Verapamil and amlodipine are calcium ion influx inhibitors of wide clinical use. They are partially charged at neutral pH and exhibit amphiphilic properties. The noncharged species can easily cross ...the lipid membrane. We have measured with solid-state NMR the structural changes induced by verapamil upon incorporation into phospholipid bilayers and have compared them with earlier data on amlodipine and nimodipine. Verapamil and amlodipine produce a rotation of the phosphocholine headgroup away from the membrane surface and a disordering of the fatty acid chains. We have determined the thermodynamics of verapamil partitioning into neutral and negatively charged membranes with isothermal titration calorimetry. Verapamil undergoes a pK-shift of ΔpKa=1.2 units in neutral lipid membranes and the percentage of the noncharged species increases from 5% to 45%. Verapamil partitioning is increased for negatively charged membranes and the binding isotherms are strongly affected by the salt concentration. The electrostatic screening can be explained with the Gouy-Chapman theory. Using a functional phosphate assay we have measured the affinity of verapamil, amlodipine, and nimodipine for P-glycoprotein, and have calculated the free energy of drug binding from the aqueous phase to the active center of P-glycoprotein in the lipid phase. By combining the latter results with the lipid partitioning data it was possible, for the first time, to determine the true affinity of the three drugs for the P-glycoprotein active center if the reaction takes place exclusively in the lipid matrix.
To date, over 20 peptides or proteins have been identified that can form amyloid fibrils in the body and are thought to cause disease. The mechanism by which amyloid peptides cause the cytotoxicity ...observed and disease is not understood. However, one of the major hypotheses is that amyloid peptides cause membrane perturbation. Hence, we have studied the interaction between lipid bilayers and the 37 amino acid residue polypeptide amylin, which is the primary constituent of the pancreatic amyloid associated with type 2 diabetes. Using a dye release assay we confirmed that the amyloidogenic human amylin peptide causes membrane disruption; however, time-lapse atomic force microscopy revealed that this did not occur by the formation of defined pores. On the contrary, the peptide induced the formation of small defects spreading over the lipid surface. We also found that rat amylin, which has 84% identity with human amylin but cannot form amyloid fibrils, could also induce similar lesions to supported lipid bilayers. The effect, however, for rat amylin but not human amylin, was inhibited under high ionic conditions. These data provide an alternative theory to pore formation, and how amyloid peptides may cause membrane disruption and possibly cytotoxicity.
Apolipoprotein A-1 (Apo A-1) plays an important role in lipid transfer and obesity. Chemical unfolding of α-helical Apo A-1 is induced with guanidineHCl and monitored with differential scanning ...calorimetry (DSC) and CD spectroscopy. The unfolding enthalpy and the midpoint temperature of unfolding decrease linearly with increasing guanidineHCl concentration, caused by the weak binding of denaturant. At room temperature, binding of 50–60 molecules guanidineHCl leads to a complete Apo A-1 unfolding. The entropy of unfolding decreases to a lesser extent than the unfolding enthalpy. Apo A-1 chemical unfolding is a dynamic multi-state equilibrium that is analysed with the Zimm-Bragg theory modified for chemical unfolding. The chemical Zimm-Bragg theory predicts the denaturant binding constant KD and the protein cooperativity σ. Chemical unfolding of Apo A-1 is two orders of magnitude less cooperative than thermal unfolding. The free energy of thermal unfolding is ~0.2 kcal/mol per amino acid residue and ~1.0 kcal/mol for chemical unfolding at room temperature. The Zimm-Bragg theory calculates conformational probabilities and the chemical Zimm-Bragg theory predicts stretches of α-helical segments in dynamic equilibrium, unfolding and refolding independently and fast.
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•Chemical unfolding of Apo A-1 is a multi-state equilibrium analysed with the Zimm-Bragg theory.•Apo A-1 unfolding enthalpy decreases linearly with guanidineHCl concentration.•Method proposed to determine the number of bound denaturant molecules.•Exothermic guanidineHCl binding decreases endothermic α-helix unfolding of Apo A-1.•Chemical Zimm-Bragg theory predicts guanidineHCl binding constant KD and protein cooperativity σ.
