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G Boheim

Publications and source records attributed to G Boheim.

At least 19 recordsLinked to original sources
PubMed1997

Modulation of the ER Ca2+ channel BCC1 from tendrils of Bryonia dioica by divalent cations, protons and H2O2.

Electrical properties of the ER Ca2+ channel BCC1 from tendrils of Bryonia dioica were analyzed after incorporation of BCC1 into black lipid bilayers. Single channel current fluctuations were modulated by divalent cations, protons and H2O2. Whereas the channel is permeable for Ca2+, Ba2+ and Sr2+, its conductance is strongly reduced in solutions containing MgCl2. Cu2+ and Zn2+ are potent inhibitors of BCC1 in micromolar concentrations. The open channel conductance of BCC1 increases with acidification of the electrolyte solution. H2O2 shows strong inhibitory effects on BCC1. The channel is almost completely closed at submillimolar concentrations of H2O2. The effects of pH and H2O2 on channel properties are directional and affect BCC1 at the Ca exit side, but not on the entry site. Thus, cytosolic pH and H2O2 levels may play an important role in the modulation of the cytoplasmic free calcium concentration through BCC1.

Calcium↗
PubMed1995

Gadolinium-sensitive, voltage-dependent calcium release channels in the endoplasmic reticulum of a higher plant mechanoreceptor organ.

The lipid bilayer technique was adapted to the functional reconstitution of ion channels from the endoplasmic reticulum of a higher plant. This was obtained at high purity from touch-sensitive tendrils of Bryonia dioica. In this preparation, a calcium-selective strongly rectifying channel is prevailing whose single-channel properties have been characterized. The single-channel conductance is 29 pS in 50 mM CaCl2. The Ca2+: K+ selectivity was determined to be approximately 6.6. The channel is voltage-gated and, more importantly, the gating voltage is strongly shifted towards more negative voltages when a transmembrane Ca2+ gradient is applied. Thus, at physiological voltages across the endoplasmic reticulum membrane, the channel's open probability will be governed largely by the chemical potential gradient of Ca2+, generated by the Ca(2+)-ATPase in that same membrane. The calcium release channel described here is effectively blocked by Gd3+ which also completely suppresses a tendril's reaction to touch, suggesting that this channel could be a key element of calcium signaling in higher plant mechanotransduction. Its molecular characteristics and inhibitor data show it to be the first known member of a hitherto unrecognized class of calcium channels.

Calcium↗
PubMed1994

Effects of polycations on ion channels formed by neutral and negatively charged alamethicins.

The effects of the peptide polycations salmon protamine (M(r) = 4332, z = +21) and poly-L-lysine (M(r) approximately equal to 100,000, z approximately equal to +775) on ion channels formed by synthetic alamethicin Alm-F30 (one negative charge), natural Alm-F50 (neutral) and phosphorylated Alm-F50 (two negative charges) reconstituted in planar lipid bilayers have been studied at the single channel level. It was observed that both polycations in micromolar concentrations transiently block ion permeation through the channels formed by each alamethicin analogue, although in case of the neutral Alm-F50 to a significantly lesser extent. Poly-L-lysine showed to be more effective than protamine in blocking these channels. If either polycation is present in the cis-compartment, blockade occurs only at cis positive membrane voltages. At constant polycation concentration, dwell times in the blocked state increase when salt concentration is lowered, and decrease at acidic pH with an apparent pK of 4.8. Mean lifetime of blockade events shortens when membrane voltage is increased, which suggests that both polycations may permeate through the oligomeric alamethicin channels if conductance levels are > 2. We suggest that blockade is caused by electrostatic binding of a single polycation molecule to the C-terminal channel mouth; in case of Alm-F30, Glu18 has to be considered as the putative binding site. Our results provide further evidence for the barrel-stave model and a parallel orientation of dipole monomers in the channel aggregate, the C-termini facing the membrane side with the more positive membrane potential.

Action Potentials↗
PubMed1994

Overexpression of algE in Escherichia coli: subcellular localization, purification, and ion channel properties.

