Common features of antigenic determinants present on mammalian nerve cell membrane structures and in cytoplasmic tetrodotoxin-sensitive proteins

Possible antigenic features common to nerve cell membrane structures and cytoplasmic tetrodotoxin-sensitive proteins were investigated by means of an immobilized immunoenzyme test. It was found that antiserum obtained by immunizing rabbits with a purified preparation of cytoplasmic tetrodotoxin-sensitive proteins can react with antigenic determinants present on the membrane fraction of bovine brain cells, on rat synaptosomes and on cells of a clonal line of mouse neuroblastoma. It was shown by means of an inhibition assay test that antibodies of the same specificity contribute to the observed response. Findings would indicate the presence of antigenic determinants common to nerve cell membrane structures and cytoplasmic tetrodotoxin-sensitive proteins. This is consistent with the hypothesis that cytoplasmic tetrodotoxin-sensitive proteins have certain features in common with membrane sodium channels.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 369–372, May–June, 1987.     

Antigenic similarity between cytoplasmic tetrodotoxin-sensitive protein and neuronal membrane proteins

Membrane proteins with a molecular weight of 290, 180, and 55 kDa were isolated using immunosorbent attached to sepharose and rabbit antibodies to cytoplasmic tetrodotoxin-sensitive protein from beef brain gray matter. A technique used for research into voltage-dependent sodium channels was applied to reconstruction of these proteins and investigation of toxin-dependent sodium flows through the lipoprotein membrane. Findings are interpreted as evidence of the similarity between cytoplasmic tetrodotoxin-sensitive protein and that of sodium channels at the cell membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev; A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 4, pp. 485–489, July–August, 1989.     

Electrically operated sodium channels in the somatic membrane of sympathetic neurons

Electrically operated sodium channels in the somatic membrane of isolated neurons from the rat superior cervical ganglion were investigated using an intracellular dialysis technique and voltage clamping. It was found that sodium currents can be conveyed along two independent systems of sodium channels in these neurons. A mathematical analysis was made of voltage-dependent tetrodotoxin-sensitive fast sodium currents within the framework of the Hodgkin-Huxley model and their kinetic properties were compared with those described in other subjects. It was also shown that the tetrodotoxin-sensitive sodium channels in the somatic membrane of sympathetic neurons have a high affinity for sodium ions. The kinetic and voltage-dependent characteristics of slow tetrodotoxin-sensitive inward sodium current are described. It is also noted that this component of the sodium current was observed in only a limited number of neurons (not more than 2%).A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 1, pp. 108–117, January–February, 1986.     

Murine neuroblastoma cells absorb anti-cytoplasmic tetrodotoxin-sensitive protein antibodies

The properties were investigated of polyclonal antibodies obtained by immunizing with a fraction of cytoplasmic glycoproteins forming sodium channels in liposomes. It was shown that these antibodies can be absorbed by intact murine neuroblastoma cells. Graphs plotting intensity of absorption against numbers of absorbant cells follow a characteristic course dependent on life of the cells in culture and serum concentration in the culture medium.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 98–105, January–February, 1988.     

Muscarinic Acetylcholine Receptors in Neuroblastoma Cells: Lack of Effect of Veratrum Alkaloids on Receptor Number

The effect of compounds that activate sodium channels on the number of muscarinic acetylcholine receptors in neuroblastoma NIE 115 cells has been investigated. The cells were used in electrically unexcitable ("control" cells) and excitable ("differentiated" cells) states. Although receptor assays using a single concentration of the radioligand [3H]scopolamine methyl chloride indicated a loss of receptors after a 6-h incubation of cells with veratrine, no true loss of receptors was seen with any of the compounds tested (veratridine, veratrine, aconitine) when full saturation analyses were performed in either control or differentiated cells. The apparent receptor loss seen with veratrine was due to a muscarinic receptor-active component of veratrine (not veratridine) occluded by the cells and released into the binding assays upon cell breakage. Veratridine and aconitine have a very low affinity for muscarinic acetylcholine receptors, and the binding of carbamoylcholine to the receptors is unaffected by tetrodotoxin, so that there is no evidence in this system for interaction between muscarinic receptors and sodium channels.     

