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1.
J Comp Neurol ; 524(12): 2479-91, 2016 08 15.
Article in English | MEDLINE | ID: mdl-26780193

ABSTRACT

In many vertebrates parallel processing in topographically ordered maps is essential for efficient sensory processing. In the active electrosensory pathway of mormyrids afferent input is processed in two parallel somatotopically ordered hindbrain maps of the electrosensory lateral line lobe (ELL), the dorsolateral zone (DLZ), and the medial zone (MZ). Here phase and amplitude modulations of the self-generated electric field were processed separately. Behavioral data indicates that this information must be merged for the sensory system to categorically distinguish capacitive and resistive properties of objects. While projections between both zones of the ELL have been found, the available physiological data suggests that this merging takes place in the midbrain torus semicircularis (TS). Previous anatomical data indicate that the detailed somatotopic representation present in the ELL is lost in the nucleus lateralis (NL) of the TS, while a rough rostrocaudal mapping is maintained. In our study we investigated the projections from the hindbrain to the midbrain in more detail, using tracer injections. Our data reveals that afferents from both maps of the ELL terminate in a detailed somatotopic manner within the midbrain NL. Furthermore, we provide data indicating that phase and amplitude information may indeed be processed jointly in the NL. J. Comp. Neurol. 524:2479-2491, 2016. © 2016 Wiley Periodicals, Inc.


Subject(s)
Brain Mapping/methods , Electric Fish/physiology , Electric Organ/physiology , Mesencephalon/physiology , Sensation/physiology , Afferent Pathways/chemistry , Afferent Pathways/physiology , Animals , Electric Organ/chemistry , Mesencephalon/chemistry , Septal Nuclei/chemistry , Septal Nuclei/physiology
2.
Proc Natl Acad Sci U S A ; 110(26): 10622-7, 2013 Jun 25.
Article in English | MEDLINE | ID: mdl-23754381

ABSTRACT

The scaffolding protein at the neuromuscular junction, rapsyn, enables clustering of nicotinic acetylcholine receptors in high concentration and is critical for muscle function. Patients with insufficient receptor clustering suffer from muscle weakness. However, the detailed organization of the receptor-rapsyn network is poorly understood: it is unclear whether rapsyn first forms a wide meshwork to which receptors can subsequently dock or whether it only forms short bridges linking receptors together to make a large cluster. Furthermore, the number of rapsyn-binding sites per receptor (a heteropentamer) has been controversial. Here, we show by cryoelectron tomography and subtomogram averaging of Torpedo postsynaptic membrane that receptors are connected by up to three rapsyn bridges, the minimum number required to form a 2D network. Half of the receptors belong to rapsyn-connected groups comprising between two and fourteen receptors. Our results provide a structural basis for explaining the stability and low diffusion of receptors within clusters.


Subject(s)
Muscle Proteins/chemistry , Receptors, Nicotinic/chemistry , Animals , Binding Sites , Cryoelectron Microscopy , Electric Organ/chemistry , Electric Organ/diagnostic imaging , Electron Microscope Tomography , Humans , Models, Molecular , Models, Neurological , Multiprotein Complexes/chemistry , Multiprotein Complexes/ultrastructure , Neuromuscular Junction/chemistry , Neuromuscular Junction/ultrastructure , Protein Structure, Quaternary , Receptors, Nicotinic/ultrastructure , Torpedo , Ultrasonography
3.
Bioorg Khim ; 37(5): 609-15, 2011.
Article in Russian | MEDLINE | ID: mdl-22332355

ABSTRACT

Lynx1 expresses in the central nervous system and plays important role in a regulation of nicotinic acetylcholine receptors. Successful milligram-quantitive expression of ws-Lynx1 was achieved only in the case of its production in the form of cytoplasm inclusion bodies. Different conditions of ws-Lynx1 refolding for yield optimization were performed. The obtained recombinant protein was characterized by means of mass spectrometry and CD spectroscopy. The binding experiments on the nAChRs from Torpedo californica membranes revealed that ws-Lynxl is biologically active and blocks muscle nAChR with IC50-20-30 microM.


