Induction of lipid flip-flop by colicin E1 - a hallmark of proteolipidic pore formation in liposome membranes.

The addition of the channel-forming domain of colicin E1 to liposomes elicited the transmembrane diffusion (flip-flop) of lipids concomitant to the release of the fluorescent dye from liposomes. Good correlation was found between kinetic and concentration dependences of the two processes. Both the liposome leakage and the lipid flip-flop were stimulated upon alkalinization of the buffer solution after colicin binding at acidic pH. These results in combination with the analysis of the data on colicin binding to liposomes provide evidence in favor of the validity of the toroidal (proteolipidic) pore model as the mechanism of colicin channel formation.

[Ion channels of various types induced in lipid membranes by gramicidin A derivatives carrying a cationic sequence at their C-termini].

The channel-forming activity of gramicidin A derivatives carrying positively charged amino acid sequences at their C-termini was studied on planar bilayer lipid membranes and liposomes. We showed previously that, at low concentrations, these peptides form classical cation-selective pores typical of gramicidin A, whereas, at high concentrations, they form large nonselective pores. The ability of the peptides to form nonselective pores, which was determined by the efflux of carboxyfluorescein, an organic dye, from liposomes, decreased substantially as the length of the gramicidin fragment in the series of cationic analogues was truncated. CD spectra showed that large pores are formed by peptides having both beta6.3 single-stranded and beta5.6 double-stranded helical conformations of the gramicidin fragment, with the C-terminal cationic sequence being extended. The dimerization of the peptides by the oxidation of the terminal cysteine promoted the formation of nonselective pores. It was shown that nonselective pores are not formed in membranes of erythrocytes, which may indicate a dependence of the channel-forming ability on the membrane type. The results may be of interest for the directed synthesis of peptides with antibacterial activity.

Lipid-mediated inactivation of colicin E1 channels by calcium ions.

Based on the model of a toroidal protein-lipid pore, the effect of calcium ions on colicin E1 channel was predicted. In electrophysiological experiments Ca2+ suppressed the activity of colicin E1 channels in membranes formed of diphytanoylphosphatidylglycerol, whereas no desorption of the protein occurred from the membrane surface. The effect of Ca2+ was not observed on membranes formed of diphytanoylphosphatidylcholine. Single-channel measurements revealed that Ca2+-induced reduction of the colicin-induced current across the negatively charged membrane was due to a decrease in the number of open colicin channels and not changes in their properties. In line with the toroidal model, the effect of Ca2+ on the colicin E1 channel-forming activity is explained by alteration of the membrane lipid curvature caused by electrostatic interaction of Ca2+ with negatively charged lipid head groups.

Chemical and photochemical modification of colicin E1 and gramicidin A in bilayer lipid membranes.

Chemical modification and photodynamic treatment of the colicin E1 channel-forming domain (P178) in vesicular and planar bilayer lipid membranes (BLMs) was used to elucidate the role of tryptophan residues in colicin E1 channel activity. Modification of colicin tryptophan residues by N-bromosuccinimide (NBS), as judged by the loss of tryptophan fluorescence, resulted in complete suppression of wild-type P178 channel activity in BLMs formed from fully saturated (diphytanoyl) phospholipids, both at the macroscopic-current and single-channel levels. The similar effect on both the tryptophan fluorescence and the electric current across BLM was observed also after NBS treatment of gramicidin channels. Of the single-tryptophan P178 mutants studied, W460 showed the highest sensitivity to NBS treatment, pointing to the importance of the water-exposed Trp460 in colicin channel activity. In line with previous work, the photodynamic treatment (illumination with visible light in the presence of a photosensitizer) led to suppression of P178 channel activity in diphytanoyl-phospholipid membranes concomitant with the damage to tryptophan residues detected here by a decrease in tryptophan fluorescence. The present work revealed novel effects: activation of P178 channels as a result of both NBS and photodynamic treatments was observed with BLMs formed from unsaturated (dioleoyl) phospholipids. These phenomena are ascribed to the effect of oxidative modification of double-bond-containing lipids on P178 channel formation. The pronounced stimulation of the colicin-mediated ionic current observed after both pretreatment with NBS and sensitized photomodification of the BLMs support the idea that distortion of membrane structure can facilitate channel formation.

Hypomyelination and increased activity of voltage-gated K(+) channels in mice lacking protein tyrosine phosphatase epsilon.

