Intracellular chloride modulates A-type potassium currents in astrocytes

Application of the GABA(A) receptor agonist muscimol to astrocytes in situ or in vitro results in a receptor-mediated Cl(-) current with a concomitant block of outward K(+) currents. The effect on K(+) current is largely selective for the inactivating A-type current. Parallel experiments with various Cl(-) pipette concentrations show a significant reduction in A-type current under low Cl(-) conditions with minimal effect on delayed current. In addition, lower Cl(-) conditions caused a depolarizing shift of steady-state inactivation (V(1/2), -68 to -57 mV) and activation (V(1/2), -5.8 to 34 mV) kinetics of A-type current only. Cl(-) had no effect on the time course of inactivation or reactivation kinetics, suggesting the Cl(-)-mediated effect is largely on activation kinetics, indirectly affecting steady-state inactivation. Muscimol application to astrocytes under perforated patch control (gramicidin) displayed a similar block of A-type current to that of conventional whole cell patch at 40 or 20 mM pipette Cl(-) concentrations. With barium application under perforated patch conditions, the study of muscimol-mediated Cl(-) current in isolation of the effect on K(+) currents was possible. This allowed estimation of intracellular Cl(-) concentration from receptor current reversal information. The average intracellular Cl(-) concentration was found to be 29 +/- 3.2 mM. The effect on activation kinetics and lack of effect on time course of inactivation or reactivation suggest that intracellular anion concentrations have an effect on the K(+) channel voltage sensor region. Cl(-) may modulate K(+) currents by altering membrane field potentials surrounding K(+) channel proteins.     

ANOMALOUS EXCHANGE KINETICS OF PEPTIDE AMIDE PROTONS IN AQUEOUS SOLUTIONS

Reports concerning anomalous rates of exchange of some amides in oxytocin, alumichrome, and gramicidin S are reexamined through systematic analysis of the exchange data as a function of pH and primary structure. It is shown that such an analysis can provide useful information on secondary structure when the degree of hydrogen bonding to both the NH undergoing exchange and the neighboring carbonyl group are taken into consideration.     

Bilirubin binding to polypeptides and chiral amines. Induced circular dichroism and fluorescence studies

Induced Cotton effects have been observed in the visible region on interaction of bilirubin with chiral mono- and diamines and poly-l -lysine. At alkaline pH distinct CD spectra are observed for bilirubin bound to the α-helical and β-sheet conformation of poly-l -lysine, which differ from that observed for the pigment bound to human serum albumin. The CD pattern observed on binding to N-acetyl-Lys-N¹-methylamide in CH₂Cl₂ and dioxane is different from that observed in the presence of l -Ala-NH-(CH₂)₆-NH-l -Ala in dioxane. The latter case resembles the spectrum observed in the presence of human serum albumin. Binding to the helical polypeptide melittin and the antiparallel β-sheet peptide, gramicidin S, in aqueous solutions results in opposite signs of the bilirubin CD bands. The quenching of tryptophan fluorescence in melittin, in aqueous solution and enhancement of bilirubin fluorescence in dioxane on binding to gramicidin S have been used to monitor pigment-peptide interactions. The results suggest the utility of bilirubin as a conformational probe.     

Effects of Ionizing Radiation on Artificial (Planar) Lipid Membranes. I. Radiation Inactivation of the Ion Channel Gramicidin A

Summary

The ion channel formed by the pentadecapeptide gramicidin A in planar lipid membranes is extremely sensitive to ionizing radiation. The membrane conductance may drop by several orders of magnitude under appropriate experimental conditions (low pH and presence of oxygen). The radiation sensitivity is strongly reduced for gramicidin M^?. This analogue has the four tryptophan residues replaced by phenylalanines. Experiments performed in the presence of various radical scavengers suggest that the inactivation of the channel is due to a combined action of OH and of HO₂ radicals at the tryptophan residues.

The shape of the inactivation curves following continuous radiolysis or pulse radiolysis were found to be in fair agreement with a simple model which assumes that the damage of a single tryptophan residue is sufficient for channel inactivation. The conductance of inactivated channels could not be resolved within the experimental accuracy. This is contrary to photolysis of gramicidin channels found by Busath and Waldbilling (1983), where a broad distribution of low conductance states was observed. The inactivation by radiolysis seems to represent an ‘all-or-none-process’ of the channel conductance.     