Subject(s)
GPI-Linked Proteins/biosynthesis , Neurotransmitter Agents/biosynthesis , Receptors, Nicotinic/metabolism , Recombinant Proteins/biosynthesis , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Base Sequence , Bungarotoxins/antagonists & inhibitors , Electric Organ/chemistry , Electric Organ/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , GPI-Linked Proteins/chemistry , GPI-Linked Proteins/pharmacology , Gene Expression , Genetic Vectors , Humans , Molecular Sequence Data , Neurotransmitter Agents/chemistry , Neurotransmitter Agents/pharmacology , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , Solubility , Torpedo
4.
Biochim Biophys Acta ; 1798(6): 1153-63, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20303928

ABSTRACT

The interaction of 18-methoxycoronaridine (18-MC) with nicotinic acetylcholine receptors (AChRs) was compared with that for ibogaine and phencyclidine (PCP). The results established that 18-MC: (a) is more potent than ibogaine and PCP inhibiting (+/-)-epibatidine-induced AChR Ca(2+) influx. The potency of 18-MC is increased after longer pre-incubation periods, which is in agreement with the enhancement of [(3)H]cytisine binding to resting but activatable Torpedo AChRs, (b) binds to a single site in the Torpedo AChR with high affinity and inhibits [(3)H]TCP binding to desensitized AChRs in a steric fashion, suggesting the existence of overlapping sites. This is supported by our docking results indicating that 18-MC interacts with a domain located between the serine (position 6') and valine (position 13') rings, and (c) inhibits [(3)H]TCP, [(3)H]ibogaine, and [(3)H]18-MC binding to desensitized AChRs with higher affinity compared to resting AChRs. This can be partially attributed to a slower dissociation rate from the desensitized AChR compared to that from the resting AChR. The enthalpic contribution is more important than the entropic contribution when 18-MC binds to the desensitized AChR compared to that for the resting AChR, and vice versa. Ibogaine analogs inhibit the AChR by interacting with a luminal domain that is shared with PCP, and by inducing desensitization.


Subject(s)
Cholinergic Antagonists/chemistry , Electric Organ/chemistry , Fish Proteins/chemistry , Ibogaine/analogs & derivatives , Receptors, Cholinergic/chemistry , Torpedo , Animals , Binding Sites , Ibogaine/chemistry , Protein Binding , Protein Structure, Tertiary
5.
Article in English | MEDLINE | ID: mdl-15711967

ABSTRACT

Elasmobranchs (sharks, skates, and rays) possess an electrosensory system with an infrastructure of canals connecting the electrosensors to the environment. The electrosensors and canals are filled with a uniform hydrogel, but the gel's function has not yet been determined. We present electrical admittance spectra collected from the hydrogel from 0.05 to 100 kHz, covering the effective range of the electrosensors. We have taken samples of this gel, postmortem, from Triaenodon obesus and Carcharodon carcharias; for purposes of comparison, we have synthesized a series of collagen-based hydrogel samples. The shark hydrogels demonstrate suppressed admittance when compared to both seawater and collagen gels. In particular, collagen hydrogels with equivalent ion concentrations are roughly 2.5 times more polarizable than the shark samples. We conclude that the shark hydrogels strongly localize ionic species, and we discuss the implications for the related roles of the gel and the canals in the electric sense. The gel-filled canals appear better suited to fostering voltage differences along their length than to providing direct electrical contact to the seawater environment.


Subject(s)
Electric Organ/chemistry , Electric Organ/physiology , Hydrogels/analysis , Hydrogels/chemistry , Sharks/physiology , Animals , Electric Capacitance , Electric Impedance , Sharks/classification , Species Specificity
6.
J Struct Biol ; 143(3): 185-200, 2003 Sep.
Article in English | MEDLINE | ID: mdl-14572474

ABSTRACT

In single-particle analysis, a three-dimensional (3-D) structure of a protein is constructed using electron microscopy (EM). As these images are very noisy in general, the primary process of this 3-D reconstruction is the classification of images according to their Euler angles, the images in each classified group then being averaged to reduce the noise level. In our newly developed strategy of classification, we introduce a topology representing network (TRN) method. It is a modified method of a growing neural gas network (GNG). In this system, a network structure is automatically determined in response to the images input through a growing process. After learning without a masking procedure, the GNG creates clear averages of the inputs as unit coordinates in multi-dimensional space, which are then utilized for classification. In the process, connections are automatically created between highly related units and their positions are shifted where the inputs are distributed in multi-dimensional space. Consequently, several separated groups of connected units are formed. Although the interrelationship of units in this space are not easily understood, we succeeded in solving this problem by converting the unit positions into two-dimensional (2-D) space, and by further optimizing the unit positions with the simulated annealing (SA) method. In the optimized 2-D map, visualization of the connections of units provided rich information about clustering. As demonstrated here, this method is clearly superior to both the multi-variate statistical analysis (MSA) and the self-organizing map (SOM) as a classification method and provides a first reliable classification method which can be used without masking for very noisy images.


