Molecular morphology of the tetrodotoxin-binding sodium channel protein from Electrophorus electricus in solubilized and reconstituted preparations.

Ellisman, Mark H.; Miller, James A.; Agnew, William S.

doi:10.1083/jcb.97.6.1834

Cited by 17 publications

(7 citation statements)

References 18 publications

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“…The evidence of Na+ channel accumulation on end bulb axolemma is consistent with our prior light and electron microscopic evidence of Na+ channel immunolocalization in neuromas in the lateral line nerve of Apteronotus using fish-specific anti-Na+ channel antibodies (Devor et al, 1989). Likewise, since Na+ channels yield large-diameter intramembranous particles (IMPS) in freeze-fracture replicas (Ellisman, 1979;Ellisman et al, 1983;Rosenbluth, 1983;Small et al, 1984;Stricharz et al, 1984) it is consistent with the observed shift of IMP sizes in rat neuroma end bulb axolemma to larger values (Fried et al, 1991).…”

Section: Neuroma End Bulbssupporting

confidence: 86%

Na+ channel immunolocalization in peripheral mammalian axons and changes following nerve injury and neuroma formation

1993

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Nerve injury frequently triggers hyperexcitability and the ectopic initiation of impulses in primary afferent axons. An important consequence is neuropathic paresthesias and pain. Electrogenesis in normal afferents depends on appropriate Na+ channel concentrations. Therefore, we have asked whether injury might trigger changes in axolemmal Na+ channel distribution that could account for neuropathic hyperexcitability. We used an Na+ channel-specific antibody, 7493, to immunolocalize Na+ channels ultrastructurally in membranes of normal rat axons, and to assess remodeling following nerve section and neuroma formation. Selective labeling of nodal axolemma and, more weakly, of Schwann cell membrane, confirmed the efficacy of our immunolabeling protocol. In neuromas at postoperative times associated with peak ectopic activity, we found clear evidence of Na+ channel accumulation. Specifically, soon after myelin was stripped from large-diameter axons, the exposed, formerly internodal axolemma became immunopositive. Small-diameter unmyelinated axons and axon sprouts in the neuroma were also marked with 7493 IgG. Activated phagocytic macrophages and endothelial cells were 7493 negative. Both large- and small-diameter axons in neuromas end in swollen, organelle-packed "end bulbs." Most, but not all, of these acquired Na+ channel immunolabeling. We propose that remodeling results from a modification of the normal process of Na+ channel turnover in neural membranes. Na+ channel protein accumulates in preterminal axolemma and neuroma end bulbs due to a combination of permissive factors (especially myelin removal) and promotional factors (removal of normal downstream targets). This accumulation is a likely precursor of afferent hyperexcitability in injured nerve.

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Section: Neuroma End Bulbssupporting

confidence: 86%

Na+ channel immunolocalization in peripheral mammalian axons and changes following nerve injury and neuroma formation

1993

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“…The concept of an evolutionarily related neuronal ion channel gene family (25) is, however, supported by a number of biochemical, pharmacological, and morphological observations: (i) the overall or subunit molecular masses of the neuronal ion channels analyzed so far are very similar (25); (ii) many different drugs are known that affect several types of channel proteins (ref. 26; unpublished data); and (iii) recent electron microscope analysis suggests an astounding similarity between the molecular morphology of the nicotinic acetylcholine receptor and the voltage-dependent sodium channel isolated from fish electric organ (27,28). In our view, all of these structural and functional relationships between the different neuronal ion channels are highly suggestive of a common evolutionary root of this class of membrane proteins (25).…”

Section: Discussionmentioning

confidence: 62%

“…2, lanes 4-7, and Table 2). In other experiments, the major common peptides obtained with V8 protease (28,15, and 11 kDa) were digested further with chymotrypsin. This resulted in the production of three or four peptide fragments of the same apparent molecular mass (19,17,11, and 9 kDa) from all three glycine receptor polypeptides (Table 2).…”

Section: Resultsmentioning

confidence: 99%

Monoclonal antibodies and peptide mapping reveal structural similarities between the subunits of the glycine receptor of rat spinal cord.

