scholarly journals A conserved tryptophan in pneumolysin is a determinant of the characteristics of channels formed by pneumolysin in cells and planar lipid bilayers

1998 ◽  
Vol 329 (3) ◽  
pp. 571-577 ◽  
Author(s):  
E. Yuri KORCHEV ◽  
C. Lindsay BASHFORD ◽  
Cecilia PEDERZOLLI ◽  
A. Charles PASTERNAK ◽  
J. Peter MORGAN ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Membrane ◽  
Molecular Mass ◽  
Lipid Bilayers ◽  
Planar Lipid Bilayers ◽  
Wild Type ◽  
Bivalent Cations ◽  
Ring Structures ◽  
Ion Conducting ◽  
Small Channels

Pneumolysin is one of the family of thiol-activatable, cytolytic toxins. Within these toxins the amino acid sequence Trp-Glu-Trp-Trp is conserved. Mutations made in this region of pneumolysin, residues 433-436 inclusive, did not affect cell binding or the formation of toxin oligomers in the target cell membrane. However, the mutations did affect haemolysis, leakage of low-molecular-mass metabolites from Lettre cells and the induction of conductance channels across planar lipid bilayers. Of eight modified pneumolysins examined, Trp-433 → Phe showed the smallest amount of haemolysis or leakage (less than 5% of wild type). Pneumolysin-induced leakage from Lettre cells was sensitive to inhibition by bivalent cations but the extent of inhibition varied depending on the modification. Leakage by the mutant Trp-433 → Phe was least sensitive to cation inhibition. The ion-conducting channels formed across planar lipid bilayers exhibit small (less than 30 pS), medium (30 pS-1 nS) and large (more than 1 nS) conductance steps. Small- and medium-sized channels were preferentially closed by bivalent cations. In contrast with wild-type toxin, which formed predominantly small channels, the modified toxin Trp-433 → Phe formed large channels that were insensitive to cation-induced closure. Polysaccharides of molecular mass more than 15 kDa inhibited haemolysis by wild-type toxin, but polysaccharide of up to 40 kDa did not prevent haemolysis by Trp-433 → Phe. Electron microscopy revealed that Trp-433 → Phe formed oligomeric arc and ring structures with dimensions identical with those of wild-type toxin, and that the ratio of arcs to rings formed was the same for wild-type toxin and the Trp-433 → Phe variant. We conclude that the change Trp-433 → Phe affects channel formation at a point subsequent to binding to the cell membrane and the formation of oligomers, and that the size of arc and ring structures revealed by electron microscopy does not reflect the functional state of the channels.

scholarly journals Annexins V and Xii Alter the Properties of Planar Lipid Bilayers Seen by Conductance Probes

2000 ◽  
Vol 115 (5) ◽  
pp. 571-582 ◽  
Author(s):  
Yuri Sokolov ◽  
William S. Mailliard ◽  
Nghia Tranngo ◽  
Mario Isas ◽  
Hartmut Luecke ◽  
...  
Keyword(s):  
Small Molecules ◽  
Lipid Bilayers ◽  
Functional Properties ◽  
Surface Potential ◽  
Annexin V ◽  
The Other ◽  
Planar Lipid Bilayers ◽  
Wild Type ◽  
Multiple Functions ◽  
Kinetics Of

Annexins are proteins that bind lipids in the presence of calcium. Though multiple functions have been proposed for annexins, there is no general agreement on what annexins do or how they do it. We have used the well-studied conductance probes nonactin, alamethicin, and tetraphenylborate to investigate how annexins alter the functional properties of planar lipid bilayers. We found that annexin XII reduces the nonactin-induced conductance to ∼30% of its original value. Both negative lipid and ∼30 μM Ca2+ are required for the conductance reduction. The mutant annexin XIIs, E105K and E105K/K68A, do not reduce the nonactin conductance even though both bind to the membrane just as wild-type does. Thus, subtle changes in the interaction of annexins with the membrane seem to be important. Annexin V also reduces nonactin conductance in nearly the same manner as annexin XII. Pronase in the absence of annexin had no effect on the nonactin conductance. But when added to the side of the bilayer opposite that to which annexin was added, pronase increased the nonactin-induced conductance toward its pre-annexin value. Annexins also dramatically alter the conductance induced by a radically different probe, alamethicin. When added to the same side of the bilayer as alamethicin, annexin has virtually no effect, but when added trans to the alamethicin, annexin dramatically reduces the asymmetry of the I-V curve and greatly slows the kinetics of one branch of the curve without altering those of the other. Annexin also reduces the rate at which the hydrophobic anion, tetraphenylborate, crosses the bilayer. These results suggest that annexin greatly reduces the ability of small molecules to cross the membrane without altering the surface potential and that at least some fraction of the active annexin is accessible to pronase digestion from the opposite side of the membrane.

