Blockade of epithelial Na⁺ channels by triamterenes — Underlying mechanisms and molecular basis

Abstract

The three subunits (α, β, γ) encoding for the rat epithelial Na⁺ channel (rENaC) were expressed in Xenopus oocytes, and the induced Na⁺ conductance was tested for its sensitivity to various triamterene derivatives. Triamterene blocked rENaC in a voltage-dependent manner, and was 100-fold less potent than amiloride at pH 7.5. At −90mV and −40mV, the IC₅₀ values were 5 μM and 10 μM, respectively. The blockage by triamterene, which is a weak base with a pK_a of 6.2, was dependent on the extracellular pH. The IC₅₀ was 1 μM at pH 6.5 and only 17 μM at pH 8.5, suggesting that the protonated compound is more potent than the unprotonated one. According to a simple kinetic analysis, the apparent inhibition constants at −90mV were 0.74 μM for the charged and 100.6 μM for the uncharged triamterene. The main metabolite of triamterene, p-hydroxytriamterene sulfuric acid ester, inhibited rENaC with an approximately twofold lower affinity. Derivatives of triamterene, in which the p-position of the phenylmoiety was substituted by acidic or basic residues, inhibited rENaC with IQ₅₀ values in the range of 0.1–20 μM. Acidic and basic triamterenes produced a rENaC blockade with a similar voltage and pH dependence as the parent compound, suggesting that the pteridinemoiety of triamterene is responsible for that characteristic. Expression of the rENaC α-subunit-deletion mutant, Δ278–283, which lacks a putative amiloride-binding site, induced a Na⁺ channel with a greatly reduced affinity for both triamterene and amiloride. In summary, rENaC is a molecular target for triamterene that binds to its binding site within the electrical field, preferably as a positively charged molecule in a voltage-and pH-dependent fashion. We propose that amiloride and triamterene bind to rENaC using very similar mechanisms.