Günther Boheim

Statistical analysis of alamethicin channels in black lipid membranes

SummaryPore formation of alamethicin has been studied by the analysis of steadystate fluctuations of single-pore conductances. An aggregation model is proposed where transitions to the next higher or lower pore state occur by uptake or release of one monomer. It is assumed that alamethicin forms an elongated loop in the bilayer. The main voltage-dependent step is the insertion of this monomer into the membrane after complexation with a cation. This mechanism is equivalent to dipole orientation in an electric field. Pore formation is restricted by the energy required to enlarge the channel in the membrane.

Alamethicin pore formation: Voltage-dependent flip-flop of α-helix dipoles

The voltage-dependency of alamethicin pore formation is explained by a flip-flop gating mechanism of single alamethicin molecules. The energetically preferred aggregate structure is changed from antiparallel to parallel molecule orientation by membrane voltage application. The electrical field is sensed by the permanent dipole of the α-helical molecule part which spans the hydrophobic membrane core. Ion conducting pore and pore states result from electrostatic repulsion of a varying number of parallel dipoles which arrange circularly. This model is consistent with published data and with two additional experimental facts, that pore state distributions are ionic strength dependent and pore state conductances depend on ionic current direction.

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 channels 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 tendrils 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.

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.

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.

Analysis of the multi-pore system of alamethicin in a lipid membrane

SummaryThe electrical properties of an alamethicin multi-pore system have been studied by voltage-jump current-relaxation experiments (this paper) and by autocorrelation and spectral analysis (following paper). With these methods a slow time constant and a fast time constant were observed which differ by about one to three orders of magnitude depending on the experimental conditions. Steady-state current and time constants were analyzed as functions of voltage, alamethicin concentration and temperature. Within experimental error the data obtained with these different methods are in good agreement. The experimentally measured relation between the voltage and alamethicin concentration dependence of the slow relaxation time fits into a model of an alamethicin pore which adopts consecutive pore states and which decays only from the lowest state. It indicates that the uptake of one alamethicin molecule by the existing pore and, in formal equivalence, the transfer of about one positive elementary charge across the membrane are associated with the transition from a given pore conductance state to the next higher state. From the voltage and alamethicin concentration dependence of the pore formation rate evidence shows that a hexameric preaggregate exists at the membrane interface out of which two to three molecules are simultaneously inserted into the membrane to form the pore nucleus. The effects of different voltage pretreatment on the experimentally determined parameters have been investigated and are discussed in detail.

Voltage-dependent channel formation by rods of helical polypeptides

SummaryThe voltage-dependence of channel formation by alamethicin and its 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 α-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 α-helical conformation forn=2–4 and studied their interaction with planar lipid bilayers. Surprisingly, despite their large difference in chain length, all four polypeptides showed qualitatively 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 Å is estimated corresponding roughly to the lumen diameter of a barrel comprised of 10 α-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. They exhibit the same concentration and voltage-dependences but lack the stabilizing principle of resolved channel states characteristic of alamethicin.

Structural and membrane modifying properties of suzukacillin, a peptide antibiotic related to alamethicin: Part A. Sequence and conformation

The primary structure and conformation of the polypeptide antibiotic suzukacillin A are investigated. Suzukacillin A is isolated from the Trichoderma viride strain 1037 and exhibits membrane modifying and lysing properties similar to those of alamethicin. A combined gas chromatographic mass spectrometric analysis of the trifluoroacetylated peptide methyl esters of partial hydrolysates revealed a tentative sequence of 23 residues including 10 2-methylalanines and one phenylalaninol, which shows many fragments known from alamethicin: Ac-Aib-Pro-Val-Aib-Val-Ala-Aib-Ala-Aib-Aib-Gln-Aib-Leu-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu(Pheol)-Gln-OH. All chiral amino acids and phenylalainol have L-configuration. Ultraviolet and infrared spectroscopy, circular dichroism in various solvents and in particular 13C nuclear magnetic resonance have been used for a comparative study of suzukacillin with alamethicin. Suzukacillin has a partially alpha-helical structure and the helix content increases largely from polar to lipophilic solvents. Suzukacillin aggregates more strongly than alamethicin in aqueous medis due to a longer alpha-helical part and higher number of aliphatic residues. A part of the alpha-helix is exceptionally stabilized due to 2-methylalanine residues shielding the peptide bonds from interactions with polar solvents. In lipophilic solvents and lecithin vesicles particularly large temperature induced reductions of the high alpha-helix content are found for alamethicin. Suzukacillin shows similar temperature coefficients in lipophilic media, however, in contrast to alamethicin a more linear change in intensity of the Cotton effects is observed.

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.

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

The structure and dynamic properties of bee venom molittin 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 α‐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.