Subject(s)
Proteins/chemistry , Proteins/ultrastructure , Algorithms , Animals , Cryoelectron Microscopy , Electric Organ/chemistry , Electrophorus , Image Processing, Computer-Assisted/statistics & numerical data , Neural Networks, Computer , Sodium Channels/chemistry , Sodium Channels/ultrastructure , Software Design
7.
Toxicon ; 41(4): 509-17, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12657321

ABSTRACT

Crotoxin, a potent neurotoxin from the South American rattlesnake Crotalus durissus terrificus, is a heterodimeric phospholipase A(2) (EC 3.1.1.4), which blocks the release of acetylcholine from peripheral neurons. We previously have suggested the existence of a 48 kDa crotoxin-binding protein in the presynaptic membranes of the electric organ of Torpedo marmorata. Here, we report the purification and characterization of this protein that we called the crotoxin acceptor protein from Torpedo (CAPT). The membranes of electric organs from Torpedo were solubilized with a detergent (4% (w/v) Triton X-100) and CAPT was isolated by affinity chromatography on a crotoxin column. SDS-PAGE showed that the purified protein was homogeneous and cross-linking studies with radioiodinated crotoxin confirmed that it had retained its toxin-binding properties. The purified CAPT has similar molecular mass as crocalbin, a crotoxin-binding protein isolated from porcine brains, yet anti-crocalbin antiserum failed to recognize CAPT. Surface plasmon resonance biosensor technology was used to measure the specific interaction between crotoxin and solubilized CAPT. Using this method, it was possible to follow CAPT throughout the purification procedure. As well, an apparent dissociation constant (K(d)(app)) of 3.4 nM was calculated for the interaction of pure CAPT and crotoxin from the dissociation rate constant (k(off)=1.2 x 10(-2)s(-1)) and the association rate constant (k(on)=3.5 x 10(6)M(-1)s(-1)).


Subject(s)
Crotoxin/metabolism , Electric Organ/chemistry , Receptors, Cell Surface/isolation & purification , Receptors, Cell Surface/metabolism , Surface Plasmon Resonance , Torpedo , Animals , Crotalus , Protein Binding , Receptors, Cell Surface/chemistry , Synaptic Membranes/metabolism
8.
J Membr Biol ; 196(1): 1-8, 2003 Nov 01.
Article in English | MEDLINE | ID: mdl-14724751

ABSTRACT

Electrocytes from the electric organ of Electrophorus electricus exhibited sodium action potentials that have been proposed to be repolarized by leak currents and not by outward voltage-gated potassium currents. However, patch-clamp recordings have suggested that electrocytes may contain a very low density of voltage-gated K(+) channels. We report here the cloning of a K(+) channel from an eel electric organ cDNA library, which, when expressed in mammalian tissue culture cells, displayed delayed-rectifier K(+) channel characteristics. The amino-acid sequence of the eel K(+) channel had the highest identity to Kv1.1 potassium channels. However, different important functional regions of eel Kv1.1 had higher amino-acid identity to other Kv1 members, for example, the eel Kv1.1 S4-S5 region was identical to Kv1.5 and Kv1.6. Northern blot analysis indicated that eel Kv1.1 mRNA was expressed at appreciable levels in the electric organ but it was not detected in eel brain, muscle, or cardiac tissue. Because electrocytes do not express robust outward voltage-gated potassium currents we speculate that eel Kv1.1 channels are chronically inhibited in the electric organ and may be functionally recruited by an unknown mechanism.