Heat shock proteins induce pores in membranes

1990 ◽  
Vol 10 (6) ◽  
pp. 509-518 ◽  
Author(s):  
G. M. Alder ◽  
B. M. Austen ◽  
C. L. Bashford ◽  
A. Mehlert ◽  
C. A. Pasternak
Keyword(s):  
Heat Shock ◽  
Human Serum Albumin ◽  
Lipid Bilayers ◽  
Diphtheria Toxin ◽  
Planar Lipid Bilayers ◽  
Bacterial Protein ◽  
Hsp 70 ◽  
Ion Conducting ◽  
Shock Proteins ◽  
Human Heat Shock Protein

Human heat shock protein (hsp) 70 and bacterial protein groEL promote leakage of calcein from liposomes induced by human serum albumin signal peptide, by S. aureus α toxin or by diphtheria toxin. Hsp 70 and groEL, as well as two mycobacterial homologues hsp 71 and hsp 65, induce ion conducting pores across planar lipid bilayers at low or neutral pH. It is concluded that hsp induce pores in membranes and that this may contribute to their action within cells.

scholarly journals Halothane modulation of skeletal muscle ryanodine receptors: dependence on Ca2+, Mg2+, and ATP

2008 ◽  
Vol 294 (4) ◽  
pp. C1103-C1112 ◽  
Author(s):  
Paula L. Diaz-Sylvester ◽  
Maura Porta ◽  
Julio A. Copello
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Genetic Disorder ◽  
Ryanodine Receptors ◽  
Volatile Anesthetic ◽  
Planar Lipid Bilayers ◽  
Wild Type ◽  
Highly Sensitive

Malignant hyperthermia (MH) susceptibility is a genetic disorder of skeletal muscle associated with mutations in the ryanodine receptor isoform 1 (RyR1) of sarcoplasmic reticulum (SR). In MH-susceptible skeletal fibers, RyR1-mediated Ca2+ release is highly sensitive to activation by the volatile anesthetic halothane. Indeed, studies with isolated RyR1 channels (using simple Cs+ solutions) found that halothane selectively affects mutated but not wild-type RyR1 function. However, studies in skeletal fibers indicate that halothane can also activate wild-type RyR1-mediated Ca2+ release. We hypothesized that endogenous RyR1 agonists (ATP, lumenal Ca2+) may increase RyR1 sensitivity to halothane. Consequently, we studied how these agonists affect halothane action on rabbit skeletal RyR1 reconstituted into planar lipid bilayers. We found that cytosolic ATP is required for halothane-induced activation of the skeletal RyR1. Unlike RyR1, cardiac RyR2 (much less sensitive to ATP) responded to halothane even in the absence of this agonist. ATP-dependent halothane activation of RyR1 was enhanced by cytosolic Ca2+ (channel agonist) and counteracted by Mg2+ (channel inhibitor). Dantrolene, a muscle relaxant used to treat MH episodes, did not affect RyR1 or RyR2 basal activity and did not interfere with halothane-induced activation. Studies with skeletal SR microsomes confirmed that halothane-induced RyR1-mediated SR Ca2+ release is enhanced by high ATP-low Mg2+ in the cytosol and by increased SR Ca2+ load. Thus, physiological or pathological processes that induce changes in cellular levels of these modulators could affect RyR1 sensitivity to halothane in skeletal fibers, including the outcome of halothane-induced contracture tests used to diagnose MH susceptibility.

scholarly journals Mechanism of calsequestrin regulation of single cardiac ryanodine receptor in normal and pathological conditions

