Modulation of Cl- secretion by benzimidazolones .1. Direct activation of a Ca2+-dependent K+ channel

We evaluated the effects of the novel benzimidazolone, 1-ethyl-2-benzimidazolinone (1-EBIO), on Cl- secretion across T84 monolayers. 1-EBIO stimulated a sustained Cl- secretory response at a half-maximal effective concentration of 490 mu M. Charybdotoxin (CTX) inhibited the 1-EBIO-induced short-circuit current (I-sc) with an inhibitory constant (K-i) of3.6 nM, whereas 293B, an inhibitor of adenosine 3',5'-cyclic monophosphate-activated K+ channels, had no effect on the current induced by 1-EBIO. In contrast, CTX failed to inhibit the 293B-sensitive forskolin-induced I-sc. The above results suggested that 1-EBIO may be activating the basolateral membrane Ca2+-dependent K+ channel (K-Ca) in these cells. This was further confirmed using nystatin to permeabilize the apical membrane in the presence of a mucosa-to-serosa K+ gradient and determining the effects of 1-EBIO on the basolateral K+ current (I-K). Under these conditions, 1-EBIO induced a large increase in I-K that was blocked by CTX. In membrane vesicles prepared from T84 cells, 1-EBIO stimulated (86)R(+) uptake in a CTX-sensitive manner; the K-i for inhibition by CTX was 3.5 nM. Similar to our intact monolayer studies, this Rb-86(+) uptake was not blocked by 293B. The effects of 1-EBIO on the K-Ca in T84 cells was determined in excised inside-out patches. 1-EBIO (100 mu M) increased the product of the number of channels and the open channel probability from 0.09 +/- 0.03 to 1.17 +/- 0.27 (n = 8); this effect on Kc, activity required a minimal level of free Ca2+. Similar to its effect on T84 cells, 1-EBIO stimulated a sustained Cl- secretory current in rat colonic epithelium, which was partially blocked by CTX. Finally, 1-EBIO stimulated a sustained Cl- secretory response in primary cultures of murine tracheal epithelium. We conclude that the benzimidazolone, 1-EBIO, stimulates Cl- secretion in secretory epithelia via the direct activation of a K-Ca. 1-EBIO is the first pharmacological opener of this important class of epithelial K+ channels to be identified.