Localization of epitopes for antibodies that differentially label sodium channels in skeletal muscle surface and T-tubular membranes

Summary We previously characterized two monoclonal antibodies, A/B2 and L/D3, that bind to the amino-terminus of the sodium channel but produce distinct immunocytochemical patterns in innervated adult skeletal muscle. Because these findings suggested the presence of several channel isoforms, we sought to define the epitopes for each antibody. Five peptides encompassing the amino-terminal 126 residues of the adult skeletal muscle sodium channel were synthesized and tested by radioimmunoassay against each antibody. Both monoclonals bound only to a peptide comprising residues 1–30 (I^1–30). A nested set of peptides within this region was then synthesized and used to compete for antibody binding to II^1–30. L/D3 binding was quantitatively inhibited by oligopeptides 1–30, 7–30, 13–30, and 19–30 but not 25–30, while binding of A/B2 was blocked only by the intact I^1–30 peptide. This data implies that the epitope for L/D3 lies within residues 19–25 while the epitope for A/B2 is contained within residues 1–6. These tentative epitope localizations were confirmed using both proteolytic cleavage of I^1–30 and immunoreactivity of a peptide corresponding to residues 1–12 with A/B2 but not L/D3. Therefore, epitopes for each monoclonal antibody are present in the SkM-1 sequence and are in close proximity in the amino-terminus of the protein. Their characteristic immunocytochemical labeling patterns may reflect differing accessibility of the epitopes in various membrane environments.We wish to thank Dr. John Lambris for helpful discussions. We also thank Ms. Candace Mello and Mr. James Hills for their expert technical assistance. This work was supported in part by NIH Grant NS 18013 (RLB) and by a grant from the W.W. Smith Charitable Trust (SAC). SAC is a Scholar of the Pfizer Scholar's Program for New Faculty.     

The coupling of Na,K-ATPase with the sodium channels in the plasma membranes of the brain synaptosomes of rats]

Effect of neurotoxins veratrine (100 micrograms/ml) and tetrodotoxin (1 microM) on the binding of 3H-ouabain (10(-8) M) with Na,K-ATPase of intact synaptosomes and isolated synaptic membranes was studied. The persistent opening of sodium channels in synaptosomes by veratrine results in an increase of specific binding of the labeled ligand by 20%. A similar effect was caused by Na/H exchanger monensin. Destruction of microtubules with vinblastine and colchicine has no influence on veratrine action, while depolymerization of microfilaments with cytochalasin B reverses the neurotoxin effect. In isolated synaptic membranes veratrine and tetrodotoxin stimulate ouabain binding, the absolute veratrine-induced increment being several times higher in the presence of ATP than in its absence. Since the closed vesicles of any type are not permeable to ATP and ouabain, it means that in the isolated membranes an interaction between sodium channels and Na,K-ATPase molecules takes place. In intact nerve endings such a mechanism may be operative along with the known ways of control of sodium pump and its ouabain-binding site.     

Tetrodotoxin-sensitive protein in the extracts from excitable tissues

The sodium permeability of liposomes preincubated with the soluble fraction of brain and heart muscle homogenates was increased veratrine. The veratrine increment was decreased by tetrodotoxin. The effect was specific for the extracts from excitable tissues. Bovine serum and soluble fraction of liver homogenate induced neither veratrine- nor tetrodotoxin-sensitivity of the liposomes. Treatment of the excitable tissue extracts by pronase and heat denaturation caused their complete inactivation. Tetrodotoxin-sensitive factor could be fractionated by ammonium sulfate precipitation and by DEAE-Servacel chromatography. On a column of Sephadex G-200 it was eluted with the void volume. It is suggested that the tetrodotoxin-sensitive factor is a protein which could be a soluble precursor of the voltage-dependent sodium channels.     