Protein tyrosine phosphatase epsilon (PTP epsilon) is strongly expressed in the nervous system; however, little is known about its physiological role. We report that mice lacking PTP epsilon exhibit hypomyelination of sciatic nerve axons at an early post-natal age. This occurs together with increased activity of delayed- rectifier, voltage-gated potassium (Kv) channels and with hyperphosphorylation of Kv1.5 and Kv2.1 Kv channel alpha-subunits in sciatic nerve tissue and in primary Schwann cells. PTP epsilon markedly reduces Kv1.5 or Kv2.1 current amplitudes in Xenopus oocytes. Kv2.1 associates with a substrate-trapping mutant of PTP epsilon, and PTP epsilon profoundly reduces Src- or Fyn-stimulated Kv2.1 currents and tyrosine phosphorylation in transfected HEK 293 cells. In all, PTP epsilon antagonizes activation of Kv channels by tyrosine kinases in vivo, and affects Schwann cell function during a critical period of Schwann cell growth and myelination.

Extracellular K(+) and opening of voltage-gated potassium channels activate T cell integrin function: physical and functional association between Kv1.3 channels and beta1 integrins.

Elevated extracellular K(+) ([K(+)](o)), in the absence of "classical" immunological stimulatory signals, was found to itself be a sufficient stimulus to activate T cell beta1 integrin moieties, and to induce integrin-mediated adhesion and migration. Gating of T cell voltage-gated K(+) channels (Kv1.3) appears to be the crucial "decision-making" step, through which various physiological factors, including elevated [K(+)](o) levels, affect the T cell beta1 integrin function: opening of the channel leads to function, whereas its blockage prevents it. In support of this notion, we found that the proadhesive effects of the chemokine macrophage-inflammatory protein 1beta, the neuropeptide calcitonin gene-related peptide (CGRP), as well as elevated [K(+)](o) levels, are blocked by specific Kv1.3 channel blockers, and that the unique physiological ability of substance P to inhibit T cell adhesion correlates with Kv1.3 inhibition. Interestingly, the Kv1.3 channels and the beta1 integrins coimmunoprecipitate, suggesting that their physical association underlies their functional cooperation on the T cell surface. This study shows that T cells can be activated and driven to integrin function by a pathway that does not involve any of its specific receptors (i.e., by elevated [K(+)](o)). In addition, our results suggest that undesired T cell integrin function in a series of pathological conditions can be arrested by molecules that block the Kv1.3 channels.

Tyrosine kinases modulate K+ channel gating in mouse Schwann cells.

1. The whole-cell configuration of the patch-clamp technique and immunoprecipitation experiments were used to investigate the effects of tyrosine kinases on voltage-dependent K+ channel gating in cultured mouse Schwann cells. 2. Genistein, a broad-spectrum tyrosine kinase inhibitor, markedly reduced the amplitude of a slowly inactivating delayed-rectifier current (IK) and, to a lesser extent, that of a transient K+ current (IA). Similar results were obtained on IK with another tyrosine kinase inhibitor, herbimycin A. Daidzein, the inactive analogue of genistein, was without effect. 3. Unlike herbimycin A, genistein produced additional effects on IA by profoundly affecting its gating properties. These changes consisted of slower activation kinetics with an increased time to peak, a positive shift in the voltage dependence of activation (by +30 mV), a decrease in the steepness of activation gating (9 mV per e-fold change) and an acceleration of channel deactivation. 4. The steepness of the steady-state inactivation was increased by genistein treatment, while the recovery from inactivation was not significantly altered. 5. The action of genistein on voltage-dependent K+ (Kv) currents was accompanied by a decrease in tyrosine phosphorylation of Kv1.4 as well as Kv1.5 and Kv2.1 encoding transient and slowly inactivating delayed-rectifier K+ channel alpha subunits, respectively. 6. In conclusion, the present study shows that tyrosine kinases markedly affect the amplitude of voltage-dependent K+ currents in Schwann cells and finely tune the gating properties of the transient K+ current component IA. These modulations may be functionally relevant in the control of K+ channel activity during Schwann cell development and peripheral myelinogenesis.

Heteromultimeric delayed-rectifier K+ channels in schwann cells: developmental expression and role in cell proliferation.

