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 Lorentzlan component in the power density spectra. With three amiloride concentrations and a linear analysis of the respective changes in the corner frequency f_c (2πf_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 μM^-1 s^-1 and k^off 15.4±4.2 s^-1. The half-maximal blocker concentration (K_mic,ami) was 0.19 μM (V_m=-60 reV). 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.