Subject(s)
Electric Organ/chemistry , Electric Organ/physiology , Electrophorus/metabolism , Membrane Potentials/physiology , Potassium Channels, Voltage-Gated , Potassium Channels/chemistry , Potassium Channels/physiology , Potassium/metabolism , Amino Acid Sequence , Animals , CHO Cells , Cloning, Molecular , Cricetinae , Cricetulus , Electrophorus/genetics , Kv1.1 Potassium Channel , Molecular Sequence Data , Organ Specificity , Potassium Channels/genetics , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Species Specificity , Tissue Distribution , Xenopus laevis
9.
J Neurobiol ; 53(3): 391-402, 2002 Nov 15.
Article in English | MEDLINE | ID: mdl-12382266

ABSTRACT

The cells of the electric organ, called electrocytes, of the weakly electric fish Sternopygus macrurus derive from the fusion of mature fast muscle fibers that subsequently disassemble and downregulate their sarcomeric components. Previously, we showed a reversal of the differentiated state of electrocytes to that of their muscle fiber precursors when neural input is eliminated. The dependence of the mature electrocyte phenotype on neural input led us to test the hypothesis that innervation is also critical during formation of electrocytes. We used immunohistochemical analyses to examine the regeneration of skeletal muscle and electric organ in the presence or absence of innervation. We found that blastema formation is a nerve-dependent process because regeneration was minimal when tail amputation and denervation were performed at the same time. Denervation at the onset of myogenesis resulted in the differentiation of both fast and slow muscle fibers. These were fewer in number, but in a spatial distribution similar to controls. However, in the absence of innervation, fast muscle fibers did not progress beyond the formation of closely apposed clusters, suggesting that innervation is required for their fusion and subsequent transdifferentiation into electrocytes. This study contributes further to our knowledge of the influence of innervation on cell differentiation in the myogenic lineage.


Subject(s)
Electric Organ/innervation , Muscle, Skeletal/innervation , Regeneration/physiology , Animals , Cell Differentiation/physiology , Electric Organ/chemistry , Electric Organ/physiology , Female , Gymnotiformes , Male , Muscle, Skeletal/chemistry , Muscle, Skeletal/physiology
10.
J Neurochem ; 82(4): 874-84, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12358793

ABSTRACT

The immunosuppressor cyclosporin A inhibits the peptidyl-prolyl-cis/trans-isomerase activity of cyclophilins and the resulting complex inhibits the phosphatase activity of calcineurin. Both enzymes were detected in peripheral nerve endings isolated from the electric organ of Torpedo and shown to be affected by 10 micro m cyclosporin A. Among the cholinergic properties studied, choline uptake was specifically inhibited by cyclosporin A to a maximum of 40%. Cyclosporin A decreased the rate of choline transport but not the binding of the non-transportable choline analogue hemicholinium-3, indicating that the number of membrane transporters was not affected. Through the use of two other immunosuppressors, FK506, which also inhibits calcineurin, and rapamycin, which does not, two different mechanisms of choline uptake inhibition were uncovered. FK506 inhibited the rate of choline transport, whereas rapamycin diminished the affinity for choline. The Torpedo homologue of the high affinity choline transporter CHT1 was cloned and its activity was reconstituted in Xenopus oocytes. Choline uptake by oocytes expressing tCHT1 was inhibited by all three immunosuppressors and also by microinjection of the specific calcineurin autoinhibitory domain A457-481, indicating that the phosphatase calcineurin regulates CHT1 activity and could be the common target of cyclosporin and FK506. Rapamycin, which changed the affinity of the transporter, may have acted through an immunophilin on the isomerization of critical prolines that are found in the tCHT1 sequence.


Subject(s)
Immunosuppressive Agents/pharmacology , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Synaptosomes/metabolism , Animals , Binding, Competitive/drug effects , Biological Transport/drug effects , Calcineurin/metabolism , Calcineurin Inhibitors , Choline/metabolism , Choline/pharmacokinetics , Cloning, Molecular , Cyclosporine/pharmacology , Electric Organ/chemistry , Enzyme Inhibitors/pharmacology , Hemicholinium 3/metabolism , Membrane Transport Proteins/drug effects , Microinjections , Models, Molecular , Molecular Sequence Data , Nerve Endings/chemistry , Nerve Endings/metabolism , Oocytes/drug effects , Oocytes/metabolism , Peptidylprolyl Isomerase/antagonists & inhibitors , Sequence Homology, Amino Acid , Sirolimus/pharmacology , Synaptosomes/chemistry , Synaptosomes/drug effects , Tacrolimus/pharmacology , Torpedo , Transfection , Xenopus laevis
11.
J Exp Zool ; 290(3): 227-33, 2001 Aug 01.
Article in English | MEDLINE | ID: mdl-11479902