Schwann cells (SCs) are responsible for myelination of nerve fibers in the peripheral nervous system. Voltage-dependent K+ currents, including inactivating A-type (KA), delayed-rectifier (KD), and inward-rectifier (KIR) K+ channels, constitute the main conductances found in SCs. Physiological studies have shown that KD channels may play an important role in SC proliferation and that they are downregulated in the soma as proliferation ceases and myelination proceeds. Recent studies have begun to address the molecular identity of K+ channels in SCs. Here, we show that a large repertoire of K+ channel alpha subunits of the Shaker (Kv1.1, Kv1.2, Kv1.4, and Kv1.5), Shab (Kv2.1), and Shaw (Kv3.1b and Kv3.2) families is expressed in mouse SCs and sciatic nerve. We characterized heteromultimeric channel complexes that consist of either Kv1.5 and Kv1.2 or Kv1.5 and Kv1.4. In postnatal day 4 (P4) sciatic nerve, most of the Kv1.2 channel subunits are involved in heteromultimeric association with Kv1.5. Despite the presence of Kv1. 1 and Kv1.2 alpha subunits, the K+ currents were unaffected by dendrotoxin I (DTX), suggesting that DTX-sensitive channel complexes do not account substantially for SC KD currents. SC proliferation was found to be potently blocked by quinidine or 4-aminopyridine but not by DTX. Consistent with previous physiological studies, our data show that there is a marked downregulation of all KD channel alpha subunits from P1-P4 to P40 in the sciatic nerve. Our results suggest that KD currents are accounted for by a complex combinatorial activity of distinct K+ channel complexes and confirm that KD channels are involved in SC proliferation.

A Ca2+/calmodulin-dependent protein kinase modulates Drosophila photoreceptor K+ currents: a role in shaping the photoreceptor potential.

Light activation of Drosophila photoreceptors leads to the generation of a depolarizing receptor potential via opening of transient receptor potential and transient receptor potential-like cationic channels. Counteracting the light-activated depolarizing current are two voltage-gated K+ conductances, IA and IK, that are expressed in these sensory neurons. Here we show that Drosophila photoreceptors IA and IK are regulated by calcium-calmodulin (Ca2+/calmodulin) via a Ca2+/calmodulin-dependent protein kinase (CaM kinase), with IK being far more sensitive than IA. Inhibition of Ca2+/calmodulin by N-(6 aminohexyl)-5-chloro-1-naphthalenesulfonamide or trifluoperazine markedly reduced the K+ current amplitudes. Likewise, inhibition of CaM kinases by KN-93 potently depressed IK and accelerated its C-type inactivation kinetics. The effect of KN-93 was specific because its structurally related but functionally inactive analog KN-92 was totally ineffective. In Drosophila photoreceptor mutant ShKS133, which allows isolation of IK, we demonstrate by current-clamp recording that inhibition of IK by quinidine or tetraethylammonium increased the amplitude of the photoreceptor potential, depressed light adaptation, and slowed down the termination of the light response. Similar results were obtained when CaM kinases were blocked by KN-93. These findings place photoreceptor K+ channels as an additional target for Ca2+/calmodulin and suggest that IK is well suited to act in concert with other components of the signaling machinery to sharpen light response termination and fine tune photoreceptor sensitivity during light adaptation.

Constitutive activation of delayed-rectifier potassium channels by a src family tyrosine kinase in Schwann cells.

In the nervous system, Src family tyrosine kinases are thought to be involved in cell growth, migration, differentiation, apoptosis, as well as in myelination and synaptic plasticity. Emerging evidence indicates that K+ channels are crucial targets of Src tyrosine kinases. However, most of the data accumulated so far refer to heterologous expression, and native K+-channel substrates of Src or Fyn in neurons and glia remain to be elucidated. The present study shows that a Src family tyrosine kinase constitutively activates delayed-rectifier K+ channels (IK) in mouse Schwann cells (SCs). IK currents are markedly downregulated upon exposure of cells to the tyrosine kinase inhibitors herbimycin A and genistein, while a potent upregulation of IK is observed when recombinant Fyn kinase is introduced through the patch pipette. The Kv1.5 and Kv2.1 K+-channel alpha subunits are constitutively tyrosine phosphorylated and physically associate with Fyn both in cultured SCs and in the sciatic nerve in vivo. Kv2.1- channel subunits are found to interact with the Fyn SH2 domain. Inhibition of Schwann cell proliferation by herbimycin A and by K+-channel blockers suggests that the functional linkage between Src tyrosine kinases and IK channels could be important for Schwann cell proliferation and the onset of myelination.

Characterization of delayed rectifier Kv channels in oligodendrocytes and progenitor cells.

