Blockade of epithelial Na+ channels by triamterenes - underlying mechanisms and molecular basis.
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.
Busch
A E
AE
Physiologisches Institut I, Eberhard-Karls-University Tübingen, Gmelinstrasse 5, D-72076 Tübingen, Germany.
Suessbrich
H
H
Kunzelmann
K
K
Hipper
A
A
Greger
R
R
Waldegger
S
S
Mutschler
E
E
Lindemann
B
B
Lang
F
F
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
D004232:Diuretics; D026941:Sodium Channel Blockers; C076984:RPH 3048; C026475:hydroxytriamterene sulfate ester; D000584:Amiloride; C047344:dimethylaminohydroxypropoxytriamterene; D014223:Triamterene