Vibrational analysis of peptides,polypeptides and proteins

Normal mode calculations have been carried out on three low-energy structures of gramicidin S obtained from conformational energy calculations. When the results on the amide modes are compared with observed bands in the infrared and Raman spectra of crystalline gramicidin S and its N-deuterated derivative, one of the structures is clearly disfavored. Of the other two, one is slightly favored, and it corresponds to the lowest-energy structure obtained from the energy calculations. Spectra from solutions in DMSO and CH₃ OH suggest that the molecular conformation is essentially retained in these solvents.     

Chloride transport by self-exchange and by KCI salt diffusion in gramicidin-treated human red blood cells

The permeability of gramicidin-treated human red blood cell membranes to K⁺ and CI^- has been measured at normal ionic strength (1) by tracer exchange at steady-state distribution of salt, and (2) by net transport of salt in the presence of a salt concentration gradient. Under both conditions KCI was the only inorganic salt in cells and medium. In the studies of self-exchanges the electrical driving force on the ions was zero. Calculation of permeability coefficients from net salt transport was simplified because the experiment was designed as a special case of the Nernst-Planck diffusion regime, i.e. the single salt case. Gramicidin altered the cell membranes from being anion to become cation selective. Gramicidin increased the potassium exchange without affecting, the chloride exchange measurably. The chloride exchange showed saturation kinetics as does chloride exchange in normal cells. The net transport of KCI in the presence of a constant concentration gradient increased to a constant value with increasing gramicidin concentration. At high gramicidin concentrations (0°C, pH 7.2) the “chloride permeability coefficient” calculated from tracer exchange (1.9×10^-6 cm/s) was 290 times the chloride permeability coefficient calculated from net salt transport (0.65×10^-8 cm/s). The latter value corresponds to a chloride conductance of 4.2×10^-6 ohm^-1 cm^-2. The chloride permeability coefficient was 2.1×10^-8 cm/s at 25°C (pH 6.8) indicating a value of 3 for the Q₂₅. It appears that normal red cells are anion selective in the sense that anion permeability exceeds cation permeability with a factor of more than a hundred between 0°C and body temperature. The anion exchange, i.e. the Hamburger shift, is a tightly coupled transport process which is several orders of magnitude faster than anion transport by salt diffusion.     

Shift from depolarizing to hyperpolarizing glycine action occurs at different perinatal ages in superior olivary complex nuclei

The inhibitory transmitters glycine and GABA undergo a developmental shift from depolarizing to hyperpolarizing action (D/H-shift). To analyse this shift in functionally related nuclei of the rat superior olivary complex (SOC), we employed voltage-sensitive dye recordings in auditory brainstem slices. Complementarily, we analysed single neurons in gramicidin perforated-patch recordings. Our results show a differential timing of the D/H-shift in the four SOC nuclei analysed. In the medial superior olive (MSO), the shift occurred at postnatal day (P) 5-9. In the superior paraolivary nucleus (SPN), it occurred between embryonic day (E) 18 and P1. No D/H-shift was observed in the medial nucleus of the trapezoid body (MNTB) until P10. This is in line with the finding that most of the patched MNTB neurons displayed glycine-induced depolarizations between P0-9. While no regional differences regarding the D/H-shift were found within the MSO, SPN, and MNTB, we observed such differences in the lateral superior olive (LSO). All LSO regions showed a D/H-shift at P4-5. However, in the high-frequency regions, hyperpolarizations were large already at P6, yet amplitudes of this size were not present until P8 in the low-frequency regions, suggesting a delayed development in the latter regions. Our physiological results demonstrate that D/H-shifts in SOC nuclei are staggered in time and occur over a period of almost two weeks. Membrane-associated immunoreactivity of the Cl- outward transporter KCC2 was found in every SOC nucleus already at times when glycine was still depolarizing. This implies that the mere presence of KCC2 does not correlate with functional Cl- outward transport.     