Changes in the ionic mechanisms of electrical excitability in the somatic membrane of rat dorsal root ganglion neurons during ontogenesis. Correlations between inward current densities

Correlations between densities of various types of inward currents in the somatic membrane of dorsal root ganglion neurons were studied in three different rat age groups: 5–9 days, 45 days, and 90 days. A linear relationship was found in neurons with "slow" tetrodotoxin-sensitive sodium current between the densities of high-threshold calcium current and "slow" sodium current (Bravias-Pearson's correlation coefficient: r=0.84 and 0.70 for n₁=16 and n₂=28, respectively). No such correlation was observed in neurons with low-threshold calcium inward current. Cells with only two types of channel — "fast" sodium and high-threshold calcium — present in their somatic membrane manifested an inverse correlation (r=–0.48, where n₄=95) between the densities of transmembrane currents passing through these channels. No inverse relationship was observed in the density of "fast" sodium and high-threshold calcium currents in neurons with tetradotoxinresistant "slow" sodium and/or low threshold calcium channels.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 6, pp. 820–827, November–December, 1986.     

Immunization with syngeneic Sendai virus-infected cells induce no MHC-restricted antibodies but antibodies specific for H-2 class I determinants

To find out whether immunoglobulins are able to recognize foreign antigens in the context of syngeneic MHC determinants, an effort was made to trigger the production of MHC-restricted antibodies by syngeneic Sendai virus (SV)-infected cells using the spleen-fragment culture technique. Antibodies were found that mimicked MHC-restricted antibodies by recognizing MHC + SV better than MHC alone. However, the binding was not specific for SV and also occurred on mitogen-stimulated (SV^–) or influenza virus-infected cells. We describe the production of H-2 class I-specific lymphocytotoxic antibodies by primary B cells responding to syngeneic SV-infected cells. No viral-specific, H-2-restricted antibodies were found.     

Binding parameters of monoclonal antibodies reacting with ovarian carcinoma ascites cells

Summary Binding parameters were determined for four mouse monoclonal antibodies reacting with three antigens on the surface of fresh human ovarian carcinoma ascites cells, under nearly physiological conditions. The object of these experiments was to aid in the selection of the optimal monoclonal antibodies for intraperitoneal immunotherapy. The number of antigenic sites per cell, the effective equilibrium association constant (affinity) and the half-life for dissociation were: for Ab MH99, 1.2×10⁶ sites/cell, (1.9–4.1)×10⁸M^–1, and 4 h; for Ab MX35, (3.2–4.1)×10⁵ sites/cell, (3.4–4.8)×10⁸M^–1, and >10 h; and for Ab MW207, 1.3×10⁵ sites/cell, (3.6–4.1)×10⁹M^–1, and 3.1 h, respectively. One of the antigens, MH99, is recognized by five different monoclonal antibodies, and competitive inhibition experiments demonstrated that two distinct determinants are present; this antigen is also recognized by the previously described Ab 17-1A. These binding data will aid the rational design of immunotherapy strategies.     

Effects of allapinine on sodium currents in neurons isolated from the rat trigeminal ganglion and cardiomyocytes

The effects were investigated of allapinine, diterpene alkaloid on ionic currents in voltage-clamped trigeminal neurons and cardiomyocytes isolated from rats. Allapinine application was found to exert an inhibitory effect on inward tetrodotoxin-sensitive sodium currents without changing their voltage dependence. Potential differences between the mechanisms of antiarrhythmic action of diterpene alkaloids and standard antiarrhythmic substances are examined.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Institute of Plant Chemistry, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Neirofiziologiya, Vol. 22, No. 2, pp. 201–206, March–April, 1990.     

