Helicobacter pylori vacuolating toxin forms anion-selective channels in planar lipid bilayers: possible implications for the mechanism of cellular vacuolation.
(9/7162)
The Helicobacter pylori VacA toxin plays a major role in the gastric pathologies associated with this bacterium. When added to cultured cells, VacA induces vacuolation, an effect potentiated by preexposure of the toxin to low pH. Its mechanism of action is unknown. We report here that VacA forms anion-selective, voltage-dependent pores in artificial membranes. Channel formation was greatly potentiated by acidic conditions or by pretreatment of VacA at low pH. No requirement for particular lipid(s) was identified. Selectivity studies showed that anion selectivity was maintained over the pH range 4.8-12, with the following permeability sequence: Cl- approximately HCO3- > pyruvate > gluconate > K+ approximately Li+ approximately Ba2+ > NH4+. Membrane permeabilization was due to the incorporation of channels with a voltage-dependent conductance in the 10-30 pS range (2 M KCl), displaying a voltage-independent high open probability. Deletion of the NH2 terminus domain (p37) or chemical modification of VacA by diethylpyrocarbonate inhibited both channel activity and vacuolation of HeLa cells without affecting toxin internalization by the cells. Collectively, these observations strongly suggest that VacA channel formation is needed to induce cellular vacuolation, possibly by inducing an osmotic imbalance of intracellular acidic compartments. (+info)
Model-independent analysis of the orientation of fluorescent probes with restricted mobility in muscle fibers.
(10/7162)
The orientation of proteins in ordered biological samples can be investigated using steady-state polarized fluorescence from probes conjugated to the protein. A general limitation of this approach is that the probes typically exhibit rapid orientational motion ("wobble") with respect to the protein backbone. Here we present a method for characterizing the extent of this wobble and for removing its effects from the available information about the static orientational distribution of the probes. The analysis depends on four assumptions: 1) the probe wobble is fast compared with the nanosecond time scale of its excited-state decay; 2) the orientational distributions of the absorption and emission transition dipole moments are cylindrically symmetrical about a common axis c fixed in the protein; 3) protein motions are negligible during the excited-state decay; 4) the distribution of c is cylindrically symmetrical about the director of the experimental sample. In a muscle fiber, the director is the fiber axis, F. All of the information on the orientational order of the probe that is available from measurements of linearly polarized fluorescence is contained in five independent polarized fluorescence intensities measured with excitation and emission polarizers parallel or perpendicular to F and with the propagation axis of the detected fluorescence parallel or perpendicular to that of the excitation. The analysis then yields the average second-rank and fourth-rank order parameters ( and ) of the angular distribution of c relative to F, and and , the average second-rank order parameters of the angular distribution for wobble of the absorption and emission transition dipole moments relative to c. The method can also be applied to other cylindrically ordered systems such as oriented lipid bilayer membranes and to processes slower than fluorescence that may be observed using longer-lived optically excited states. (+info)
The vacuolating toxin from Helicobacter pylori forms hexameric pores in lipid bilayers at low pH.
(11/7162)
Pathogenic strains of Helicobacter pylori secrete a cytotoxin, VacA, that in the presence of weak bases, causes osmotic swelling of acidic intracellular compartments enriched in markers for late endosomes and lysosomes. The molecular mechanisms by which VacA causes this vacuolation remain largely unknown. At neutral pH, VacA is predominantly a water-soluble dodecamer formed by two apposing hexamers. In this report, we show by using atomic force microscopy that below pH approximately 5, VacA associates with anionic lipid bilayers to form hexameric membrane-associated complexes. We propose that water-soluble dodecameric VacA proteins disassemble at low pH and reassemble into membrane-spanning hexamers. The surface contour of the membrane-bound hexamer is strikingly similar to the outer surface of the soluble dodecamer, suggesting that the VacA surface in contact with the membrane is buried within the dodecamer before protonation. In addition, electrophysiological measurements indicate that, under the conditions determined by atomic force microscopy for membrane association, VacA forms pores across planar lipid bilayers. This low pH-triggered pore formation is likely a critical step in VacA activity. (+info)
Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors.
(12/7162)
COPI-coated vesicle budding from lipid bilayers whose composition resembles mammalian Golgi membranes requires coatomer, ARF, GTP, and cytoplasmic tails of putative cargo receptors (p24 family proteins) or membrane cargo proteins (containing the KKXX retrieval signal) emanating from the bilayer surface. Liposome-derived COPI-coated vesicles are similar to their native counterparts with respect to diameter, buoyant density, morphology, and the requirement for an elevated temperature for budding. These results suggest that a bivalent interaction of coatomer with membrane-bound ARF[GTP] and with the cytoplasmic tails of cargo or putative cargo receptors is the molecular basis of COPI coat assembly and provide a simple mechanism to couple uptake of cargo to transport vesicle formation. (+info)
A single conductance pore for chloride ions formed by two cystic fibrosis transmembrane conductance regulator molecules.
