was found in epithelial cells from the endolymphatic sac (ES) epithelia of guinea-pigs. In the current-clamp configuration, the average resting membrane potential was -41.7+/-8.4 mV (n = 22). Application of amiloride at a concentration of 20 microM elicited a decrease in cation conductance that was responsible for a membrane hyperpolarization by 17.9+/-6.0 mV (n = 22). Substitution of N-methyl d-glucamine chloride (NMDG-Cl) for external NaCl led to a more significant membrane hyperpolarization by 28.4+/-8.3 mV (n = 22). At holding potential of -70 mV, amiloride and ethylisopropylamiloride (EIPA) blocked the inward current in a concentration-dependent manner over the range of concentrations of between 0.1 microM and 50 microM, with an inhibitory constant (Ki) of 1.3+/-0.4 microM (n = 7) and 1.5+/-0.3 microM (n = 5), respectively. In the voltage-clamp configuration, substitution of NMDG-Cl for external NaCl significantly reduced the inward current (n = 9), indicating that the whole-cell conductance has a high permeability for Na+. Superfusion with 20 microM amiloride induced a significant reduction of the inward current, shifted the reversal potential from -39.4+/-8.8 mV to -60.4+/-10.5 mV (n = 12), and decreased the inward conductance from 5.0+/-1.3 nS to 3.7+/-1.5 nS (n = 12). The permeability ratio of Na+ over K+, calculated from the difference in reversal potential between the currents before and after application of amiloride, was approximately 5:1. Additionally, the conductance was not activated by application of forskolin, 3-isobutyl-1-methylxanthine (IBMX) and 8-bromo-cAMP (8-Br-cAMP). These findings suggest that a low-amiloride-affinity Na+ channel localized in the ES epithelial cells may be involved in uptake of Na+ in the ES.