Effect of phosphorylation on the functional properties of the sodium channel reconstituted into an artificial membrane

The phosphorylation by protein kinase A of cytoplasmic protein of bovine brain, reconstituted as liposome membrane, is studied. The reconstituted protein imitates the potential-dependent sodium channel. The effect of phosphorylation is shown to depend on the way in which the protein is reconstructed into the artificial membrane. Phosphorylation of channels reconstructed in the open state inhibits the rate of sodium ion flux, while phosphorylation of channels reconstructed in the closed state enhances the activating effect of veratrine and scorpion toxin.A. A. Bogomolets Institute of Physiology, Ukrainian Academy of Sciences, Kiev. A. V. Palladin Institute of Biochemistry, Ukrainian Academy of Sciences, Kiev. Translated from Neirofiziologiya, Vol. 24, No. 2, pp. 192–198, March–April, 1992.     

Binding of monoclonal antibodies to cerebral cytoplasmic tetrodotoxin-sensitive protein to human lymphocytes

To examine whether lymphocytes express antigenic determinants of brain cytoplasmic tetrodotoxin-sensitive protein (CTSP), anti-CTSP monoclonal antibody (Bab) binding to human peripheral blood lymphocytes was studied using ELISA assay. It was shown that the Mab bound human lymphocytes in a dose-dependent fashion. Halph-maximal binding of this Mab was significantly enhanced if test-cells were pretreated with concanavalin A or with mixed lymphocyte culture supernatant. Results are discussed from the point of view of the hypothesis that CTSP are metabolic precursors of membrane sodium channels.     

Characterization of monoclonal antibodies to protoplast membranes of Nicotiana tabacum identified by an enzyme-linked immunosorbent assay

Murine monoclonal antibodies to protoplast membrne antigens were generated using mouse myelomas and spleen cells from mice immunized with Nicotiana tabacum L. leaf protoplasts. For selecting antibody-secreting clones, a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for monoclonal antibody binding to immobilized cellular membrane preparations or immobilized protoplasts was developed. With intact protoplasts as immobilized antigen, the ELISA is selective for antibodies that bind to plasma-membrane epitopes present on the external surface of protoplasts. Using the membrane ELISA, a total of 24 hybridoma lines were identified that secreted antibodies to plant membrane epitopes. The protoplast ELISA and subsequent immunofluorescence studies identified four hybridoma lines as secreting antibodies which bound to the external surface of protoplasts and cells. The corresponding antigens were not species- or tissue-specific, were periodatesensitive, and were located in membranes which equilibrated broadly throughout a linear sucrose gradient. When protein blots of electrophoretically separated membrane proteins were probed with these antibodies, a band of M_r 14 kilodaltons (kDa) and a smear of bands of M_r 45–120 kDa were labeled. An additional set of three antibodies appeared by immunofluorescence to bind to the plasma membrane of broken but not intact protoplasts and labeled membranes equilibrating at a density of approx. 1.12 kg·l^-1 in a linear sucrose density gradient. These classes of monoclonal antibodies enlarge the library of monoclonal antibodies (Norman et al. 1986, Planta 167, 452–459) available for the study of plant plasma-membrane structure and function.Abbreviations ELISA Enzyme-linked immunosorbent assay - Ig immunoglobulin - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Changes in the ionic mechanisms of the electrical excitability of rat sensory neuronal somatic membrane during ontogenesis. Distribution of ionic channels carrying inward currents

The distribution of different types of ionic channels carrying inward currents was investigated in the somatic membranes of spinal ganglion neurons in rats belonging to three different age groups: at 5–9 days, 45 days, and 3 months. A decrease was found in the number of neuronal membranes operating all four types of inward current channels simultaneously: "fast" (tetrodotoxin-sensitive), "slow" (tetrodotoxin-resistant) sodium currents and low- and high-threshold calcium currents. There were 14.5% of such neurons in the first age group, 5% in the second, and 1% on the third. It was found that this change was related to the disappearance of "slow" (tetrodotoxin-resistant) sodium and high-threshold calcium channels from the membrane. The number of neuronal somatic membranes with only two types of inward current channels ("fast" sodium and high-threshold calcium channels) increased proportionately.A. A. Bogomolets Institute of Technology, Academy of Sciences of the Ukrainian SSR, Kiev Translated from Neirofiziologiya, Vol. 18, No. 6, pp. 813–820, November–December, 1986.     