It has recently been suggested that substrate recognition patterns for human P-glycoprotein encoded by mdr1 consist of two electron donor groups with a spatial separation of 2.5 +/- 0.3 A (type I ...units) or three electron donor groups with a spatial separation of the two outer groups of 4.6 +/- 0.6 A (type II units) Seelig 1998. Since P-gp and the multidrug resistance-associated protein (MRP1) have overlapping substrate specificity, we screened the chemical structures of 21 compounds, previously tested as MRP1 substrates, for electron donor units. In addition, we searched the putative transmembrane domains (TMD 1-12) of P-gp and (TMD 6-17) of MRP1 for amino acid side chains having the potential to interact with the respective substrates.
The three-dimensional structures of potential MRP1 substrates were modeled with a force-field approach and were then screened for electron donor units. Helical wheel projections of the 12 putative transmembrane domains of P-gp (1-12) and MRP (6-17) were analyzed for their content of amino acid residues with hydrogen bonding side chains, charged amino acid residues, and amino acid residues with pi-electron systems.
MRP1 recognizes compounds with type I and type II units. At least one electrically neutral together with either one negatively charged type I unit or two electrically neutral type I units are required for the compound to be bound and transported. Transport increases with increasing number of electron donor units. Compounds which carry exclusively electrically neutral type I units (P-gp substrates) are transported only weakly by MRP1, and compounds with cationic type I units (P-gp substrates) are not transported at all. An analysis of the putative transmembrane alpha-helices of MRP1 and P-gp reveals that the amino acid residues with hydrogen-bond donor side chains are arranged preferentially on one side of the helix and amino acid residues with inert (non-hydrogen-bonding) side chains on the other side. In the case of MRP1, the hydrogen-bonding face also contains several cationic residues whereas, in the case of P-gp, it contains clusters of amino acid residues with beta-electron systems.
We propose that P-gp and MRP1 recognize type I or type II units in chemical compounds having diverse structures, and that these transporters bind their substrates via hydrogen bond formation. Furthermore, we propose that transport of anionic substrates by MRP1 is facilitated by cationic amino acid residues present in the transmembrane helices of MRP1, whereas the transport of cationic substrates by P-gp is facilitated by a beta-electron slide guide.
Cell-penetrating peptides (CPPs) traverse cell membranes of cultured cells very efficiently by a mechanism not yet identified. Recent theories for the translocation suggest either the binding of the ...CPPs to extracellular glycosaminoglycans or the formation of inverted micelles with negatively charged lipids. In the present study, the binding of the protein transduction domains (PTD) of human (HIV-1) and simian immunodeficiency virus (SIV) TAT peptide (amino acid residues 47−57, electric charge z p = +8) to membranes containing various proportions of negatively charged lipid (POPG) is characterized. Monolayer expansion measurements demonstrate that TAT-PTD insertion between lipids requires loosely packed monolayer films. For densely packed monolayers (π > 29 mN/m) and lipid bilayers, no insertion is possible, and binding occurs via electrostatic adsorption to the membrane surface. Light scattering experiments show an aggregation of anionic lipid vesicles when the electric surface charge is neutralized by TAT-PTD, the observed stoichiometry being close to the theoretical value of 1:8. Membrane binding was quantitated with isothermal titration calorimetry and three further methods. The reaction enthalpy is ΔH° ≈ −1.5 kcal/mol peptide and is almost temperature-independent with Δ ∼0 kcal/(mol K), indicating equal contributions of polar and hydrophobic interactions to the reaction heat capacity. The binding of TAT-PTD to the anionic membrane is described by an electrostatic attraction/chemical partition model. The electrostatic attraction energy, calculated with the Gouy−Chapman theory, accounts for ∼80% of the binding energy. The overall binding constant, K app, is ∼103−104 M-1. The intrinsic binding constant (K p), corrected for electrostatic effects and describing the partitioning of the peptide between the lipid−water interface and the membrane, is small and is K p ∼1−10 M-1. Deuterium and phosphorus-31 nuclear magnetic resonance demonstrate that the lipid bilayer remains intact upon TAT-PTD binding. The NMR data provide no evidence for nonbilayer structures and also not for domain formation. This is further supported by the absence of dye efflux from single-walled lipid vesicles. The electrostatic interaction between TAT-PTD and anionic phosphatidylglycerol is strong enough to induce a change in the headgroup conformation of the anionic lipid, indicating a short-lived but distinct correlation between the TAT-PTD and the anionic lipids on the membrane outside. TAT-PTD has a much lower affinity for lipid membranes than for glycosaminoglycans, making the latter interaction a more probable pathway for CPP binding to biological membranes.