(13/7162)
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase (PKA)- and ATP-regulated chloride channel, whose gating process involves intra- or intermolecular interactions among the cytosolic domains of the CFTR protein. Tandem linkage of two CFTR molecules produces a functional chloride channel with properties that are similar to those of the native CFTR channel, including trafficking to the plasma membrane, ATP- and PKA-dependent gating, and a unitary conductance of 8 picosiemens (pS). A heterodimer, consisting of a wild type and a mutant CFTR, also forms an 8-pS chloride channel with mixed gating properties of the wild type and mutant CFTR channels. The data suggest that two CFTR molecules interact together to form a single conductance pore for chloride ions. (+info)
Properties of a new calcium-permeable single channel from tracheal microsomes.
(14/7162)
After the incorporation of the tracheal microsomal membrane into bilayer lipid membrane (BLM), a new single channel permeable for calcium was observed. Using the BLM conditions, 53 mM Ca2+ in trans solution versus 200 nM Ca2+ in cis solution, the single calcium channel current at 0 mV was 1.4-2.1 pA and conductance was 62-75 pS. The channel Ca2+/K+ permeability ratio was 4.8. The open probability (P-open) was in the range of 0.7-0.97. The P-open, measured at -10 mV to +30 mV (trans-cis), was not voltage dependent. The channel was neither inhibited by 10-20 microM ruthenium red, a specific blocker of ryanodine calcium release channel, nor by 10-50 microM heparin, a specific blocker of IP3 receptor calcium release channel, and its activity was not influenced by addition of 0.1 mM MgATP. We suggest that the observed new channel is permeable for calcium, and it is neither identical with the known type 1 or 2 ryanodine calcium release channel, nor type 1 or 2 IP3 receptor calcium release channel. (+info)
Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers.
(15/7162)
In any lipid bilayer membrane, there is an upper limit on the cholesterol concentration that can be accommodated within the bilayer structure; excess cholesterol will precipitate as crystals of pure cholesterol monohydrate. This cholesterol solubility limit is a well-defined quantity. It is a first-order phase boundary in the phospholipid/cholesterol phase diagram. There are many different solubility limits in the literature, but no clear picture has emerged that can unify the disparate results. We have studied the effects that different sample preparation methods can have on the apparent experimental solubility limit. We find that artifactual demixing of cholesterol can occur during conventional sample preparation and that this demixed cholesterol may produce artifactual cholesterol crystals. Therefore, phospholipid/cholesterol suspensions which are prepared by conventional methods may manifest variable, falsely low cholesterol solubility limits. We have developed two novel preparative methods which are specifically designed to prevent demixing during sample preparation. For detection of the cholesterol crystals, X-ray diffraction has proven to be quantitative and highly sensitive. Experiments based on these methods yield reproducible and precise cholesterol solubility limits: 66 mol% for phosphatidylcholine (PC) bilayers and 51 mol% for phosphatidylethanolamine (PE) bilayers. We present evidence that these are true, equilibrium values. In contrast to the dramatic headgroup effect (PC vs. PE), acyl chain variations had no effect on the cholesterol solubility limit in four different PC/cholesterol mixtures. (+info)
Hydrocarbon chain packing and the effect of ethanol on the thermotropic phase behavior of mixed-chain phosphatidylglycerols.
(16/7162)
Previous studies in this laboratory have delineated the relationship between the acyl chain asymmetry of mixed-chain phosphatidylcholines and the effect of ethanol concentration ([EtOH]) on their melting behavior (Li et al., Biophys J., 70 (1996) 2784-2794). This present investigation extends these findings to another phospholipid family by using high-resolution differential scanning calorimetry (DSC) to characterize the effect of ethanol concentration on the main phase transition temperature (Tm) of five molecular species of mixed-chain phosphatidylglycerol (PG). For C(14):C(18)PG, C(15):C(17)PG, C(16):C(16)PG, and C(17):C(15)PG, a biphasic profile in the Tm versus [EtOH] plot was observed, and the minimum in the plot for each PG occurred at 33, 15, 19, and 36 mg/ml, respectively. This biphasic behavior is typical of phospholipids whose acyl chain asymmetry is fairly small. For C(18):C(14)PG, only a linear decrease in the Tm was observed as a function of ethanol concentration; this effect is characteristic of highly asymmetric phospholipids. Our DSC results obtained with mixed-chain PG in the presence of ethanol demonstrate that the acyl chain asymmetry of the five lipids studied can be ranked as follows: C(15):C(17)PG+info)