Alginate-producing (mucoid) strains of Pseudomonas aeruginosa possess a 54-kDa outer membrane (OM) protein (AlgE) which is missing in nonmucoid bacteria. The coding region of the algE gene from mucoid P. aeruginosa CF3/M1 was subcloned in the expression vector pT7-7 and expressed in Escherichia coli. The level of expression of recombinant AlgE was seven times higher than that of the native protein in P. aeruginosa. Recombinant AlgE was found mainly in the OM. A putative precursor protein (56 kDa) of AlgE could be immunologically detected in the cytoplasmic membrane (CM). Surface exposition of AlgE in the OM of E. coli was indicated by labeling lysine residues with N-hydroxysuccinimide-biotin. Secondary-structure analysis suggested that AlgE is anchored in the OM by 18 membrane-spanning beta-strands, probably forming a beta-barrel. Recombinant AlgE was purified, and isoelectric focusing revealed a pI of 4.4. Recombinant AlgE was spontaneously incorporated into planar lipid bilayers, forming ion channels with a single-channel conductance of 0.76 nS in 1 M KCl and a mean lifetime of 0.7 ms. Single-channel current measurements in the presence of other salts as well as reversal potential measurements in salt gradients revealed that the AlgE channel was strongly anion selective. For chloride ions, a weak binding constant (Km = 0.75 M) was calculated, suggesting that AlgE might constitute an ion channel specific for another particular anion, e.g., polymannuronic acid, which is a precursor of alginate. Consistent with this idea, the open-state probability of the channel decreased when GDP-mannuronic acid was added. The AlgE channel was inactivated when membrane voltages higher than +85 mV were applied. The electrophysiological characteristics of AlgE, including its rectifying properties, are quite different from those of typical porins.

Amino Acid Sequence↗
PubMed1993

Structural fluctuations between two conformational states of a transmembrane helical peptide are related to its channel-forming properties in planar lipid membranes.

Putative transmembrane helices of membrane proteins in general and channel proteins in particular often contain proline residues which may induce a bend into an otherwise regular helical structure. Here we show by fluorescence-energy-transfer measurements and molecular-dynamics calculations that, in the case of synthetic bilayer-spanning helical polypeptides, a proline-induced bend in a helix acts as a flexible element mediating rigid body motions of the helical segments. Most important, such structural fluctuations in the transmembrane helices seem to play a functional role in the formation of ionic channels in planar lipid bilayers and biological membranes.

Amino Acid Sequence↗
PubMed1991

Contribution of proline-14 to the structure and actions of melittin.

The structure and dynamic properties of bee venom melittin and a synthetic analogue, [Ala14]-melittin (melittin P14A), are compared, using high resolution 1H nuclear magnetic resonance (NMR) spectroscopy and amide exchange measurements in methanol. P14A is shown to adopt a regular, stable alpha-helical conformation in solution without the flexibility around the Pro-14 residue found in melittin. P14A has twice the hemolytic activity of melittin but is less able to induce voltage-dependent ion conductance in planar bilayers. The results indicate that helix flexibility afforded by the Pro-14 residue promotes the ability of melittin to adopt the transbilayer associates thought to underlie ion translocation.

Alanine↗
PubMed1990

Molecular basis of altered excitability in Shaker mutants of Drosophila melanogaster.

Mutations in the Shaker (Sh) locus of Drosophila melanogaster have differing effects on action potential duration and repolarization in neurons as well as on A-type K+ channels (IA) in muscle. The molecular basis of three exemplary Sh alleles (ShKS133, ShE62 and Sh5) has been identified. They are point mutations in the Sh transcription unit expressing aberrant voltage-gated A-type K+ channels. Replicas of each mutation have been introduced by in vitro mutagenesis into Sh cDNA. The expression of in vitro transcribed mutant Sh cRNA in Xenopus laevis oocytes reproduced the specific phenotypic traits of each Sh allele. The lack of IA in ShKS133 is due to a missense mutation within a sequence motif occurring in all hitherto characterized voltage-gated K+ channel forming proteins. The reduction of IA in ShE62 is due to a mutation in an AG acceptor site. The intervening sequence between exons 19 and 20 is not spliced in ShE62 RNA. As a consequence, ShE62 flies do not contain the full complement of Sh K+ forming proteins. Finally, the Sh5 mutation leads to an altered voltage dependence of K+ channel activation and inactivation as well as to an accelerated rate of recovery from inactivation. This is due to a missense mutation altering the amino acid sequence of the proposed transmembrane segment S5 of the Sh K+ channels. Segment S5 is located adjacently to the presumed voltage sensor of voltage-gated ion channels. The results explain the altered properties of excitable cells in Sh mutants and provide a general model for the possible role of A-type K+ channels in modulating action potential profiles.

Action Potentials↗
PubMed1986

Voltage-dependent channel formation by rods of helical polypeptides.

