Artemisinins are extracted from sweet wormwood (Artemisia annua) and are the most potent antimalarials available, rapidly killing all asexual stages of Plasmodium falciparum. Artemisinins are sesquiterpene lactones widely used to treat multidrug-resistant malaria, a disease that annually claims 1 million lives. Despite extensive clinical and laboratory experience their molecular target is not yet identified. Activated artemisinins form adducts with a variety of biological macromolecules, including haem, translationally controlled tumour protein (TCTP) and other higher-molecular-weight proteins. Here we show that artemisinins, but not quinine or chloroquine, inhibit the SERCA orthologue (PfATP6) of Plasmodium falciparum in Xenopus oocytes with similar potency to thapsigargin (another sesquiterpene lactone and highly specific SERCA inhibitor). As predicted, thapsigargin also antagonizes the parasiticidal activity of artemisinin. Desoxyartemisinin lacks an endoperoxide bridge and is ineffective both as an inhibitor of PfATP6 and as an antimalarial. Chelation of iron by desferrioxamine abrogates the antiparasitic activity of artemisinins and correspondingly attenuates inhibition of PfATP6. Imaging of parasites with BODIPY-thapsigargin labels the cytosolic compartment and is competed by artemisinin. Fluorescent artemisinin labels parasites similarly and irreversibly in an Fe2+-dependent manner. These data provide compelling evidence that artemisinins act by inhibiting PfATP6 outside the food vacuole after activation by iron.

The Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum exhibits complex kinetics of activation with respect to ATP. ATPase activity is pH-dependent, with similar pH-activity profiles at high and low concentrations of ATP. Low concentrations of Ca2+ in the micromolar range activate the ATPase, whereas activity is inhibited by Ca2+ at millimolar concentrations. The pH-dependence of this Ca2+ inhibition and the effect of the detergent C12E8 (dodecyl octaethylene glycol monoether) on Ca2+ inhibition are similar to those observed on activation by low concentrations of Ca2+. On the basis of these and other studies we present a kinetic model for the ATPase. The ATPase is postulated to exist in one of two conformations: a conformation (E1) of high affinity for Ca2+ and MgATP and a conformation (E2) of low affinity for Ca2+ and MgATP. Ca2+ binding to E2 and to the phosphorylated form E2P are equal. Proton binding at the Ca2+-binding sites in the E1 and E2 conformations explains the pH-dependence of Ca2+ effects. Binding of MgATP to the phosphorylated intermediate E1'PCa2 and to E2 modulate the rates of the transport step E1'PCa-E2'PCa2 and the return of the empty Ca2+ sites to the outside surface of the sarcoplasmic reticulum, as well as the rate of dephosphorylation of E2P. Only a single binding site for MgATP is postulated.

In addition to the annular or boundary lipids that surround the transmembrane surface of the potassium channel KcsA from Streptomyces lividans, x-ray crystallographic studies have detected one anionic lipid molecule bound at each protein-protein interface in the homotetrameric structure, at sites referred to as nonannular sites. The binding constant for phosphatidylglycerol at the nonannular sites has been determined using fluorescence quenching methods with a mutant of KcsA lacking the normal three lipid-exposed Trp residues. Binding is weak, with a binding constant of 0.42 +/- 0.06 in units of mol fraction, implying that the nonannular sites will only be approximately 70% occupied in bilayers of 100% phosphatidylglycerol. However, the nonannular sites show high selectivity for anionic lipids over zwitterionic lipids, and it is suggested that a change in packing at the protein-protein interface leads to a closing of the nonannular binding site in the unbound state. Increasing the anionic lipid content of the membrane leads to a large increase in open channel probability, from approximately 2.5% in the presence of 25 mol % phosphatidylglycerol to approximately 62% in 100 mol % phosphatidylglycerol. The relationship between open channel probability and phosphatidylglycerol content shows cooperativity. The data are consistent with a model in which three or four of the four nonannular sites in the KcsA homotetramer have to be occupied by anionic lipid for the channel to open. The conductance of the open channel increases with increasing concentration of anionic lipid, an effect possibly due to effects of anionic lipid on the concentration of K(+) close to the membrane surface.