ABSTRACT

Myosin light and heavy chains from skeletal and cardiac muscles and from the electric organ of Electrophorus electricus (L.) were characterised using biochemical and immunological methods, and compared with myosin extracted from avian, reptilian, and mammalian skeletal and cardiac muscles. The results indicate that the electric tissue has a myosin light chain 1 (LC1) and a muscle-specific myosin heavy chain. We also show that monoclonal antibody F109-12A8 (against LC1 and LC2) recognizes LC1 of myosin from human skeletal and cardiac muscles as well as those of rabbit, lizard, chick, and electric eel. However, only cardiac muscles from humans and rabbits have LC2, which is recognized by antibody F109-16F4. The data presented confirm the muscle origin of the electric tissue of E. electricus. This electric tissue has a profile of LC1 protein expression that resembles the myosin from cardiac muscle of the eel more than that from eel skeletal muscle. This work raises an interesting question about the ontogenesis and differentiation of the electric tissue of E. electricus.


Subject(s)
Electric Organ/physiology , Electrophorus/physiology , Myosin Heavy Chains/biosynthesis , Myosin Light Chains/biosynthesis , Animals , Antibodies, Monoclonal , Cell Differentiation , Electric Organ/chemistry , Heart/physiology , Humans , Muscle, Skeletal/physiology , Vertebrates/physiology
12.
Eur J Neurosci ; 13(2): 221-9, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11168526

ABSTRACT

Accumulating evidence points to the participation of dystroglycan in the clustering of nicotinic acetylcholine receptors at the neuromuscular junction [Côté et al. (1999) Nature Genet., 3, 338--342]. Dystroglycan is part of a multimolecular complex, either associated with dystrophin (the dystrophin-associated protein complex) at the sarcolemma or with utrophin (the utrophin-associated protein complex) at the neuromuscular junction. Understanding the assembly of this complex at the developing synapse led us to investigate, in Torpedo electrocyte, the intracellular routing and the targeting of several of its components, including dystroglycan, syntrophin, dystrophin and dystrobrevin. We previously demonstrated that acetylcholine receptors and rapsyn, the 43-kDa receptor-associated protein at the synapse, are cotargeted to the postsynaptic membrane via the exocytic pathway [Marchand et al. (2000) J. Neurosci., 20, 521--528]. Using cell fractionation, immunopurification and immuno-electron microscope techniques, we show that beta-dystroglycan, an integral glycoprotein that constitutes the core of the dystrophin-associated protein complex localized at the innervated membrane, is transported together with acetylcholine receptor and rapsyn in post-Golgi vesicles en route to the postsynaptic membrane. Syntrophin, a peripheral cytoplasmic protein of the complex, associates initially with these exocytic vesicles. Conversely, dystrophin and dystrobrevin were absent from these post-Golgi vesicles and associate directly with the postsynaptic membrane. This study provides the first evidence for a separate targeting of the various components of the dystrophin-associated protein complex and a step-by-step assembly at the postsynaptic membrane.


Subject(s)
Dystrophin-Associated Proteins , Dystrophin/metabolism , Membrane Proteins/metabolism , Muscle Proteins/metabolism , Receptors, Nicotinic/metabolism , Synaptic Vesicles/metabolism , Animals , Dystrophin/analysis , Electric Organ/chemistry , Electric Organ/cytology , Electric Organ/metabolism , Exocytosis/physiology , Membrane Proteins/analysis , Microscopy, Immunoelectron , Muscle Proteins/analysis , Neuropeptides/analysis , Neuropeptides/metabolism , Receptors, Nicotinic/analysis , Synaptic Vesicles/chemistry , Synaptic Vesicles/ultrastructure , Torpedo
13.
An Acad Bras Cienc ; 72(3): 341-51, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11028099

ABSTRACT

The electric eel Electrophorus electricus is a fresh water teleost showing an electrogenic tissue that produces electric discharges. This electrogenic tissue is distributed in three well-defined electric organs which may be found symmetrically along both sides of the eel. These electric organs develop from muscle and exhibit several biochemical properties and morphological features of the muscle sarcolema. This review examines the contribution of the cytoskeletal meshwork to the maintenance of the polarized organization of the electrocyte, the cell that contains all electric properties of each electric organ. The cytoskeletal filaments display an important role in the establishment and maintenance of the highly specialized membrane model system of the electrocyte. As a muscular tissue, these electric organs expresses actin and desmin. The studies that characterized these cytoskeletal proteins and their implications on the electrophysiology of the electric tissues are revisited.


