Little or no effect of crofelemer on apical membrane cation channels and intracellular cAMP and calcium signaling. A, left, short-circuit current in primary cultures of CFTR-deficient human bronchial epithelial cells without versus with pretreatment with 50 μM crofelemer in the luminal solution. Where indicated, amiloride (10 μM) and UTP (100 μM) were added. Right, summary of differences in short-circuit current after amiloride and UTP additions (S.E., n = 3; ∗, P < 0.05). B, apical membrane K+ current in human bronchial epithelial cells after basolateral membrane permeabilization with 20 μM amphotericin B and in the presence of a K+ gradient (apical [K+], 5 mM; basolateral [K+], 150 mM). C, cAMP levels in T84 cell homogenates under basal conditions and at 10 min after treatment with 20 μM forskolin. Differences ± crofelemer were not significant. D, calcium signaling measured by fura-2 fluorescence in T84 cells under basal conditions and after ATP addition (100 μM). Where indicated, cells were pretreated with 50 μM crofelemer. Inset, peak ATP increase in fura-2 fluorescence ratio (S.E., n = 4). Difference was not significant.