Pfeiffer¹,

Simler²,

Grenningloh³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

301

273

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The glycine receptor of rat spinal cord is an oligomeric membrane glycoprotein of molecular mass 250,000 daltons that contains three polypeptides of 48,000, 58,000, and 93,000 daltons. Monoclonal antibodies (mAbs) were prepared against the affinity-purified glycine receptor protein by using '5I-labeled receptor preparations for the detection of positive hybrids. From nine monoclonal antibodies obtained, six recognized denatured receptor polypeptides blotted to nitrocellulose paper. Two of these antibodies bound to more than one glycine receptor polypeptide: mAb GlyR 4a stained the 48,000-and 58,000-dalton polypeptides, and mAb GlyR 7a stained the 48,000-and 93,000-dalton polypeptides. Common antigenic determinants thus are shared by the different subunits of the glycine receptor. Complementary results were obtained by peptide mapping of '251-labeled glycine receptor polypeptides with various proteases. A set of peptide fragments of the same apparent molecular mass was produced from the different glycine receptor polypeptides by using V8 protease, chymotrypsin, and elastase. These data suggest that the subunits of the glycine receptor have significant homology within their primary structure and may have evolved from a common ancestor receptor polypeptide.The amino acid glycine is a major inhibitory neurotransmitter in the spinal cord and other regions of the central nervous system of vertebrates (1). Upon binding to its receptor, glycine increases the chloride conductance of the postsynaptic membrane and thus produces a hyperpolarization-i.e., inhibition of neuronal firing (2, 3). This hyperpolarizing action of glycine can be antagonized by the plant alkaloid strychnine, a potent convulsive poison in man and animals (1-4). Radiolabeled strychnine has been widely used to study and localize the glycine receptor in the nervous systems of different species (5, 6). We have employed strychnine as a tool in the purification and biochemical analysis of the glycine receptor (reviewed in ref. 7).From electrophysiological and biochemical studies, it was concluded that the glycine receptor is an integral membrane protein that contains a chemically gated chloride ion channel. Upon solubilization with nonionic detergents, the glycine receptor behaves as a large glycoprotein (molecular mass -250 kDa) whose native conformation depends on the presence of exogenous phospholipids (8, 9). After affinitypurification on aminostrychnine-agarose, three polypeptides of 48, 58, and 93 kDa are detected (9); all three polypeptides are thought to be subunits of the glycine receptor (7, 9). The 48-kDa polypeptide can be covalently labeled with [3H]-strychnine and thus harbors the antagonist binding site of the glycine receptor (10,11). The function of the other polypeptides is presently unknown.To further explore the membrane topology and functional sites of the different glycine receptor subunits, we have produced monoclonal antibodies (mAbs) against this neuronal membrane protein. Here, we report that two of these mAbs recognize mor...

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“…The size distribution and freeze-fracture pattern suggest that some of these particles may be the (Na + +K+)ATPase (Skriver et al, 1980) or the voltagesensitive sodium channel (Ellisman et al, 1983), two macromolecules concentrated along normal nodal axolemma (Ellisman & Levinson, 1982;Ariyasu et al, 1985).…”

Section: Introductionmentioning

confidence: 98%

The distribution of (Na+ + K+)ATPase is continuous along the axolemma of unensheathed axons from spinal roots of ‘dystrophic’ mice

Ariyasu

Ellisman

1987

J Neurocytol

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(Na+ + K+)ATPase-like immunoreactivity along the axolemma of sensory and motor neurons and the plasmalemma of Schwann cells from spinal roots of dystrophic mice (129 ReJ Dy/Dy) was determined using polyclonal antibodies specific for guinea pig renal (Na+ + K+)ATPase (GP-17), along with polyclonal (439-2) and monoclonal (9A5) antibodies specific for rat renal (Na+ + K+)ATPase. In normal and dystrophic mice, (Na+ + K+)ATPase-like immunoreactivity was observed along the axolemma at nodes of Ranvier using GP-17 and 439-2, each of which binds to isozymes of (Na+ + K+)ATPase composed of the alpha and alpha + forms of the catalytic subunit. Staining was not seen along the nodal axolemma with 9A5, a preparation that binds to the alpha form of the catalytic subunit. The terminal processes and microvilli of Schwann cells were stained using all three antibody probes. The axolemma of unensheathed axons in dystrophic mice was continuously and uniformly labelled with GP-17 and 439-2, but not 9A5. Concentrations of (Na+ + K+)ATPase-like immunoreactivity along Schwann cell processes were observed most often in areas adjacent to unensheathed axolemma. At heminodes, staining abruptly decreased along Schwann cell processes in areas that were separated from the unensheathed axolemma by other intervening Schwann cell processes. It was concluded from these data that in dystrophic mice (Na+ + K+)ATPase is uniformly distributed along unensheathed portions of axons without evidence of detectable focal concentrations of the enzyme, and that the catalytic subunit of (Na+ + K+)ATPase along unensheathed axons is distinct from the alpha form found in Schwann cells and other organs. In addition, (Na+ + K+)ATPase is concentrated along the plasmalemma of Schwann cells in regions of close apposition to axolemmal areas associated with large ionic fluxes.

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Molecular morphology of the tetrodotoxin-binding sodium channel protein from Electrophorus electricus in solubilized and reconstituted preparations.

Cited by 17 publications

References 18 publications

Na+ channel immunolocalization in peripheral mammalian axons and changes following nerve injury and neuroma formation

Na+ channel immunolocalization in peripheral mammalian axons and changes following nerve injury and neuroma formation

Monoclonal antibodies and peptide mapping reveal structural similarities between the subunits of the glycine receptor of rat spinal cord.

The distribution of (Na+ + K+)ATPase is continuous along the axolemma of unensheathed axons from spinal roots of ‘dystrophic’ mice

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