J. Malcolm East

Interactions of phospholipids with the potassium channel KcsA.

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.

A diversity of SERCA Ca2+ pump inhibitors

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.

Sarco(endo)plasmic reticulum calcium pumps: recent advances in our understanding of structure/function and biology (Review)

This review examines the structure and function of the sarco(endo)plasmic reticulum calcium pump (SERCA1a) in the light of the recent publication of the 2.6 Å resolution structure of this protein, and looks at the increasing awareness of the key role played by SERCAs in calcium signalling. The roles played by the calcium pump isoforms, SERCA1a/b, SERCA2a/b and SERCA3a/b/c in cellular function are discussed, and the modulation of SERCA activity by phospholamban, sarcolipin and other modulatory influences is examined. The recent discoveries of human SERCA mutations leading to disease states is reviewed, and the insights into SERCA function using transgenic approaches are outlined.

The inhibitors thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone favour the E2 form of the Ca2+, Mg2+-ATPase

2,5‐Di(tert‐butyl)‐1,4‐benzohydroquinone has been shown to inhibit the Ca2+, Mg2+‐ATPase of sarcoplasmic reticulum with an affinity of 0.4 μM. It has been shown to shift the E2‐E1 equilibrium for the ATPase towards E2, as shown previously for the inhibitor thapsigargin. The shift towards E2 results in a decrease in affinity for Ca2+, as also observed for thapsigargin. A marked decrease in the rate of the E2‐E1 transition is observed for both BHQ and thapsigargin. A decrease in the equilibrium level of phosphorylation by Pi and of the steady‐state lever of phosphorylation by ATP are consistent with a decrease in the equilibrium constant for phosphorylation by Pi and an increase in the rate of dephosphorylation.

The Presence of Sarcolipin Results in Increased Heat Production by Ca2+-ATPase

Skeletal muscle sarcoplasmic reticulum of large mammals such as rabbit contains sarcolipin (SLN), a small peptide with a single transmembrane α-helix. When reconstituted with the Ca2+-ATPase from skeletal muscle sarcoplasmic reticulum into sealed vesicles, the presence of SLN leads to a reduced level of accumulation of Ca2+. Heats of reaction of the reconstituted Ca2+-ATPase with ATP were measured using isothermal calorimetry. The heat released increased linearly with time over 30 min and increased with increasing SLN content. Rates ATP hydrolysis by the reconstituted Ca2+-ATPase were constant over a 30-min time period and were the same when measured in the presence or absence of an ATP-regenerating system. The calculated values of heat released per mol of ATP hydrolyzed increased with increasing SLN content and fitted to a simple binding equation with a dissociation constant for the SLN·ATPase complex of 6.9 × 10–4 ± 2.9 × 10–4 in units of mol fraction per monolayer. It is suggested that the interaction between Ca2+-ATPase and SLN in the sarcoplasmic reticulum could be important in thermogenesis by the sarcoplasmic reticulum.

Selectivity in Lipid Binding to the Bacterial Outer Membrane Protein OmpF

The outer membrane porin OmpF from Escherichia coli has been reconstituted into lipid bilayers of defined composition, and fluorescence spectroscopy is used to characterize its interaction with the surrounding lipid. OmpF is a trimer within the membrane. It contains two Trp residues per monomer, Trp(214) at the lipid-protein interface and Trp(61) at the trimer interface. The fluorescence of Trp-214 in the mutant W61F is quenched by dibromostearoylphosphatidylcholine (di(Br(2)C18:0)PC), whereas the fluorescence of Trp(61) in the mutant W214F is not quenched by di(Br(2)C18:0)PC when fluorescence is excited directly through the Trp rather than through the Tyr residues. Measurements of relative fluorescence quenching for OmpF reconstituted into mixtures of lipid X and di(Br(2)C18:0)PC have been analyzed to give the binding constant of lipid X for OmpF, relative to that for dioleoylphosphatidylcholine (di(C18:1)PC). The phosphatidylcholine showing the strongest binding to OmpF is dimyristoyloleoylphosphatidylcholine (di(C14:1)PC) with binding constants decreasing with either increasing or decreasing fatty acyl chain length. Comparison with various theories for hydrophobic matching between lipids and proteins suggests that in the chain length range from C14 to C20, hydrophobic matching is achieved largely by distortion of the lipid bilayer around the OmpF, whereas for chains longer than C20, distortion of both the lipid bilayer and of the protein is required to achieve hydrophobic matching. Phosphatidylcholine and phosphatidylethanolamine bind with equal affinity to OmpF, but the affinity for phosphatidylglycerol is about half that for phosphatidylcholine.

Importance of direct interactions with lipids for the function of the mechanosensitive channel MscL