No abstract

Trp fluorescence spectroscopy is a powerful tool to study the structures of membrane proteins and their interactions with the surrounding lipid bilayer. Many membrane proteins contain more than one Trp residue, making analysis of the fluorescence data more complex. The mechanosensitive channels MscL's of Mycobacterium tuberculosis (TbMscL) and Escherichia coli (EcMscL) contain no Trp residues. We have therefore introduced single Trp residues into the transmembrane regions of TbMscL and EcMscL to give the Trp-containing mutants F80W-TbMscL and F93W-EcMscL, respectively, which we show are highly suitable for measurements of lipid binding constants. In vivo cell viability assays in E. coli show that introduction of the Trp residues does not block function of the channels. The Trp-containing mutants have been reconstituted into lipid bilayers by mixing in cholate followed by dilution to re-form membranes. Cross-linking experiments suggest that the proteins retain their pentameric structures in phosphatidylcholines with chain lengths between C14 and C24, phosphatidylserines, and phosphatidic acid. Quenching of Trp fluorescence by brominated phospholipids suggests that the Trp residue in F80W-TbMscL is more exposed to the lipid bilayer than the Trp residue in F93W-EcMscL. Binding constants for phosphatidylcholines change with changing fatty acyl chain length, the strongest interaction for both TbMscL and EcMscL being observed with a chain of length C16, corresponding to a bilayer of hydrophobic thickness ca. 24 A, compared to a hydrophobic thickness for TbMscL of about 26 A estimated from the crystal structure. Lipid binding constants change by only a factor of 1.5 in the chain length range from C12 to C24, much less than expected from theories of hydrophobic mismatch in which the protein is treated as a rigid body. It is concluded that MscL distorts to match changes in bilayer thickness. The binding constants for dioleoylphosphatidylethanolamine for both TbMscL and EcMscL relative to those for dioleoylphosphatidylcholine are close to 1. Quenching experiments suggest a single class of binding sites for phosphatidylserine, phosphatidylglycerol, and cardiolipin on TbMscL; binding constants are greater than those for phosphatidylcholine and decrease with increasing ionic strength, suggesting that charge interactions are important in binding these anionic phospholipids. Quenching experiments suggest two classes of lipid binding sites on TbMscL for phosphatidic acid, binding of phosphatidic acid being much less dependent on ionic strength than binding of phosphatidylserine.

The potassium channel KcsA from Streptomyces lividans has been reconstituted into bilayers of phosphatidylcholines and fluorescence spectroscopy has been used to characterize the response of KcsA to changes in bilayer thickness. The Trp residues in KcsA form two bands, one on each side of the membrane. Trp fluorescence emission spectra and the proportion of the Trp fluorescence intensity quenchable by I(-) hardly vary in the lipid chain length range C10 to C24, suggesting efficient hydrophobic matching between KcsA and the lipid bilayer over this range. Measurements of fluorescence quenching for KcsA reconstituted into mixtures of brominated and nonbrominated phospholipids have been analyzed to give binding constants of lipids for KcsA, relative to that for dioleoylphosphatidylcholine (di(C18:1)PC). Relative lipid binding constants increase by only a factor of three with increasing chain length from C10 to C22 with a decrease from C22 to C24. Strongest binding to di(C22:1)PC corresponds to a state in which the side chains of the lipid-exposed Trp residues are likely to be located within the hydrocarbon core of the lipid bilayer. It is suggested that matching of KcsA to thinner bilayers than di(C24:1)PC is achieved by tilting of the transmembrane alpha-helices in KcsA. Measurements of fluorescence quenching of KcsA in bilayers of brominated phospholipids as a function of phospholipid chain length suggest that in the chain length range C14 to C18 the Trp residues move further away from the center of the lipid bilayer with increasing chain length, which can be partly explained by a decrease in helix tilt angle with increasing bilayer thickness. In the chain length range C18 to C24 it is suggested that the Trp residues become more buried within the hydrocarbon core of the bilayer.

The ability of fish oil to decrease TNF-alpha production is influenced by inherent TNF-alpha production and by polymorphisms in the TNF-alpha and lymphotoxin alpha genes.

The SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) is probably the most extensively studied membrane protein transporter. There is a vast array of diverse inhibitors for the Ca2+ pump, and many have proved significant in helping to elucidate both the mechanism of transport and gaining conformational structures. Some SERCA inhibitors such as thapsigargin have been used extensively as pharmacological tools to probe the roles of Ca2+ stores in Ca2+ signalling processes. Furthermore, some inhibitors have been implicated in the cause of diseases associated with endocrine disruption by environmental pollutants, whereas others are being developed as potential anticancer agents. The present review therefore aims to highlight some of the wide range of chemically diverse inhibitors that are known, their mechanisms of action and their binding location on the Ca2+ ATPase. Additionally, some ideas for the future development of more useful isoform-specific inhibitors and anticancer drugs are presented.