title: Blockade of epithelial Na+ channels by triamterenes - underlying mechanisms and molecular basis.
abstract: The three subunits (alpha, beta, gamma) 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 -90 mV and -40 mV, the IC50 values were 5 microM and 10 microM, respectively. The blockage by triamterene, which is a weak base with a pKa of 6.2, was dependent on the extracellular pH. The IC50 was 1 microM at pH 6.5 and only 17 microM 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 -90 mV were 0.74 microM for the charged and 100.6 microM 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 IC50 values in the range of 0.1-20 microM. 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 alpha-subunit-deletion mutant, Delta278-283, 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.
authors.affiliation: Physiologisches Institut I, Eberhard-Karls-University Tübingen, Gmelinstrasse 5, D-72076 Tübingen, Germany.
authors.forename: A E
authors.initials: AE
authors.lastname: Busch
authors.forename: H
authors.initials: H
authors.lastname: Suessbrich
authors.forename: K
authors.initials: K
authors.lastname: Kunzelmann
authors.forename: A
authors.initials: A
authors.lastname: Hipper
authors.forename: R
authors.initials: R
authors.lastname: Greger
authors.forename: S
authors.initials: S
authors.lastname: Waldegger
authors.forename: E
authors.initials: E
authors.lastname: Mutschler
authors.forename: B
authors.initials: B
authors.lastname: Lindemann
authors.forename: F
authors.initials: F
authors.lastname: Lang
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D004232:Diuretics / Q000494:pharmacology*; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008433:Mathematics; D008564:Membrane Potentials; D051381:Rats; D026941:Sodium Channel Blockers; D014223:Triamterene / Q000031:analogs & derivatives / Q000494:pharmacology*; D014982:Xenopus laevis
chemical_list: D004232:Diuretics; D026941:Sodium Channel Blockers; C076984:RPH 3048; C026475:hydroxytriamterene sulfate ester; D000584:Amiloride; C047344:dimethylaminohydroxypropoxytriamterene; D014223:Triamterene