Subject(s)
Cytoskeleton/chemistry , Electric Organ/chemistry , Electrophorus/anatomy & histology , Actin Cytoskeleton/chemistry , Actin Cytoskeleton/physiology , Actin Cytoskeleton/ultrastructure , Animals , Cytoskeleton/metabolism , Cytoskeleton/ultrastructure , Densitometry , Electric Organ/physiology , Electric Organ/ultrastructure , Electrophoresis, Gel, Two-Dimensional , Electrophorus/physiology , Microscopy, Electron
14.
An. acad. bras. ciênc ; 72(3): 341-51, Sept. 2000. ilus, tab
Article in English | LILACS | ID: lil-269386

ABSTRACT

The electric eel Electrophorus electricus is a fresh water teleost showing an electrogenic tissue that produces electric discharges. This electrogenic tissue is distributed in three well-defined electric organs which may be found symmetrically along both sides of the eel. These electric organs develop from muscle and exhibit several biochemical properties and morphological features of the muscle sarcolema. This review examines the contribution of the cytoskeletal meshwork to the maintenance of the polarized organization of the electrocyte, the cell that contains all electric properties of each electric organ. The cytoskeletal filaments display an important role in the establishment and maintenance of the highly specialized membrane model system of the electrocyte. As a muscular tissue, these electric organs expresses actin and desmin. The studies that characterized these cytoskeletal proteins and their implications on the electrophysiology of the electric tissues are revisited.


Subject(s)
Animals , Cytoskeleton/chemistry , Electric Organ/chemistry , Electrophorus/physiology , Actin Cytoskeleton/chemistry , Actin Cytoskeleton/physiology , Actin Cytoskeleton/ultrastructure , Cytoskeleton/metabolism , Cytoskeleton/ultrastructure , Densitometry , Electric Organ/physiology , Electric Organ/ultrastructure , Electrophoresis, Gel, Two-Dimensional , Microscopy, Electron
15.
Neurosci Lett ; 291(1): 21-4, 2000 Sep 08.
Article in English | MEDLINE | ID: mdl-10962144

ABSTRACT

Asymmetric phospholipid distribution between the outer and inner monolayers of cholinergic synaptosomal membranes at rest and their redistribution upon depolarization-induced acetylcholine (ACh) release were investigated. Translocation of phospholipids between the monolayers was measured using fluorescence-labeled phospholipid probes, NBD-PS, NBD-PE, and NBD-PC. The percentage of probes in the inner leaflet at equilibrium in synaptosomes at rest was estimated to be 63% (NBD-PS), 36% (NBD-PE), and 31% (NBD-PC). Depolarization-induced exocytosis induced rapid redistribution of these probes. Approximately 35% of PS and PC in the inner leaflet moved to the outer leaflet, whereas only 16% of PE moved. To further elucidate the mechanism of exocytosis and membrane fusion at presynaptic nerve terminals in the future, the rapid phospholipid translocation associated with exocytosis must be taken into account.


Subject(s)
Cholinergic Fibers/metabolism , Lipid Bilayers/metabolism , Neurotransmitter Agents/metabolism , Phospholipids/metabolism , Synaptosomes/metabolism , 4-Chloro-7-nitrobenzofurazan/analogs & derivatives , 4-Chloro-7-nitrobenzofurazan/analysis , 4-Chloro-7-nitrobenzofurazan/metabolism , Acetylcholine/metabolism , Animals , Biological Transport , Cadmium/pharmacology , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Electric Organ/chemistry , Exocytosis/drug effects , Exocytosis/physiology , Fluorescence , Fluorescent Dyes , Membrane Fusion/physiology , Phosphatidylcholines/analysis , Phosphatidylcholines/metabolism , Phosphatidylethanolamines/analysis , Phosphatidylethanolamines/metabolism , Phosphatidylserines/analysis , Phosphatidylserines/metabolism , Phospholipids/analysis , Potassium Chloride/pharmacology , Presynaptic Terminals/metabolism , Skates, Fish/metabolism , Sonication , Synaptic Membranes/metabolism , Synaptosomes/chemistry , Synaptosomes/drug effects
16.
Int J Biochem Cell Biol ; 32(4): 427-33, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10762068