2013 ◽  
Vol 142 (2) ◽  
pp. 127-136 ◽  
Author(s):  
Haiyan Chen ◽  
Giorgia Valle ◽  
Sandra Furlan ◽  
Alma Nani ◽  
Sandor Gyorke ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Contractile Function ◽  
Sudden Cardiac Arrest ◽  
Polymorphic Ventricular Tachycardia ◽  
Planar Lipid Bilayers ◽  
Wild Type ◽  
Model Studies ◽  
Pathological Conditions ◽  
Ryr2 Channel

Release of Ca2+ from the sarcoplasmic reticulum (SR) drives contractile function of cardiac myocytes. Luminal Ca2+ regulation of SR Ca2+ release is fundamental not only in physiology but also in physiopathology because abnormal luminal Ca2+ regulation is known to lead to arrhythmias, catecholaminergic polymorphic ventricular tachycardia (CPVT), and/or sudden cardiac arrest, as inferred from animal model studies. Luminal Ca2+ regulates ryanodine receptor (RyR)2-mediated SR Ca2+ release through mechanisms localized inside the SR; one of these involves luminal Ca2+ interacting with calsequestrin (CASQ), triadin, and/or junctin to regulate RyR2 function. CASQ2-RyR2 regulation was examined at the single RyR2 channel level. Single RyR2s were incorporated into planar lipid bilayers by the fusion of native SR vesicles isolated from either wild-type (WT), CASQ2 knockout (KO), or R33Q-CASQ2 knock-in (KI) mice. KO and KI mice have CPVT-like phenotypes. We show that CASQ2(WT) action on RyR2 function (either activation or inhibition) was strongly influenced by the presence of cytosolic MgATP. Function of the reconstituted CASQ2(WT)–RyR2 complex was unaffected by changes in luminal free [Ca2+] (from 0.1 to 1 mM). The inhibition exerted by CASQ2(WT) association with the RyR2 determined a reduction in cytosolic Ca2+ activation sensitivity. RyR2s from KO mice were significantly more sensitive to cytosolic Ca2+ activation and had significantly longer mean open times than RyR2s from WT mice. Sensitivity of RyR2s from KI mice was in between that of RyR2 channels from KO and WT mice. Enhanced cytosolic RyR2 Ca2+ sensitivity and longer RyR2 open times likely explain the CPVT-like phenotype of both KO and KI mice.

scholarly journals The Amino-Terminal Region of Vpr from Human Immunodeficiency Virus Type 1 Forms Ion Channels and Kills Neurons

1999 ◽  
Vol 73 (5) ◽  
pp. 4230-4238 ◽  
Author(s):  
S. C. Piller ◽  
G. D. Ewart ◽  
D. A. Jans ◽  
P. W. Gage ◽  
G. B. Cox
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Cell Death ◽  
Ion Channels ◽  
Ion Channel ◽  
Lipid Bilayers ◽  
Planar Lipid Bilayers ◽  
Wild Type ◽  
Immunodeficiency Virus

ABSTRACT We have previously reported that the accessory protein Vpr from human immunodeficiency virus type 1 forms cation-selective ion channels in planar lipid bilayers and is able to depolarize intact cultured neurons by causing an inward sodium current, resulting in cell death. In this study, we used site-directed mutagenesis and synthetic peptides to identify the structural regions responsible for the above functions. Mutations in the N-terminal region of Vpr were found to affect channel activity, whereas this activity was not affected by mutations in the hydrophobic region of Vpr (amino acids 53 to 71). Analysis of mutants containing changes in the basic C terminus confirmed previous results that this region, although not necessary for ion channel function, was responsible for the observed rectification of wild-type Vpr currents. A peptide comprising the first 40 N-terminal amino acids of Vpr (N40) was found to be sufficient to form ion channels similar to those caused by wild-type Vpr in planar lipid bilayers. Furthermore, N40 was able to cause depolarization of the plasmalemma and cell death in cultured hippocampal neurons with a time course similar to that seen with wild-type Vpr, supporting the idea that this region is responsible for Vpr ion channel function and cytotoxic effects. Since Vpr is found in the serum and cerebrospinal fluids of AIDS patients, these results may have significance for AIDS pathology.

scholarly journals The high-affinity calcium–calmodulin-binding site does not play a role in the modulation of type 1 inositol 1,4,5-trisphosphate receptor function by calcium and calmodulin