We examined the molecular identity of K+ channel genes underlying the delayed rectifier (IK) in differentiated cultured oligodendrocytes (OLGs) and oligodendrocyte progenitor (OP) cells. Using reverse transcription-PCR cloning, we found that OP cells and OLGs expressed multiple Kv transcripts, namely Kv1.2, Kv1.4, Kv.1.5, and Kv1.6. Immunocytochemical and Western blot analyses revealed that Kv1.5 and Kv1.6 as well as Kv1.2 and Kv1.4 channel proteins could be detected in these cells, but definitive evidence for functional K+ channel expression was obtained only for the Kv1.5 channel. In addition, mRNA and immunoreactive protein levels of both Kv1.5 and Kv1.6 channels were significantly lower in differentiated OLGs when compared with levels in OP cells. Proliferation of OP cells was inhibited by K+ channel blockers, but not by incubation with either Kv1.5 or Kv1.6 antisense oligonucleotides. We conclude that (1) IK in OP cells and OLGs is encoded partly by Kv1.5 subunits, possibly forming heteromultimeric channels with Kv1.6 or other Kv subunits; and (2) inhibition of Kv1.5 or Kv1.6 channel expression alone does not prevent mitogenesis. Concomitant inhibition of other Kv channels underlying IK may be necessary for OP cells to exit from cell cycle.

[Thymus-dependent nature of the antigens of enteropathogenic viruses]. / Timus-zavisimaia priroda antigenov énteropatogennykh virusov.

The nature of antigens in enteropathogenic animal viruses--coronavirus--an agent of transmissive gastroenteritis of pigs (TGE) and pig enterovirus of serotype VI was studied. Basing on the results of the histomorphometric study of the white spleen pulp under the individual introduction of viruses and in combination with the lymphoid chalone it was established that the developing immune response has a thymus-dependent induction mechanism. The pharmacological stimulation of the T-system of animals suffering from virus gastroenteritis provides a positive therapeutic effect. The selective stimulation of the animal T-system may be considered as a promising trend in therapy of intestinal infections and as one of the possible ways to increase the immune response to the antigens of viral vaccines.

[Development of specific technics for the prevention of mycoplasma infections in swine]. / K razrabotke metodov spetsificheskoi profilaktiki mikoplazmoza svinei.

Four experimental series were run in 2 experiments with 44 unweaned piglets to test non-inactivated vaccine from ts-mutant M-60 of Mycoplasma (M.) arginini and from attenuated strains of CH-2 M. hyorhinis, EP-29 M. hyosynoviae, M. suipneumoniae, and B-1 Acholeplasma laidlawii. Similar deviations of clinical and immunological parameters were recorded from piglets inoculated with the above vaccine and infected with pathogenic mycoplasma cultures. These deviations, however, were less strongly pronounced in animals which had been inoculated. Mycoplasma species were re-isolated from bronchial lymph nodes and lungs of 62.5% of inoculated piglets. Lasting residual virulence was recorded from the attenuated mycoplasma strains. That residual virulence had no substantial impact upon growth and development of the piglets under laboratory conditions, throughout the period of observation. The above results are likely to suggest the advisability of further studies for the development of a vaccine from ts-mutants and attenuated strains of pathogens of mycoplasmosis in swine.

Acyclic analogs of nucleosides. Synthesis and in vitro antiviral activity of hydroxyalkyl-2-(trifluoromethylthiomethyl) benzimidazoles.

In a search for novel antiviral compounds of the 'doubly modified' nucleoside type, we have prepared 1-(4-hydroxy-2-oxabutyl)-, 1-(4-hydroxy-3-hydroxymethyl-2-oxabutyl)-, 1-(4-hydroxy-1-hydroxymethy1-2-oxabutyl)-, 1-(4-hydroxy-1-methyl-2-oxabutyl), 1-(4,5-dihydroxy-2-oxapentyl)-, 1-(5-hydroxy-2-oxapentyl), 1-(5-hydroxy-1-chloromethyl-2-oxapentyl)-, and 1-(6-hydroxy-1-chloromethyl-2-oxahexyl)-2-(trifluoromethylthiomethyl)benzimidazole. They were obtained by condensing the trimethylsilyl derivative of 2-(trifluoromethylthiomethyl) benzimidazole with alkylating agents in the presence of an equimolar mixture of trifluoromethanesulfonic acid and trimethylchlorosilane. These nucleoside analogs showed moderate antiviral activity against some RNA viruses.

Acyclic analogs of nucleosides. Synthesis of hydroxyalkyl derivatives of 2-trifluoromethyl- and 2-trifluoromethylthiobenzimidazole.

1-(2,3-Dihydroxypropyl)-, 1-(4-hydroxy-2-oxabutyl)-, 1-(3-hydroxymethyl-4-hydroxy-2-oxabutyl)-, 1-(1,5-dihydroxy-3-oxa-2-pentyl)-, 1-(5-hydroxy-3-oxa-2-pentyl)-, and 1-(4,5-dihydroxy-2-oxapentyl)-2-trifluoromethyl- and -2-trifluoromethylthiobenzimidazoles were obtained by condensation of trimethylsilyl derivatives of 2-substituted benzimidazoles with alkylating agents in the presence of SnCl4, or by direct alkylation of the sodium salts of the heterocycles.