ABSTRACT

The present investigation deals with the purification and the partial characterization of the soluble creatine kinase (CK) isoenzyme, isolated from the electric organ electrocyte of Electrophorus electricus (L.). Purification was performed by precipitation of the enzyme in the crude extract with ammonium sulfate (80%). The precipitate obtained was analyzed on an ion exchange column of diethylaminoethyl cellulose-52 (DEAE) followed by gel filtration on Superose 12 in a Fast Protein Liquid Chromatography (FPLC) system. Electrophoretic mobility of the active peak confirmed previous results identifying the hybrid isoenzyme MB in the electrocyte cytoplasm. Electrocyte CK is a dimeric enzyme with two identical subunits of approximately 40 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The sequence analysis of the N-terminal peptide (14 amino acids) of the 40 kDa subunit showed homology with other CK enzymes from electric fish (Torpedo) and human muscle type CK.


Subject(s)
Creatine Kinase/isolation & purification , Electric Organ/chemistry , Animals , Creatine Kinase/chemistry , Electrophoresis, Polyacrylamide Gel , Electrophorus , Isoenzymes/chemistry , Isoenzymes/isolation & purification , Sequence Analysis, Protein
17.
Brain Res ; 845(2): 185-91, 1999 Oct 23.
Article in English | MEDLINE | ID: mdl-10536197

ABSTRACT

Miniature end-plate potentials (MEPPs) were focally recorded from the cytoplasmic surface of electrocytes in isolated columns of the Torpedo electric organ. Double electrode studies showed that the junctional area was restricted to 12 micron2. MEPP frequencies ranging from 1/min to 400/s were controlled with electrode advancement against the cytoplasmic surface. Stable membrane potentials and noise levels indicated constant intracellular, focal recording conditions. Focal MEPPs are only 1-3 mV and MEPP amplitudes smoothly decreased with an increase in MEPP frequency which demonstrates a process that meters quantal size at moment of release. Thus, release if not from a prepackaged store. MEPP interval analyses showed that events are weakly interactive at low frequencies and periodic at higher frequencies. The interdependency of MEPP amplitudes and intervals indicates that the mechanism of release controls both rate and quantal size. We propose that the amplitude and frequency dependencies of MEPPs at the Torpedo nerve-electrocyte junction are best described by a membrane channel (e.g., mediatophore, Israël and Dunant, Neurochem. Int. 28 (1996) 1-9) that meters transmitter from a presynaptic store.


Subject(s)
Electric Organ/physiology , Exocytosis/physiology , Motor Endplate/physiology , Neurotransmitter Agents/metabolism , Periodicity , Animals , Electric Organ/chemistry , Electrolytes/analysis , Electrophysiology , Logistic Models , Membrane Potentials/physiology , Presynaptic Terminals/chemistry , Presynaptic Terminals/physiology , Torpedo
18.
Cell Mol Neurobiol ; 19(6): 745-57, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10456235

ABSTRACT

1. Phencyclidine (PCP) is an inhibitor of the nicotinic acetylcholine receptor (AChR) with characteristics of an open-channel blocker. The location of PCP binding site on the AChR molecule is unknown. 2. PCP inhibits the AChR from electric organ with a higher potency than muscle AChR. To find the molecular basis of this difference, we expressed the two native and six hybrid receptors, and two receptors containing mutated mouse gamma subunits in Xenopus laevis oocytes. The inhibition of ACh-induced current in these receptors by PCP was studied using whole-cell voltage-clamp. All hybrid receptors generated robust ACh-induced currents, while incomplete receptors (gamma-less or delta-less) did not. 3. PCP potency was higher on hybrids containing Torpedo beta and gamma subunits regardless of the alpha and delta subunit origin. A mouse gamma subunit containing the asparagine 6' to the serine mutation in the M2 segment conferred a high sensitivity to PCP. 4. These results support the conclusion that the amino acid residues at the position 6' of the M2 segments contribute to the PCP potency difference between Torpedo and mouse receptors. 5. Another noncompetitive inhibitor of the AChR, the cembranoid eupalmerin acetate (EUAC), also inhibited the electric organ receptor with a somewhat higher potency than muscle AChR. However, the IC50 values for EUAC inhibition of hybrid receptors did not follow the pattern observed for PCP. Therefore, these two inhibitors interact differently with the AChR molecule.