2002 ◽  
Vol 365 (3) ◽  
pp. 659-667 ◽  
Author(s):  
Elena NOSYREVA ◽  
Tomoya MIYAKAWA ◽  
Zhengnan WANG ◽  
Lyuba GLOUCHANKOVA ◽  
Akiko MIZUSHIMA ◽  
...  
Keyword(s):  
Binding Site ◽  
Lipid Bilayers ◽  
Expression System ◽  
Planar Lipid Bilayers ◽  
Wild Type ◽  
Direct Role ◽  
Mammalian Expression ◽  
High Affinity ◽  
Inositol 1,4,5 Trisphosphate

Modulation of the inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3R) by cytosolic calcium (Ca2+) plays an essential role in Ca2+ signalling, but structural determinants and mechanisms responsible for the InsP3R regulation by Ca2+ are poorly understood. In the present study, we expressed rat InsP3R type 1 (InsP3R1) in Spodoptera frugiperda cells using a baculovirus-expression system and reconstituted the recombinant InsP3R1 into planar lipid bilayers for functional analysis. We observed only minor effects of 0.5mM of calmodulin (CaM) antagonist W-7 on the Ca2+ dependence of InsP3R1. Based on a previous analysis of mouse InsP3R1 [Yamada, Miyawaki, Saito, Nakajima, Yamamoto-Hino, Ryo, Furuichi and Mikoshiba (1995) Biochem J. 308, 83–88], we generated the Trp1577→Ala (W1577A) mutant of rat InsP3R1 which lacks the high-affinity Ca2+—CaM-binding site. We found that the W1577A mutant displayed a bell-shaped Ca2+ dependence similar to the wild-type InsP3R1 in planar lipid bilayers. Activation of B cell receptors resulted in identical Ca2+ signals in intact DT40 cells lacking the endogenous InsP3R and transfected with the wild-type InsP3R1 or the W1577A mutant cDNA subcloned into a mammalian expression vector. In the planar lipid bilayer experiments, we showed that both wild-type InsP3R1 and W1577A mutant were equally sensitive to inhibition by exogenous CaM. From these results, we concluded that the interaction of CaM with the high-affinity Ca2+—CaM-binding site in the coupling domain of the InsP3R1 does not play a direct role in biphasic modulation of InsP3R1 by cytosolic Ca2+ or in InsP3R1 inhibition by CaM.

scholarly journals Expression of a Gene for a Porin-Like Protein of the OmpA Family from Mycobacterium tuberculosisH37Rv

1998 ◽  
Vol 180 (14) ◽  
pp. 3541-3547 ◽  
Author(s):  
Ryan H. Senaratne ◽  
Hamid Mobasheri ◽  
K. G. Papavinasasundaram ◽  
Peter Jenner ◽  
Edward J. A. Lea ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Specific Activity ◽  
Signal Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rabbit Antiserum ◽  
Intact Protein ◽  
Planar Lipid Bilayers

ABSTRACT An open reading frame in the genomic database ofMycobacterium tuberculosis H37Rv was identified as having homology with an outer membrane protein. We found that the gene specified a protein belonging to the OmpA family, which includes some porins of gram-negative organisms. The gene was amplified by PCR and cloned into Escherichia coli. Overexpression of the gene was toxic to the host, but limited amounts could be purified from cells before growth ceased. A truncated gene devoid of the code for a presumed signal sequence was well expressed, but the protein had no pore-forming activity in the liposome swelling assay. However, the intact protein, OmpATb, behaved as a porin of low specific activity, with a pore diameter of 1.4 to 1.8 nm, and was also active in planar lipid bilayers, showing a single-channel conductance of 700 pS. The protein had a molecular mass of about 38 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal rabbit antiserum raised to the truncated protein recognized a protein of similar molecular mass in detergent extracts of broken M. tuberculosis cells. Reverse transcription-PCR confirmed that the gene for OmpATb was expressed in M. tuberculosis cells growing in culture. Comparison of the purified protein with that in the detergent-extracted preparation using liposomes and planar lipid bilayers showed that the two materials had similar pore-forming properties. OmpATb is different from either of the mycobacterial porins described so far. This is the first report of a porin-like molecule from M. tuberculosis; the porin is likely to be important in controlling the access of hydrophilic molecules to the bacterial cell.