We have studied the effects of lipid structure on the function of the mechanosensitive channel of large conductance (MscL) from Escherichia coli to determine whether effects follow from direct interaction between the lipids and protein or whether they follow indirectly from changes in the curvature stress in the membrane. The G22C mutant of MscL was reconstituted into sealed vesicles containing the fluorescent molecule calcein, and the release of calcein from the vesicles was measured following opening of the channel by reaction with [2-(triethylammonium)ethyl] methanethiosulfonate (MTSET), which introduces five positive charges into the region of the pore constriction. The presence of anionic lipids in the vesicle membrane changed the rates and amplitudes of calcein release, the effects not correlating with calculated changes in lipid spontaneous curvature. Mutation of charged residues in the Arg-104, Lys-105, Lys-106 cluster removed high-affinity binding of anionic lipids to MscL, and the presence of anionic lipid no longer affected calcein flux through MscL. Changing the zwitterionic lipid from phosphatidylcholine to phosphatidylethanolamine resulted in a large decrease in the rate of calcein release, the change in rate varying linearly with lipid composition, as expected if spontaneous curvature affected the rate of release. However, rates of release of calcein measured in the presence of phosphatidylethanolamine- N-methyl and phosphatidylethanolamine- N, N-dimethyl did not fit the correlation between rate and curvature established for the phosphatidylcholine/phosphatidylethanolamine mixtures. Rather, the effects of zwitterionic lipid headgroup on calcein flux suggested that what was important was the presence of a proton in the headgroup, able to take part in hydrogen bonding to MscL. We conclude that the function of MscL is likely to be modulated by direct interaction with the surrounding, annular phospholipids that contact the protein in the membrane.

Anionic phospholipids affect the rate and extent of flux through the mechanosensitive channel of large conductance MscL.

The mechanosensitive channel of large conductance MscL from Escherichia coli has been reconstituted into sealed vesicles, and the effects of lipid structure on the flux of the fluorescent molecule calcein through the open channel have been studied. The channel was opened by reaction of the G22C mutant of MscL with the reagent [2-(triethylammonium)ethyl]methanethiosulfonate (MTSET) which introduces five positive charges within the pore constriction. Flux through the channel was small when the lipid was phosphatidylcholine, but addition of the anionic lipids phosphatidylglycerol, phosphatidic acid, or cardiolipin up to 50 mol % resulted in increases in the amplitudes and rates of release of calcein. Similar effects were seen when either wild-type MscL or the G22C mutant was opened by osmotic pressure difference; rates of release of calcein were very slow in the absence of anionic lipid but increased with increasing concentrations of phosphatidylglycerol to 50 mol %. The observed partial release of trapped calcein following activation of MscL was attributed to the formation of a long-lived subconductance state of MscL following channel opening. Effects of anionic lipid were attributed to an increase in the rate of the transition from closed to fully open state and to a decrease in the rate of the transition from the fully open state to the subconductance state. Higher concentrations of anionic lipid led to a decrease in the rate and amplitude of release of calcein, possibly due to a decreased rate of flux through the open channel. In mixtures with anionic lipids, phosphatidylethanolamine resulted in lower rates and amplitude of release than phosphatidylcholine.

Interactions of Anionic Phospholipids and Phosphatidylethanolamine with the Potassium Channel KcsA

Fluorescence quenching methods have been used to study interactions of anionic phospholipids with the potassium channel KcsA from Streptomyces lividans. Quenching of the Trp fluorescence of KcsA reconstituted into mixtures of dioleoylphosphatidylcholine (DOPC) and an anionic phospholipid with dibromostearoyl chains is more marked at low mole fractions of the brominated anionic phospholipid than is quenching in mixtures of dibromostearoylphosphatidylcholine and nonbrominated anionic lipid. The quenching data are consistent with two classes of binding site for lipid on KcsA, one set corresponding to annular binding sites around KcsA to which DOPC and two-chain anionic phospholipids bind with similar affinities, the other set (non-annular sites) corresponding to sites at which anionic phospholipids can bind but from which DOPC is either excluded or binds with very low affinity. The binding constant for tetraoleoylcardiolipin at the annular sites is significantly less than that for DOPC, being comparable to that for dioleoylphosphatidylethanolamine. Tetraoleoylcardiolipin binds with highest affinity to the non-annular sites, the affinity for dioleoylphosphatidylglycerol being the lowest. The affinity for dioleoylphosphatidylserine decreases at high ionic strength, suggesting that electrostatic interactions between the anionic phospholipid headgroup and positively charged residues on KcsA are important for binding at the non-annular site. The effect of ionic strength on the binding of phosphatidic acid is less marked than on phosphatidylserine. The value of the binding constant for the non-annular site depends on the extent of Trp fluorescence quenching following from binding at the non-annular site. It is suggested that the non-annular site to which binding is detected in the fluorescence quenching experiments corresponds to the binding site for phosphatidylglycerol detected at monomer-monomer interfaces in x-ray diffraction studies.

Characterisation of a novel Ca2+ pump inhibitor (bis-phenol) and its effects on intracellular Ca2+ mobilization.

Bis-phenol, a phenolic antioxidant, is an inhibitor of sarcoplasmic reticulum (SR), endoplasmic reticulum (ER) and plasma membrane Ca2+ ATPases. The concentration of bis-phenol giving half-maximal inhibition of the SR Ca(2+)-ATPase is 2 microM. On binding to the SR Ca(2+)-ATPase it shifts the E2 to E1 transition towards the E2 state and slows the transition between E2 to E1. Bis-phenol completely inhibits Ca(2+)-dependent ATP hydrolysis and Ca2+ uptake by rat cerebellar microsomes at a concentration of 30 microM. The plasma membrane Ca(2+)-ATPase is also completely inhibited at similar concentrations, however, the Na+/K(+)-ATPase is only marginally affected. Other inhibitors of the ER Ca(2+)-ATPases, thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ), inhibit Ca2+ uptake by approximately 75%. Bis-phenol therefore inhibits all types of ER Ca(2+)-ATPases present in cerebellum. This inhibitor is also able to mobilize Ca2+ from intracellular Ca2+ stores, including those sensitive to InsP3, in intact HL-60 cells.