2,3-butanedione monoxime affects cystic fibrosis transmembrane conductance regulator channel function through phosphorylation-dependent and phosphorylation-independent mechanisms: the role of bilayer material properties.

2,3-Butanedione monoxime (BDM) is widely believed to act as a chemical phosphatase. We therefore examined the effects of BDM on the cystic fibrosis transmembrane regulator (CFTR) Cl(-) channel, which is regulated by phosphorylation in a complex manner. In guinea pig ventricular myocytes, forskolin-activated whole-cell CFTR currents responded biphasically to external 20 mM BDM: a rapid approximately 2-fold current activation was followed by a slower (tau approximately 20 s) inhibition (to approximately 20% of control). The inhibitory response was abolished by intracellular dialysis with the phosphatase inhibitor microcystin, suggesting involvement of endogenous phosphatases. The BDM-induced activation was studied further in Xenopus laevis oocytes expressing human epithelial CFTR. The concentration for half-maximal BDM activation (K(0.5)) was state-dependent, approximately 2 mM for highly and approximately 20 mM for partially phosphorylated channels, suggesting a modulated receptor mechanism. Because BDM modulates many different membrane proteins with similar K(0.5) values, we tested whether BDM could alter protein function by altering lipid bilayer properties rather than by direct BDM-protein interactions. Using gramicidin channels of different lengths (different channel-bilayer hydrophobic mismatch) as reporters of bilayer stiffness, we found that BDM increases channel appearance rates and lifetimes (reduces bilayer stiffness). At 20 mM BDM, the appearance rates increase approximately 4-fold (for the longer, 15 residues/monomer, channels) to approximately 10-fold (for the shorter, 13 residues/monomer channels); the lifetimes increase approximately 50% independently of channel length. BDM thus reduces the energetic cost of bilayer deformation, an effect that may underlie the effects of BDM on CFTR and other membrane proteins; the state-dependent changes in K(0.5) are consistent with such a bilayer-mediated mechanism.

Hydroxyl radical activation of a Ca(2+)-sensitive nonselective cation channel involved in epithelial cell necrosis.

In a previous work the involvement of a fenamate-sensitive Ca(2+)-activated nonselective cation channel (NSCC) in free radical-induced rat liver cell necrosis was demonstrated (5). Therefore, we studied the effect of radical oxygen species and oxidizing agents on the gating behavior of a NSCC in a liver-derived epithelial cell line (HTC). Single-channel currents were recorded in HTC cells by the excised inside-out configuration of the patch-clamp technique. In this cell line, we characterize a 19-pS Ca(2+)-activated, ATP- and fenamate-sensitive NSCC nearly equally permeable to monovalent cations. In the presence of Fe(2+), exposure of the intracellular side of NSCC to H(2)O(2) increased their open probability (P(o)) by approximately 40% without affecting the unitary conductance. Desferrioxamine as well as the hydroxyl radical (.OH) scavenger MCI-186 inhibited the effect of H(2)O(2), indicating that the increase in P(o) was mediated by.OH. Exposure of the patch membrane to the oxidizing agent 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) had a similar effect to.OH. The increase in P(o) induced by.OH or DTNB was not reverted by preventing formation or by DTNB washout, respectively. However, the reducing agent dithiothreitol completely reversed the effects on P(o) of both.OH and DTNB. A similar increase in P(o) was observed by applying the physiological oxidizing molecule GSSG. Moreover, GSSG-oxidized channels showed enhanced sensitivity to Ca(2+). The effect of GSSG was fully reversed by GSH. These results suggest an intracellular site(s) of action of oxidizing agents on cysteine targets on the fenamate-sensitive NSCC protein implicated in epithelial cell necrosis.