Single-channel characteristics of a purified bovine renal amiloride-sensitive Na+ channel in planar lipid bilayers

1993 ◽  
Vol 264 (6) ◽  
pp. C1489-C1499 ◽  
Author(s):  
Y. Oh ◽  
D. J. Benos
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Na Channel ◽  
Planar Lipid Bilayers ◽  
Dependent Manner ◽  
Na Channels ◽  
Channel Characteristics ◽  
Large Channel ◽  
Small Channels ◽  
Small Conductance

We have purified an amiloride-inhibitable Na+ channel protein from bovine renal papillae using ion-exchange and immunoaffinity chromatography. In the present study, these purified Na+ channels were reconstituted into planar lipid bilayers, and their single-channel characteristics were studied. We observed both large- and small-conductance Na(+)-selective ion channels in planar lipid bilayers. Single-channel conductance for the large- and small-conductance channels saturated as a function of Na+ concentration. These relations could be fitted by a simple Langmuir isotherm with a Michaelis constant of 55 and 45 mM and a maximum open-state conductance of 56 or 8.4 pS, respectively. Both channels were perfectly cation selective, with a Na(+)-to-K+ permeability ratio of 6.7:1 for the large channel and 7.8:1 for the small channel, and their open single-channel current-voltage relations were linear when bathed with symmetrical Na+ solutions. The percent open time of the reconstituted large or small channels varied between 10 and 50% or 1 and 20%, respectively. After application of amiloride, both the large- and small-conductance Na+ channels were inhibited in a dose-dependent manner.

High-voltage electron microscopy of patch-clamped membranes

1987 ◽  
Vol 45 ◽  
pp. 582-583
Author(s):  
F. Sachs ◽  
M. J. Song
Keyword(s):  
Electron Microscopy ◽  
Cell Membrane ◽  
Patch Clamp ◽  
High Voltage Electron Microscopy ◽  
Small Patch ◽  
Cellular Electrophysiology ◽  
Voltage Electron ◽  
Electron Microscopes ◽  
Patch Technique ◽  
Membrane Patch

Cellular electrophysiology has been revolutionized by the introduction of patch clamp techniques. The patch clamp records current from a small patch of the cell membrane which has been sucked into a glass pipette. The membrane patch, a few micons in diameter, is attached to the glass by a seal which is electrically, diffusionally and mechanically tight. Because of the tight electrical seal, the noise level is low enough to record the activity of single ion channels over a time scale extending from 10μs to days. However, although the patch technique is over ten years old, the patch structure is unknown. The patch is inside a glass pipette where it has been impossible to see with standard electron microscopes. We show here that at 1 Mev the glass pipette is transparent and the membrane within can be seen with a resolution of about 30 A.

Normal Synaptonemal Complex and Abnormal Recombination Nodules in Two Alleles of the Drosophila Meiotic Mutant mei-W68

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1337-1356 ◽  
Author(s):  
Adelaide T C Carpenter
Keyword(s):  
Electron Microscopy ◽  
Synaptonemal Complex ◽  
High Frequency ◽  
Serial Section ◽  
The Other ◽  
Wild Type ◽  
Recombination Nodules ◽  
Section Electron ◽  
Serial Section Electron Microscopy ◽  
Section Electron Microscopy

Abstract The meiotic phenotypes of two mutant alleles of the mei-W68 gene, 1 and L1, were studied by genetics and by serial-section electron microscopy. Despite no or reduced exchange, both mutant alleles have normal synaptonemal complex. However, neither has any early recombination nodules; instead, both exhibit high numbers of very long (up to 2 μm) structures here named “noodles.” These are hypothesized to be formed by the unchecked extension of identical but much shorter structures ephemerally seen in wild type, which may be precursors of early recombination nodules. Although the mei-W68L1 allele is identical to the mei-W681 allele in both the absence of early recombination nodules and a high frequency of noodles (i.e., it is amorphic for the noodle phene), it is hypomorphic in its effects on exchange and late recombination nodules. The differential effects of this allele on early and late recombination nodules are consistent with the hypothesis that Drosophila females have two separate recombination pathways—one for simple gene conversion, the other for exchange.

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