Influence of voltage and extracellular Na+ on amiloride block and transport kinetics of rat epithelial Na+ channel expressed in Xenopus oocytes

We expressed the three subunits of the epithelial amiloride-sensitive Na+ channel (ENaC) from rat distal colon heterologously in oocytes of Xenopus laevis and analysed blocker-induced fluctuations in current using conventional dual-microelectrode voltage-clamp. To minimize Na+ accumulation we performed all experiments in low-Na+ solutions (15 mM). Noise analysis revealed that control or ENaC-injected oocytes did not exhibit spontaneous relaxation noise. However, in ENaC-expressing oocytes, amiloride induced a distinct Lorentzian component in the power density spectra. With three amiloride concentrations and a linear analysis of the respective changes in the corner frequency f(c) (2pif(c) plot) we determined the rate constants k(on) and k(off) for the amiloride-ENaC interaction. At a clamp potential (V-m) of -60 mV k(on) was 80.8+/-5.1 muM(-1) s(-1) and k(off) 15.4+/-4.2 s(-1). The half-maximal blocker concentration (K-mic.ami) was 0.19 muM (V-m=-60 mV). While k(on) was voltage-independent in the range -50 to -100 mV, k(off) and K-mic.ami decreased significantly with increasing membrane hyperpolarization, resulting in an increased affinity of amiloride for its binding site on ENaC. Increasing extracellular [Na+] ([Na+](o)) led to saturation of ENaC. Subsequent noise analysis revealed that single-channel current increased non-linearly with [Na+](o) and that saturation was not due to a reduction in the number of open channels. The apparent affinity of Na+ for its binding site on the channel was voltage dependent and increased with hyperpolarization. Noise analysis revealed that k(on) and k(off) for amiloride decreased with increasing [Na+](o), while the affinity of the amiloride-binding site did not change. These findings show that the affinity of rat intestinal ENaC for amiloride is voltage dependent and is influenced non-competitively by [Na+](o), indicating that Na+ and amiloride do not compete for the same binding site at the channel.