Phytochemicals perturb membranes and promiscuously alter protein function.

A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding.

Phosphoinositides alter lipid bilayer properties.

Phosphatidylinositol-4,5-bisphosphate (PIP2), which constitutes ∼1% of the plasma membrane phospholipid, plays a key role in membrane-delimited signaling. PIP2 regulates structurally and functionally diverse membrane proteins, including voltage- and ligand-gated ion channels, inwardly rectifying ion channels, transporters, and receptors. In some cases, the regulation is known to involve specific lipid-protein interactions, but the mechanisms by which PIP2 regulates many of its various targets remain to be fully elucidated. Because many PIP2 targets are membrane-spanning proteins, we explored whether the phosphoinositides might alter bilayer physical properties such as curvature and elasticity, which would alter the equilibrium between membrane protein conformational states-and thereby protein function. Taking advantage of the gramicidin A (gA) channels' sensitivity to changes in lipid bilayer properties, we used gA-based fluorescence quenching and single-channel assays to examine the effects of long-chain PIP2s (brain PIP2, which is predominantly 1-stearyl-2-arachidonyl-PIP2, and dioleoyl-PIP2) on bilayer properties. When premixed with dioleoyl-phosphocholine at 2 mol %, both long-chain PIP2s produced similar changes in gA channel function (bilayer properties); when applied through the aqueous solution, however, brain PIP2 was a more potent modifier than dioleoyl-PIP2. Given the widespread use of short-chain dioctanoyl-phosphoinositides, we also examined the effects of diC8-phosphoinositol (PI), PI(4,5)P2, PI(3,5)P2, PI(3,4)P2, and PI(3,4,5)P3. The diC8 phosphoinositides, except for PI(3,5)P2, altered bilayer properties with potencies that decreased with increasing head group charge. Nonphosphoinositide diC8 phospholipids generally were more potent bilayer modifiers than the polyphosphoinositides. These results show that physiological increases or decreases in plasma membrane PIP2 levels, as a result of activation of PI kinases or phosphatases, are likely to alter lipid bilayer properties, in addition to any other effects they may have. The results further show that exogenous PIP2, as well as structural analogues that differ in acyl chain length or phosphorylation state, alters lipid bilayer properties at the concentrations used in many cell physiological experiments.

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.