Synthesis and characterization of retro gramicidin A-dAla-gramicidin A,a 31-residue-long gramicidin analogue

A 31-residue peptide, retro gramicidin A-d Ala-gramicidin A, mimicking a gramicidin A dimer was prepared by solid phase method using a 4-(oxymethyl)-Pam resin and BOC as α amino protecting group. To avoid any possible formation of gramicidin A, couplings of fragments were used together with stepwise growing of the peptide chain. After purification by silica gel chromatography, this peptide was shown to form long living channels in lipid bilayer. Preliminary physicochemical investigations suggest that the peptide does not adopt the expected π^6.3_DL helical conformation.     

Convenient preparation of [Orn(Tfa)2]- and [Orn(Boc)2, Orn(Tfa)2′]gramicidin S,versatile unsymmetrically protected derivatives of gramicidin S

Abstract: Treatment of gramicidin S (GS) with trifluoroacetic anhydride afforded a derivative in which only one of the two Orn side chains was trifluoroacetylated in 72% yield, furnishing the first efficient method for the preparation of a monoprotected derivative of GS. The mono(Tfa) derivative [Orn(Tfa)²]GS was treated with di-tert-butyl dicarbonate to yield dually protected derivative [Orn(Boc)²,Orn(Tfa)^2′]GS from which another monoprotected derivative [Orn(Boc)²]GS was prepared in high yield. These unsymmetrically protected GS derivatives are versatile starting materials for the preparation of various other GS derivatives. As an example of application of the unsymmetrically protected derivatives, a dimeric GS derivative was prepared via a singly p-nitrobenzenesulfonyl(NBS)-activated derivative [Orn(Boc)²,Orn(NBS)^2′]GS.     

Synthesis and ionic channels of a linear gramicidin containing naphthylalanine instead of tryptophan

Naphthylalanine^9,11,13,15 gramicidin A was prepared by the solid phase method using an aminopolyacrylic resin after optical resolution of (D, L) naphthylalanine by enzymatic methods. Removal of the peptide from the resin was achieved by transesterification of the succinic ester linkage. Infrared spectroscopy indicated that the presence of naphthylalanine strongly modifies the monomer-dimer equilibrium. Single-channel measurements suggested that the conductance of the gramicidin channel can be governed by the dipole moment of the aromatic side-chains.     

Role of ring size on the secondary structure and antibiotic activity of gramicidin S

In order to investigate the contribution of ring size to the secondary structure and antibiotic activity of gramicidin S, many analogs consisting of 6, 7, 8, 9, 11, 12, 13, and 14 amino acid residues were synthesized. In the analogs with smaller ring sizes than that of gramicidin S, only des-Val¹-, des-Leu³- and des-Pro⁵-gramicidin S showed weak activity against the gram-positive micro-organisms tested. On the other hand, all analogs having larger rings, in which L- or D-Leu residues were inserted, were active. The activity of the analogs consisting of 10, 11 and 12 amino acid residues was stronger than those of the analogs with other ring sizes, and the activity of endo-Leu^2a-gramicidin S against S. epidermidis SP-al-1 and B. subtilis ATCC 6633 was twice of that of gramicidin S.     

Interaction of gramicidin S analogs with lipid bilayer membrane

One of the side chains of Orn residues in gramicidin S (GS) was connected with alanine (AGS), sarcosine (SGS), or histidine (HGS) residue, aiming at developing membrane-active artificial enzymes by virtue of the membrane-associating property of GS. The conformation of the GS analogs was similar to that of GS. However, the affinity of GS and its analogs for dipalmitoylphosphatidylcholine (DPPC) vesicles decreased in the order of GS > SGS > HGS ? AGS. The addition of GS analogs at 10 μ to DPPC vesicles decreased the membrane fluidity, indicating that GS analogs did not disrupt the vesicular structure of DPPC vesicles. On the other hand, GS analogs enhanced carboxyfluorescein-leakage from DPPC vesicles. It was therefore considered that the GS analogs induced the phase-separation of the lipid bilayer membrane. Hydrolytic reactions of HGS in the presence of DPPC vesicles were studied using N-methylindoxyl alkanoate as substrate. HGS reacted only with N-methylindoxyl hexanoate below the phase-transition temperature of the membrane. The substrate specificity of HGS was ascribed to the condensation of HGS in the neighbourhood of the substrate in the lipid bilayer membrane due to the phase-separation below the phase-transition temperature of the membrane.