Differences between cation-chloride co-transporter functions in the visual cortex of pigmented and albino rats

Albinism in mammals is accompanied by specific morphological and functional alterations of the visual system. To understand their cellular basis we studied the physiological characteristics and transmembrane currents of pyramidal neurons in 350-microm-thick slices of visual cortex from pigmented and albino rats using whole-cell and gramicidin perforated patch-clamp recordings. The resting membrane potential was significantly more positive and the rheobase was significantly lower in neurons of layers II/III and V in albinos as compared with pigmented rats. No significant differences were found in the input resistance, time constant and chronaxy. Whereas the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated currents were not significantly different, the maximum gamma-aminobutyric acid (GABA)(A) receptor (GABA(A)R)-mediated currents and miniature inhibitory postsynaptic currents showed significantly lower amplitudes in neurons of layer V in visual cortex of albinos as compared with pigmented rats. The reversal potential of the GABA(A)R-mediated currents (E(GABA)) was significantly shifted to more positive values in albinos. Pharmacological experiments showed that this shift could be caused by an increased action of the inward chloride co-transporter NKCC1 and reduced action of the outward chloride co-transporter KCC2 in albino rats. This difference seems to be restricted to the visual cortex because in pyramidal neurons from frontal cortex E(GABA) was not significantly different in albinos as compared with pigmented rats. These results are discussed in relation to functional alterations in the albino visual system.     

Channel-Forming Bacterial Toxins in Biosensing and Macromolecule Delivery

To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on “Intracellular Traffic and Transport of Bacterial Protein Toxins”, reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their “second life” in a variety of developing medical and technological applications.     

Synthesis and characterization of (1 - 13C) Phe9 gramicidin A. Effects of side chain variations

The synthesis of (1-¹³C)-Phe⁹-gramicidin (90% enriched) was carried out by the solid phase method. The peptide was removed from the resin by treatment with ethanolamine, deblocked, formylated and purified by preparative t.l.c. to obtain the gramicidin analog in an overall yield of 24%. The peptide was verified and characterized by high pressure liquid chromatography, carbon-13 nuclear magnetic resonance, circular dichroism and single channel currents. Single channel conductances were found to be similar to those of (1-¹³C)-Phe¹¹-GB but significantly lower than that of gramicidin A. When this gramicidin analog was incubated with phospholipid, the characteristic channel spectrum was not obtained and interaction with sodium ion was not observed. A possible explanation for this behavior is discussed.     

Effect of kainate on the membrane conductance of hilar glial precursor cells recorded in the perforated-patch configuration

The effects of kainate on membrane current and membrane conductance were investigated in presumed hilar glial precursor cells of juvenile rats. The perforated-patch configuration was used also to reveal possible second-messenger effects. Kainate evoked an inward current that was accompanied by a biphasic change in membrane conductance in 69% of the cells. An initial conductance increase with a time course similar to that of the inward current was followed by a second delayed conductance increase. This second conductance was absent in whole-cell-clamp recordings, suggesting that it was mediated by a second messenger effect. Analysis of the reversal potentials of the membrane current during both phases of the kainate-induced conductance change revealed that the first conductance increase reflected the activation of AMPA receptors. Several lines of evidence suggest that the delayed second conductance increase was due to the indirect activation of Ca²⁺-dependent K⁺ channels via Ca²⁺-influx through AMPA receptors. (1) the delayed second conductance increase was blocked by Ba²⁺ and the reversal of its underlying current was significantly shifted towards E_K+, suggesting that it is due to the activation of K⁺ channels. (2) The delayed second conductance increase disappeared in a Ca²⁺-free saline buffered with BAPTA, indicating that it depended on Ca²⁺-influx. (3) Co²⁺, Cd²⁺ and nimodipine failed to block the delayed second conductance increase excluding a major contribution of voltage-dependent Ca²⁺ channels. (4) The involvement of metabotropic glutamate receptors also appeared unlikely, because the kainate-induced delayed second conductance increase could not be blocked by a depletion of the intracellular Ca²⁺ stores with the Ca²⁺-ATPase inhibitor thapsigargin, and t-ACPD exerted no effect on membrane current and conductance. We conclude that kainate activates directly AMPA receptors in presumed hilar glial precursor cells. This results in a Ca²⁺ influx that could lead indirectly to the activation of Ca²⁺-dependent K⁺ channels. GLIA 23:35–44, 1998. © 1998 Wiley-Liss, Inc.     

