1-palmitoyl-2-oleoylphosphatidylcholine and tetrabutylammonium

KcsA is a pH-dependent potassium channel that is activated at acidic pH. The channel undergoes global conformational changes upon activation. We hypothesized that the open-close conformational changes of the transmem brane region could promote the flip-flop of phosphoiipids. Based on this hypothesis, we measured the flip-flop ofNBD-labeled phospholipids in KcsA-incorporated proteoliposomes. Both flip and flop rates of ~NBD-PC were significantly enhanced in the presence of KcsA and were several times higher at pH 4.0 than at pH 7.4, suggesting that KcsA promotes the phospholipid flip in a conformation-dependent manner. Phospholipids were nonselectively flipped with respect to the glycerophospholipid structure. In the active state of KcsA channel,tetrabutylammonium locks the channel in the open conformation at acidic pH; however, it did not alter the fliprate of CGNBD-PC. Thus, the open-close transition of the transmembrane region did not affect the flip-flop of phospholipids. In addition, the KcsA mutant that lacked anN-terminal amphipathic helix (MO-helix) was found to show reduced ability to fl ip C6NBD-phospholipids at acidic pH. The closed conformation is stabilized in the absence of MO-heli x, and thus the attenuated flip could be explained by the reduced prevalence of the open conformation.These results suggest that the open conformation of KcsA can disturb the bilayer integrity and facilitate the flip-flop of phospholipids.

Topics: 4-Chloro-7-nitrobenzofurazan; Bacterial Proteins; Cholesterol; Hydrogen-Ion Concentration; Lipid Bilayers; Models, Molecular; Mutation; Phosphatidylcholines; Phospholipids; Potassium Channels; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Quaternary Ammonium Compounds

The synthesis and anion transport properties of a series of transmembrane anion transporters based on an isophthalamide scaffold with phenyl, naphthyl or anthracenyl central rings are reported. Anion transport studies using POPC vesicles, showed that the compounds have Hill coefficients >1. This is indicative of higher order complex formation, evidence that leads us to suggest that the compounds are not functioning solely as mobile carriers but rather that a cooperative transport mechanism is being observed. Fluorescence spectroscopy was used to show that the compounds aggregate in the phospholipid bilayer, which provides evidence that these compounds function as a self-assembled anion-conducting aggregate.

Topics: Anions; Bicarbonates; Biological Transport; Chlorides; Cholesterol; Diffusion; Lipid Bilayers; Organic Chemicals; Phosphatidylcholines; Phospholipids; Phthalic Acids; Protein Binding; Quaternary Ammonium Compounds; Solutions; Solvents; Spectrometry, Fluorescence

Water-filled hydrophobic cavities in channel proteins serve as gateways for transfer of ions across membranes, but their properties are largely unknown. We determined water distributions along the conduction pores in two tetrameric channels embedded in lipid bilayers using neutron diffraction: potassium channel KcsA and the transmembrane domain of M2 protein of influenza A virus. For the KcsA channel in the closed state, the distribution of water is peaked in the middle of the membrane, showing water in the central cavity adjacent to the selectivity filter. This water is displaced by the channel blocker tetrabutyl-ammonium. The amount of water associated with the channel was quantified, using neutron diffraction and solid state NMR. In contrast, the M2 proton channel shows a V-shaped water profile across the membrane, with a narrow constriction at the center, like the hourglass shape of its internal surface. These two types of water distribution are therefore very different in their connectivity to the bulk water. The water and protein profiles determined here provide important evidence concerning conformation and hydration of channels in membranes and the potential role of pore hydration in channel gating.

Topics: Bacterial Proteins; Cell Membrane; Escherichia coli; Gene Expression; Influenza A virus; Ion Channel Gating; Ion Transport; Lipid Bilayers; Models, Molecular; Phosphatidylcholines; Phosphatidylglycerols; Potassium; Potassium Channel Blockers; Potassium Channels; Protein Conformation; Protein Multimerization; Protons; Quaternary Ammonium Compounds; Recombinant Proteins; Streptomyces lividans; Viral Matrix Proteins; Water