The voltage-dependence of channel formation by alamethicin and it natural analogues can be described by a dipole flip-flop gating model, based on electric field-induced transbilayer orientational movements of single molecules. These field-induced changes in orientation result from the large permanent dipole moment of alamethicin, which adopts alpha-helical conformation in hydrophobic medium. It was, therefore, supposed that the only structural requirement for voltage-dependent formation of alamethicin-type channels might be a rigid lipophilic helical segment of minimum length. In order to test this hypothesis we synthesized a family of lipophilic polypeptides--Boc-(Ala-Aib-Ala-Aib-Ala)n-OMe, n = 1-4--which adopt alpha-helical conformation for n = 2-4 and studied their interaction with planar lipid bilayers. Surprisingly, despite their large difference in chain length, all four polypeptides showed quantitatively similar behavior. At low field strength of the membrane electric field these polypeptides induce a significant, almost voltage-independent increase of the bilayer conductivity. At high field strength, however, a strongly voltage-dependent conductance increase occurs similar to that observed with alamethicin. It results from the opening of a multitude of ion translocating channels within the membrane phase. The steady-state voltage-dependent conductance depends on the 8th-9th power of polypeptide concentration and involves the transfer of 4-5 formal elementary charges. From the power dependences on polypeptide concentration and applied voltage of the time constants in voltage-jump current-relaxation experiments, it is concluded that channels could be formed from preexisting dodecamer aggregates by the simultaneous reorientation of six formal elementary charges. Channels exhibit large conductance values of several nS, which become larger towards shorter polypeptide chain length. A mean channel diameter of 19 A is estimated corresponding roughly to the lumen diameter of a barrel comprised of 10 alpha-helical staves. Similar to experiments with the N-terminal Boc-derivative of alamethicin we did not observe the burst sequence of nonintegral conductance steps typical of natural (N-terminal Ac-Aib)-alamethicin. Saturation in current/voltage curves as well as current inactivation in voltage-jump current-relaxation experiments are found. This may be understood by assuming that channels are generated as dodecamers but, while reaching the steady state, reduce their size to that of an octamer or nonamer. We conclude that the overall behavior of these synthetic polypeptides is very similar to that of alamethicin.(ABSTRACT TRUNCATED AT 400 WORDS)

Alamethicin↗
PubMed1985

Phallolysin. A mushroom toxin, forms proton and voltage gated membrane channels.

Phallolysin, a water soluble protein of Mr 34,000 produced by the poisonous mushroom Amanita phalloides, causes lysis of various mammalian cell types. Lysis is thought to be initiated by the formation of ion permeable membrane channels. We therefore studied the interaction of phallolysin with solvent-free planar lipid bilayers. In the presence of low phallolysin concentrations (10-100 nM) single channel current fluctuations were observed. Unit channel conductances are 44 pS in 500 mM NaCl and 77 pS in 1 M NaCl. Although the channel does not significantly discriminate between alkali cations, its permeability to Cl- is lower (PK+/PCl- = 4/1). Gating kinetics display a pronounced bursting behavior and a dependence on membrane voltage, cis side pH-value, and on membrane lipid composition. An equivalence relation between membrane voltage and proton concentration was found, i.e. a pH change of one unit is equivalent to a corresponding voltage change of 130 mV. Dependence on the amount of negatively charged lipids is explained by changes of the actual pH due to surface charge effects.

Amanitins↗
PubMed1984

Reconstitution of highly purified saxitoxin-sensitive Na+-channels into planar lipid bilayers.

Highly purified Na+-channels isolated from rat brain have been reconstituted into virtually solvent-free planar lipid bilayer membranes. Two different types of electrically excitable channels were detected in the absence of any neurotoxins. The activity of both channels was blocked by saxitoxin. The first channel type is highly selective for Na+ over K+ (approximately 10:1), it shows a bursting behavior, a conductance of 25 pS in Na+-Ringer and undergoes continuous opening and closing events for periods of minutes within a defined range of negative membranes voltages. The second channel type has a conductance of 150 pS and a lower selectivity for Na+ and K+ (2.2:1); only a few opening and closing events are observed with this channel after one voltage jump. The latter type of channel is also found with highly purified Na+-channel from Electrophorus electricus electroplax. A qualitative analysis of the physicochemical and pharmacological properties of the high conductance channel has been carried out. Channel properties are affected not only by saxitoxin but also by a scorpion (Centruroides suffusus suffusus) toxin and a sea anemone (Anemonia sulcata) toxin both known to be selective for the Na+-channel. The spontaneous transformation of the large conductance channel type into the small one has been considered; the two channel types may represent the expression of activity of different conformational states of the same protein.

Animals↗
PubMed1984

New scorpion toxins with a very high affinity for Na+ channels. Biochemical characterization and use for the purification of Na+ channels.