Effects of water-soluble carbodiimide on "fast" sodium channel gating in the rat sensory neuron membrane

Currents through "fast" (tetrodotoxin-sensitive) channels before and after external application of solutions containing 100 mM 1-ethyl-3-dimethylaminopropyl)carbodiimide-HCl (WSC) were measured during volage clamping at the membrane of dialyzed neurons of rat spinal ganglia. Treating the membrane with WSC (pH 4.8–4.9) led to a 5 to 20-fold reduction in sodium conductance, a 1.5–2.5-fold deceleration in the dynamics of current increase, and less abrupt voltage-dependent sodium channel activation curves. The shifted effective charge of activation was normally halved. The WSC produced no effect on activation parameters at normal pH (7.6). It was deduced that the changes observed resulted from WSC reacting with carboxyl groups located on the outer surface of the membrane. These groups are thought to be involved in the system of charge movements of the sodium channel gating mechanism.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 46–53, January–February, 1987.     

Interaction between sea anemone toxin BgTX8 and ionic channels of neurons isolated from mammals

The action of the toxin BgTX₈ separated from the sea actiniaBunodosoma granolifera on transient tetrodotoxin-sensitive sodium and outward potassium currents of units isolated from rat sensory ganglia was investigated using techniques of voltage clamping at the membrane and intracellular perfusion. It was found that BgTX₈ decelerates the inactivation kinetics but has little effect on activation kinetics of sodium current. At the same time, a 5–10% increase in the amplitude of inward current was often observed at holding potentials of about –100 to –120 mV at the membrane. The dissociation constant of the receptor-toxin equals 4×10^–6 M and is adequately described by Langmuir's isotherm. It was also established that intracellular perfusion of neurons with anemone toxin-containing solution leads to a reduction in the amplitude of sodium current and decelerates its inactivation process. Suppression of outward potassium current was also noted.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Institute of Brain Research, Academy of Sciences, Havana, Cuba. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 32–37, January–February, 1988.     

New HLA-A2 variants defined by monoclonal antibodies and cytotoxic T lymphocytes

Three HLA-A2 variants, A2-DW, A2-KC, and A2-Lee, were identified in three Chinese donors using a panel of monoclonal antibodies. A2-DW was negative with two of the ten HLA-A2 monoclonal antibodies tested, whereas A2-KC was negative with five of the ten and A-2 Lee was negative with one.Epstein-Barr virus-specific cytotoxic T cells generated from the A2-DW donor recognized and killed target cells prepared from the A2-KC donor, but did not recognize target cells from HLA-A2.1, –A2.2, or –A2.4 donors. In isoelectric focusing studies, A2-DW and A2-KC focus in identical positions more acidic than the other HLA-A2 antigens tested.     

Monoclonal antibodies raised against post-translational domains of the electroplax sodium channel

Summary Eleven monoclonal antibodies were identified that recognized eel electroplax sodium channels. All the monoclonal antibodies specifically immunostained the mature TTX-sensitive sodium channel (M _r 265,000) on immunoblots. None of the monoclonal antibodies would precipitate the in vitro translated channel core polypeptide in solution. One monoclonal antibody, 3G4, was found to bind to an epitope involving terminal polysialic acids. Extensive digestion of the channel by the exosialidase, neuraminidase, or partial polysialic acid removal bythe endosialidase, endo-N-acetylneuraminidase, destroy the 3G4 epitope, 3G4 is, therefore, a highly selective probe for the post-translationally attached polysialic acids. Except for this monoclonal antibody, the epitopes recognized by the remaining antibodies were highly resistant to extensive N-linked deglycosylation. Thus, the monoclonal antibodies may be directed against unique post-translationally produced domains of the electroplax sodium channel, presumably sugar groups that are abundant on this protein (Miller, J.A., Agnew, W.S., Levinson, S.R. 1983.Biochemistry 22:462–470). These monoclonal antibodies should prove useful as tools to study discrete post-translational processing events in sodium channel biosynthesis.