title: Influence of voltage and extracellular Na(+) on amiloride block and transport kinetics of rat epithelial Na(+) channel expressed in Xenopus oocytes.
abstract: 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) (2 pi f(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 microM(-1) s(-1) and k(off) 15.4 +/- 4.2 s(-1). The half-maximal blocker concentration (K(mic,ami)) was 0.19 microM (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.
authors.affiliation: Laboratory of Physiology, KU Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium.
authors.forename: Andrei
authors.initials: A
authors.lastname: Segal
authors.forename: Mouhamed S
authors.initials: MS
authors.lastname: Awayda
authors.forename: Jan
authors.initials: J
authors.lastname: Eggermont
authors.forename: Willy
authors.initials: W
authors.lastname: Van Driessche
authors.forename: Wolf-Michael
authors.initials: WM
authors.lastname: Weber
mesh_terms: D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D016477:Artifacts; D004232:Diuretics / Q000494:pharmacology*; D025802:Electric Capacitance; D053503:Epithelial Sodium Channels; D005260:Female; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D051381:Rats; D012964:Sodium / Q000493:pharmacokinetics*; D015222:Sodium Channels / Q000187:drug effects / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D004232:Diuretics; D053503:Epithelial Sodium Channels; D015222:Sodium Channels; D000584:Amiloride; D012964:Sodium