BLOCKADE OF EPITHELIAL NA- UNDERLYING MECHANISMS AND MOLECULAR-BASIS(CHANNELS BY TRIAMTERENES )

The three subunits (alpha, beta, gamma) encoding for the rat epithelia l Na+ channel (rENaC) were expressed in Xenopus oocytes, and the induc ed Na+ conductance was tested for its sensitivity to various triamtere ne derivatives. Triamterene blocked rENaC in a voltage-dependent manne r, and was 100-fold less potent than amiloride at pH 7.5. At -90 mV an d -40 mV, the IC50 values were 5 mu M and 10 mu M, respectively. The b lockage by triamterene, which is a weak base with a pK(a) of 6.2, was dependent on the extracellular pH. The IC50 was 1 mu M at pH 6.5 and o nly 17 mu M at pH 8.5, suggesting that the protonated compound is more potent than the unprotonated one. According to a simple kinetic analy sis, the apparent inhibition constants at -90 mV were 0.74 mu M for th e charged and 100.6 mu M for the uncharged triamterene. The main metab olite of triamterene, p-hydroxytriamterene sulfuric acid ester, inhibi ted rENaC with an approximately twofold lower affinity. Derivatives of triamterene, in which the p-position of the phenylmoiety was substitu ted by acidic or basic residues, inhibited rENaC with IC50 values in t he range of 0.1-20 mu M. Acidic and basic triamterenes produced a rENa C blockade with a similar voltage and pH dependence as the parent comp ound, suggesting that the pteridinemoiety of triamterene is responsibl e for that characteristic. Expression of the rENaC alpha-subunit-delet ion mutant, Delta 278-283, which lacks a putative amiloride-binding si te, induced a Na+ channel with a greatly reduced affinity for both tri amterene and amiloride. In summary, rENaC is a molecular target for tr iamterene that binds to its binding site within the electrical field, preferably as a positively charged molecule in a voltage- and pH-depen dent fashion. We propose that amiloride and triamterene bind to rENaC using very similar mechanisms.