K+-dependent paradoxical membrane depolarization and Na+ overload, major and reversible contributors to weakness by ion channel leaks

Normal resting potential (P1) of myofibers follows the Nernst equation, exhibiting about −85 mV at a normal extracellular K⁺ concentration ([K⁺]_o) of 4 mM. Hyperpolarization occurs with decreased [K⁺]_o, although at [K⁺]_o < 1.0 mM, myofibers paradoxically depolarize to a second stable potential of −60 mV (P2). In rat myofiber bundles, P2 also was found at more physiological [K⁺]_o and was associated with inexcitability. To increase the relative frequency of P2 to 50%, [K⁺]_o needed to be lowered to 1.5 mM. In the presence of the ionophore gramicidin, [K⁺]_o reduction to only 2.5 mM yielded the same effect. Acetazolamide normalized this increased frequency of P2 fibers. The findings mimic hypokalemic periodic paralysis (HypoPP), a channelopathy characterized by hypokalemia-induced weakness. Of myofibers from 7 HypoPP patients, up to 25% were in P2 at a [K⁺]_o of 4 mM, in accordance with their permanent weakness, and up to 99% were in P2 at a [K⁺]_o of 1.5 mM, in accordance with their paralytic attacks. Of 36 HypoPP patients, 25 had permanent weakness and myoplasmic intracellular Na⁺ ([Na⁺]_i) overload (up to 24 mM) as shown by in vivo ²³Na-MRI. Acetazolamide normalized [Na⁺]_i and increased muscle strength. HypoPP myofibers showed a nonselective cation leak of 12–19.5 μS/cm², which may explain the Na⁺ overload. The leak sensitizes myofibers to reduced serum K⁺, and the resulting membrane depolarization causes the weakness. We postulate that the principle of paradoxical depolarization and loss of function upon [K⁺]_o reduction may apply to other tissues, such as heart or brain, when they become leaky (e.g., because of ischemia).     

Gramicidin S is active against both gram-positive and gram-negative bacteria

Four linear and four cyclic analogs of gramicidin S (GS) in which d -Phe was replaced with either d -His, d -Ser, d -Tyr or D-Asn have been prepared by solid-phase peptide synthesis and characterized with respect to antibacterial, antifungal and hemolytic activity. Unlike previous reports, GS and a number of cyclic analogs were found to be active against gram-positive as well as gram-negative bacteria. GS showed MICs ranging from 3 to 12.5 μg/mL for gram-negative bacteria, compared to MICs of 3 μg/mL for gram-positive bacteria. Furthermore, these analogs were also found to exhibit antifungal activity. Unlike the cyclic analogs, all linear analogs were found to be inactive against a wide range of microorganisms tested, and showed low levels of hemolytic activity. The antibacterial activity was found to be highly dependent on the type of assay used, with solution-based assays showing greater activity against gram-negative bacteria than agar-based assays. The GS cyclic analogs were all less toxic than GS itself, with the analog containing the d -Phe to d -Tyr substitution showing the greatest activity of the synthetic analogs. Hemolytic activity in solution against human and sheep red blood cells paralleled antibiotic activity, with those peptides exhibiting greater antibiotic activity generally showing greater hemolytic activity. Membrane destabilization as monitored using the hydrophobic probe N-phenyl-1-naphthylamine was also found to parallel antibacterial and hemolytic activity of cyclic and linear analogs. These results indicate that GS and certain related analogs may have applications as broad-spectrum antibiotics and should be reevaluated for such purposes. © Munksgaard 1996.