Biochemical characterization of the Tityus gamma toxin receptor associated with the voltage-sensitive Na+ channel was carried out in different tissue preparations with the use of an iodinated toxin derivative. The affinity of the toxin for the receptor is high with a dissociation constant of 4 X 10(-12) M for rat synaptosomes. The density of binding sites is in the range of 0.3 to 2 pmol/mg of protein. Toxin gamma does not seem to bind to Na+ channels located on transverse-tubule membranes of skeletal muscle, but only to Na+ channels located on the sarcolemma. Both affinity labelling and radiation inactivation analysis indicate a molecular weight for the toxin receptor of 270 000 daltons. The same molecular weight is found using the tetrodotoxin. Only one single major protein component of the Na+ channel was purified from Electrophorus electroplax, rat brain membranes and chick heart membrane using the toxin gamma as a marker. The molecular weight of this component is 230 000-270 000 daltons. Reconstitution of the purified Na+ channel into planar lipid bilayers has been carried out. Two different types of electrically excitable channels with conductances of 150 and 25 pS were detected. The activity of both channels is blocked by saxitoxin.

Animals↗
PubMed1983

Melittin and a chemically modified trichotoxin form alamethicin-type multi-state pores.

The bee venom constituent, melittin, is structurally and functionally related to alamethicin. By forming solvent-free planar bilayers of small area (approx. 100 microns 2) on the tip of fire-polished glass pipettes we could observe single melittin pores in these membranes. An increase in the applied voltage induced further non-integral conductance levels. This indicates that melittin forms multi-level pores similar to those formed by alamethicin. Trichotoxin A40, an antibiotic analogue of alamethicin, also induces a voltage-dependent bilayer conductivity, but no stable pore states are resolved. However, chemical modification of the C-terminal molecule part by introduction of a dansyl group leads to a steeper voltage-dependence and pore state stabilization. Comparing structure and activity of several natural and synthetic amphiphilic polypeptides, we conclude that a lipophilic, N-terminal alpha-helical part of adequate length (dipole moment) and a large enough hydrophilic, C-terminal region are sufficient prerequisites for voltage-dependent formation of multi-state pores.

Alamethicin↗
PubMed1982

The gating of single calcium-dependent potassium channels is described by an activation/blockade mechanism.

Single calcium dependent potassium channels from cultured rat myoballs have been studied with the patch clamp technique, and current records subjected to statistical analysis. From the dependence of the mean open state probability on the internal calcium concentration, two calcium ions are required to open the channel. The open state and closed state lifetime distributions reveal that the usual activation model is not applicable to these channels. They are consistent with a two step gating mechanism that involves both activation by calcium and blockade by a calcium-sensitive gate.

Animals↗
PubMed1981

A new method for membrane reconstitution: fusion of protein-containing vesicles with planar bilayer membranes below lipid phase transition temperature.

A new method of membrane reconstitution was developed by fusion of channel protein containing vesicles with planar bilayer membranes. The fusion process only occurred below and near the phase transition temperature of the lipid used. We obtained the following results: 1. Our system is solvent-free and vesicles do not come into contact with the air/water interface. This obviates a possible denaturation of hydrophobic proteins. 2. Channel forming proteins and protein complexes, respectively, are active in a frozen lipid matrix. 3. We detected an unknown channel in cilia fragments. 4. Purified acetylcholine receptors form fluctuating channels in a membrane consisting of a pure synthetic lecithin (two component system).

Animals↗
PubMed1981

Agonist-activated ionic channels in acetylcholine receptor reconstituted into planar lipid bilayers.

Planar lipid bilayers were formed with the mixed chain phospholipid 1-stearoyl-3-myristolglycero-2-phosphocholine. Acetylcholine receptor membrane fragments or the purified receptor protein was incorporated into these bilayers by fusing receptor-containing vesicles with the planar membranes a few degrees below the lipid phase transition temperature. Single-channel currents activated by nicotinic agonists in the reconstituted system resembled those observed in intact rat and frog muscle membrane as measured by the patch clamp technique. The observed channel characteristics did not depend on the degree of receptor purification. Thus, the receptor-enriched fragments and those depleted of nonreceptor peripheral peptides, the purified receptor monomer/dimer mixtures, and the isolated receptor monomer as defined by gel electrophoresis all shared similar electrochemical properties in the synthetic lipid bilayer. The agonist-activated ionic channel seems, therefore, to be contained within the receptor monomer.

Acetylcholine↗
PubMed1980

The lowest conductance state of the alamethicin pore.

High resolution experiments of the alamethicin pore demonstrate the existence of a further pore state at low conductance values. This lowest resolved conductance state is found at 19 pS in 1 M KCl at room temperature. The value differs from that of the next higher conductance state by a factor of 14--15 and is approx. 20% lower than the gramicidin A pore conductance. The lowest conductance state seems to be impermeable to Ca2+, Cl-, Tris-H+ and Hepes-, whereas the higher conductance states are not.

Alamethicin↗