Subject(s)
Electric Organ/chemistry , Muscle Proteins/drug effects , Muscle, Skeletal/chemistry , Nerve Tissue Proteins/drug effects , Nicotinic Antagonists/pharmacology , Phencyclidine/pharmacology , Receptors, Nicotinic/drug effects , Acetylcholine/pharmacology , Amino Acid Sequence , Amino Acid Substitution , Animals , Atropine/pharmacology , Binding Sites , DNA, Complementary/genetics , Diterpenes/pharmacology , Dose-Response Relationship, Drug , Ion Channel Gating/drug effects , Ion Channels/drug effects , Kinetics , Mice , Molecular Sequence Data , Muscle Proteins/chemistry , Muscle Proteins/isolation & purification , Mutagenesis, Site-Directed , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/isolation & purification , Patch-Clamp Techniques , Point Mutation , Protein Multimerization , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/genetics , Receptors, Nicotinic/isolation & purification , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Torpedo , Xenopus laevis
19.
J Neurosci ; 18(23): 9924-35, 1998 Dec 01.
Article in English | MEDLINE | ID: mdl-9822748

ABSTRACT

The electric organ (EO) of the weakly electric fish Sternopygus macrurus derives from striated myofibers that fuse and suppress many muscle properties. Mature electrocytes are larger than muscle fibers, do not contain sarcomeres, or express myosin heavy chain (MHC) or tropomyosin. Furthermore, electrocytes express keratin, a protein not expressed in muscle. In S. macrurus the EO is driven continuously at frequencies higher than those of the intermittently active skeletal muscle. The extent to which differences in EO and muscle phenotype are accounted for by activity patterns, or innervation per se, was determined by assessing the expression of MHC, tropomyosin, and keratin 2 and 5 weeks after the elimination of (1) activity patterns by spinal transection or (2) all synaptic input by denervation. Immunohistochemical analyses showed no changes in muscle fiber phenotypes after either experimental treatment. In contrast, the keratin-positive electrocytes revealed an upregulation of MHC and tropomyosin. Nearly one-third of all electrocytes expressed MHC (35%) and tropomyosin (25%) 2 weeks after spinal transection, whereas approximately two-thirds (61%) expressed MHC 2 weeks after denervation. After 5 weeks of denervation or spinal transection, all electrocytes contained MHC and tropomyosin. Newly formed sarcomere clusters also were observed in denervated electrocytes. The MHC expressed in electrocytes corresponded to that present in a select population of muscle fibers, i.e., type II fibers. Thus, the elimination of electrical activity or all synaptic input resulted in a partial reversal of the electrocyte phenotype to an earlier developmental stage of its myogenic lineage.


Subject(s)
Electric Fish/physiology , Electric Organ/chemistry , Electric Organ/innervation , Myosin Heavy Chains/analysis , Tropomyosin/analysis , Animals , Atrophy , Cell Division/physiology , Cells, Cultured , Electric Organ/pathology , Female , Gene Expression/physiology , Keratins/analysis , Keratins/genetics , Male , Muscle Denervation , Muscle Fibers, Skeletal/chemistry , Muscle Fibers, Skeletal/cytology , Muscle, Skeletal/chemistry , Muscle, Skeletal/innervation , Muscle, Skeletal/pathology , Myosin Heavy Chains/genetics , Phenotype , Sarcomeres/chemistry , Spinal Cord/surgery , Tropomyosin/genetics
20.
Article in English | MEDLINE | ID: mdl-9787763

ABSTRACT

Desmin, the intermediate filament protein of muscle, is present in the electric organs of Electrophorus electricus L. as five isovariants, instead of the one to two isovariants found in muscle. We analyzed the isodesmin pattern in the three different electric organs using densitometry of Coomassie blue-stained bands in electrofocusing polyacrylamide gel electrophoresis. We were able to compare the relative amount of each of the five desmin isovariants in an isodesmin pattern characteristic of each electric organ. These patterns proved to be, in some cases, statistically different. Desmin in each electric organ could have slightly different functions in order to correlate with the organ-specific isovariant patterns.


Subject(s)
Desmin/chemistry , Electric Organ/chemistry , Electrophorus/metabolism , Animals , Densitometry , Intermediate Filaments/chemistry , Isoelectric Focusing
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