Received for publication May 2, 2006.
Revised August 14, 2006.
Accepted for publication September 8, 2006.

2,3 Butanedione Monoxime Affects CFTR Channel Function Through Phosphorylation-Dependent and Phosphorylation-Independent Mechanisms. The role of bilayer material properties

Abstract

2,3-butanedione monoxime (BDM) is widely believed to act as a chemical phosphatase. We therefore examined BDM's effects on the cystic fibrosis transmembrane conductance 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 ~2-fold current activation was followed by a slower ( ~20 s) inhibition (to ~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 oocytes expressing human epithelial CFTR. The concentration for half-maximal BDM activation (K_0.5) was statedependent, ~2 mM for highly and ~20 mM for partially phosphorylated channels, suggesting a modulated receptor mechanism. Because BDM modulates many different membrane proteins with similar K_0.5s, 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 ~4 fold (for the longer, 15 residues/monomer, channels) to ~10 fold (for the shorter, 13 residues/monomer, channels); the lifetimes increase ~50% independent of channel length. BDM thus reduces the energetic cost of bilayer deformation, an effect that may underlie BDM's effects on CFTR and other membrane proteins; the state-dependent changes in K_0.5 are consistent with such a bilayer-mediated mechanism.

Key words: Ion channel regulation, Ion transporters (SERCA, Na/K ATPase, CFTR)