Talin, an actin-binding protein, is assumed to anchor at the membrane via an intrinsic amino acid sequence. Three N-terminal talin fragments, 21–39 (S19), 287–304 (H18), and 385–406 (H17) have been ...proposed as potential membrane anchors. The interaction of the corresponding synthetic peptides with lipid model systems was investigated with CD spectroscopy, isothermal titration calorimetry, and monolayer expansion measurements. The membrane model systems were neutral or negatively charged small unilamellar vesicles or monolayers with a lateral packing density of bilayers (32 mN/m). S19 partitions into charged monolayers/bilayers with a penetration areaAp = 140 ± 30 Å2 and a free energy of binding of ΔG0 = −5.7 kcal/mol, thereby forming a partially α-helical structure. H18 does not interact with lipid monolayers or bilayers. H17 penetrates into neutral and charged monolayers/bilayers with Ap = 148 ± 23 Å2 and Ap = 160 ± 15 Å2, respectively, forming an α-helix in the membrane-bound state. Membrane partitioning is mainly entropy-driven. Under physiological conditions the free energy of binding to negatively charged membranes is ΔG0 = −9.4 kcal/mol with a hydrophobic contribution of ΔGh = −7.8 kcal/mol, comparable to that of post-translationally attached membrane anchors, and an electrostatic contribution of ΔGh = −1.6 kcal/mol. The latter becomes more negative with decreasing pH. We show that H17 provides the binding energy required for a membrane anchor.
We compared the P-glycoprotein ATPase activity in inside–out plasma membrane vesicles and living NIH-MDR1-G185 cells with the aim to detect substrate transport. To this purpose we used six substrates ...which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside–out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside–out plasma membrane vesicles, because their concentrations in the cytosolic leaflets were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside–out vesicles. Their concentration was significantly lower in the cytosolic leaflet of cells than in the cytosolic leaflet of inside-out membrane vesicles, indicating that P-glycoprotein could cope with passive influx. P-glycoprotein thus transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). However, it prevented substrate entry into the cytosol only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux.
Recent studies have suggested that inositol-1,4,5-trisphosphate-receptor (IP3R)-mediated Ca2+ release plays an important role in the modulation of excitation-contraction coupling (ECC) in atrial ...tissue and the generation of arrhythmias, specifically chronic atrial fibrillation (AF). IP3R type-2 (IP3R2) is the predominant IP3R isoform expressed in atrial myocytes. To determine the role of IP3R2 in atrial arrhythmogenesis and ECC, we generated IP3R2-deficient mice. Our results revealed that endothelin-1 (ET-1) stimulation of wild-type (WT) atrial myocytes caused an increase in basal Ca2+i, an enhancement of action potential (AP)-induced Ca2+i transients, an improvement of the efficacy of ECC (increased fractional SR Ca2+ release), and the occurrence of spontaneous arrhythmogenic Ca2+ release events as the result of activation of IP3R-dependent Ca2+ release. In contrast, ET-1 did not alter diastolic Ca2+i or cause spontaneous Ca2+ release events in IP3R2-deficient atrial myocytes. Under basal conditions the spatio-temporal properties (amplitude, rise-time, decay kinetics, and spatial spread) of Ca2+i transients and fractional SR Ca2+ release were not different in WT and IP3R2-deficient atrial myocytes. WT and IP3R2-deficient atrial myocytes also showed a significant and very similar increase in the amplitude of AP-dependent Ca2+i transients and Ca2+ spark frequency in response to isoproterenol stimulation, suggesting that both cell types maintained a strong inotropic reserve. No compensatory changes in Ca2+ regulatory protein expression (IP3R1, IP3R3, RyR2, NCX, SERCA2) or morphology of the atria could be detected between WT and IP3R2-deficient mice. These results show that lack of IP3R2 abolishes the positive inotropic effect of neurohumoral stimulation with ET-1 and protects from its arrhythmogenic effects.
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