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.

AUTOSOMAL MODIFIERS OF THE BOBBED PHENOTYPE ARE A MAJOR COMPONENT OF THE rDNA MAGNIFICATION PARADOX IN DROSOPHILA MELANOGASTER

Genetics ◽  
1986 ◽  
Vol 113 (2) ◽  
pp. 305-319
Author(s):  
Craig H Marcus ◽  
Anne E Zitron ◽  
David A Wright ◽  
R Scott Hawley
Keyword(s):  
Drosophila Melanogaster ◽  
Epistatic Effect ◽  
The Other ◽  
Wild Type ◽  
The Stability ◽  
Kinetics Of

ABSTRACT rDNA magnification in Drosophila melanogaster is defined experimentally as the ability of bb/Ybb  - males to produce exceptional progeny that are wild type with respect to rDNA associated phenotypes. Here, we show that some of these bobbed-plus progeny result not from genetic reversion at the bb locus but rather from variants at two or more autosomal loci that ameliorate the bobbed phenotype of rDNA deficient males in Drosophila. In doing so we resolve several aspects of a long-standing paradox concerning the phenomenon of rDNA magnification. This problem arose from the use of two genetic assays, which were presumed to be identical, but paradoxically, produced conflicting data on both the kinetics of reversion and the stability of magnified bb  + chromosomes. We resolve this problem by demonstrating that in one assay bobbed-plus progeny arise primarily by genetic reversion at the bobbed locus, whereas in the other assay bobbed-plus progeny arise both by reversion and by an epistatic effect of autosomal modifiers on the bobbed phenotype. We further show that such modifiers can facilitate the appearance of phenotypically bobbed-plus progeny even under conditions where genetic reversion is blocked by magnification defective mutants. Finally, we present a speculative model relating the action of these modifiers to the large increases in rDNA content observed in males undergoing magnification.

scholarly journals Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae.

Genetics ◽  
1989 ◽  
Vol 122 (3) ◽  
pp. 519-534 ◽  
Author(s):  
N Rudin ◽  
E Sugarman ◽  
J E Haber
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
The Other ◽  
Crossing Over ◽  
Physical Analysis ◽  
Wild Type ◽  
Inverted Orientation ◽  
Mating Type Switching ◽  
A Site ◽  
Kinetics Of

Abstract We have investigated HO endonuclease-induced double-strand break (DSB) recombination and repair in a LACZ duplication plasmid in yeast. A 117-bp MATa fragment, embedded in one copy of LACZ, served as a site for initiation of a DSB when HO endonuclease was expressed. The DSB could be repaired using wild-type sequences located on a second, promoterless, copy of LACZ on the same plasmid. In contrast to normal mating-type switching, crossing-over associated with gene conversion occurred at least 50% of the time. The proportion of conversion events accompanied by exchange was greater when the two copies of LACZ were in direct orientation (80%), than when inverted (50%). In addition, the fraction of plasmids lost was significantly greater in the inverted orientation. The kinetics of appearance of intermediates and final products were also monitored. The repair of the DSB is slow, requiring at least an hour from the detection of the HO-cut fragments to completion of repair. Surprisingly, the appearance of the two reciprocal products of crossing over did not occur with the same kinetics. For example, when the two LACZ sequences were in the direct orientation, the HO-induced formation of a large circular deletion product was not accompanied by the appearance of a small circular reciprocal product. We suggest that these differences may reflect two kinetically separable processes, one involving only one cut end and the other resulting from the concerted participation of both ends of the DSB.

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 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.

Surface potential determination in planar lipid bilayers: A simplification of the conductance-ratio method

2007 ◽  
Vol 70 (3) ◽  
pp. 515-518 ◽  
Author(s):  
Fernando Abdulkader ◽  
Manoel Arcisio-Miranda ◽  
Rui Curi ◽  
Joaquim Procopio
Keyword(s):  
Lipid Bilayers ◽  
Surface Potential ◽  
Ratio Method ◽  
Planar Lipid Bilayers ◽  
Conductance Ratio
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