Purification of the tetrodotoxin-binding component associated with the voltage-sensitive sodium channel from Electrophorus electricus electroplax membranes.

Agnew, William S.; Levinson, S. Rock; Brabson, John S.; Raftery, Michael A.

doi:10.1073/pnas.75.6.2606

Cited by 248 publications

(176 citation statements)

References 22 publications

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“…This value is in good agreement with estimates by irradiation inactivation (4) and sedimentation studies (22,23). Further, the longest axis of the particle is consistent with the Stokes radius of the detergent-solubilized molecule measured by gel exclusion chromatography (95 A) (18). For instance, if lipid and detergent were bound along the circumference of the rod, an approximately spherical particle of these dimensions would be expected.…”

Section: Discussionsupporting

confidence: 77%

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Electron microscopic visualization of the tetrodotoxin-binding protein from Electrophorus electricus.

Ellisman¹,

Agnew²,

Miller³

et al. 1982

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

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Preparations of highly purified tetrodotoxinbinding protein (sodium channel) from the electric organ ofthe eel Electrophorus electricu were examined in negatively stained preparations. Structures observed in preparations exhibiting the highest tetrodotoxin binding tended to aggregate into ordered clusters with a unique ribbon-like conformation. The individual particles of these aggregates are elongated or rod-shaped, approximately 40 A wide and 170 A long. Stereoscopic imaging ofthe three-dimensional aspects of the structures revealed that the rodlike image is not an edge view of a flattened disc but represents a cylindrical structure. Individual rods in nonclustered forms were also observed but with greater frequency in preparations with lower specific activity. The dimensions ofthe particles suggest that they represent a protein core formed by perhaps one copy of the large glycopeptide previously identified as being part of the sodium channel. The structure of the sodium channel component visualized by negative staining is discussed in the context of the excitable properties it contributes to biological membranes.The electrical excitability of the membranes of both nerve and muscle cells is a consequence ofrapid and precisely controlled changes in the membrane conductance ofspecific ions, notably Na' and KV. The electrical potential across the membrane is a consequence ofthe relative permeabilities to these ions (1-3), which are, in turn, modulated by the action of highly specialized ion channels (4,5). Electrophysiological and pharmacological properties of the channels have been well studied (6, 7), but little is known of the structural and mechanistic characteristics of these complex protein structures (4, 5). Indeed, these "electrically excitable channels" represent a previously uncharacterized class of membrane proteins. The voltage-sensitive sodium channel forms a cation-selective pore that spans the membrane and transports sodium in preference to other ions present in physiological solutions (8 There are a variety of pharmacological agents that interfere with the complex function ofthe sodium channel. Among these, the neurotoxins tetrodotoxin (TTX) and saxitoxin reversibly block sodium movement at nanomolar concentrations, apparently by occluding the pathway through which the ions pass (9). The remarkable specificity of these toxins has resulted in their application as biochemical markers for quantitation of the sodium channel in tissues, disrupted membranes, and solubilized preparations (6,10).We have been engaged in the biochemical isolation of the voltage-sensitive sodium channel from the electroplax of the South American eel Electrophorus electricus. We have previously described the purification ofthe TTX-and saxitoxin-binding protein from the electroplax membrane and some of its physicochemical characteristics (11). It is a glycoprotein ofs2O,, = 8 S and Stokes radius = 95 A, and, purified, it is an aggregate particle of protein-detergent (Lubrol-PX) and phospholipid (phosphatidylcholine)....

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Section: Discussionsupporting

confidence: 77%

“…The purification of the TTXR was similar to that reported by Agnew et aL (18). Medium-sized eels (1-1.5 m) were killed by hypothermia, and the main organs were removed and stored at -800C.…”

mentioning

confidence: 52%

Electron microscopic visualization of the tetrodotoxin-binding protein from Electrophorus electricus.

Ellisman¹,

Agnew²,

Miller³

et al. 1982

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

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“…The detailed protocol for the Lubrol-PX purification procedure has been described previously (1,3) . In addition, we have recently developed a method of purifying the protein with sodium cholate as the solubilizing detergent .…”

Section: Methodsmentioning

confidence: 99%

“…These membranes served as the starting material for both the Lubrol-PX and the sodium cholate purification procedures. Material solubilized in either detergent was assayed by the G-50 gel filtration system described by Levinson and co-workers (1,9) with the modification that the G-50 Sephadex was equilibrated in buffer containing 0 .1 % Lubrol-PX.…”

Section: Methodsmentioning

confidence: 99%

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

Ellisman

Miller

Agnew³

1983

The Journal of Cell Biology

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The appearance of detergent-solubilized voltage-regulated sodium channel protein was recently characterized by this laboratory. Negative-staining revealed rod-shaped particles measuring 40 X 170 A. Further studies have suggested that the actual configuration of this protein may be quite different from the rod-shaped structures. Freeze-fracture and freeze-etch images of the protein in reconstituted membranes indicated that the channel is cylindrical with a diameter of 100 A and a minimum length of 80 A. Experiments with two detergent systems (Lubrol-PX and sodium cholate) enabled us to explain the discrepancy between this structure and the rod-shaped particles visualized earlier. Negative staining in either detergent at low pH (4.5) produced rod-shaped structures. As the pH was increased, doughnut-shaped particles, consistent with the structure of the protein in freeze-etch, appeared in negative stain. The tendency of the protein to change shape under different pH conditions appears to be a peculiar property of this protein.

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Type I and type II Na+ channel ?-subunit polypeptides exhibit distinct spatial and temporal patterning, and association with auxiliary subunits in rat brain

et al. 1999

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Here we investigate differences in the temporal and spatial patterning, and subunit interactions of two of the major Na+ channel α‐subunit isoforms in mammalian brain, the type I and type II Na+ channels. By using subtype‐specific antibodies, we find that both isoforms are abundant in adult rat brain, where both interact with the covalently bound β2 auxiliary subunit. Immunoblot analysis reveals complementary levels of type I and type II in different brain regions, with the highest levels of type I in brainstem, cortex, substantia nigra, and caudate, where it is found predominantly on the soma of neurons, and the highest levels of type II in globus pallidus, hippocampus and thalamus, where it is preferentially localized to axons. Developmentally, type I Na+ channel polypeptide expression in brain increases dramatically during the third postnatal week, peaks at the end of the first postnatal month, and then decreases such that adult levels are ≈ 50% of those at peak. Type II Na+ channel polypeptide expression in brain also undergoes large increases in the third postnatal week, but levels continue to increase such that peak expression levels are maintained in adult animals. Type I Na+ channels are found associated with the auxiliary β2 subunit at all ages, whereas free type II Na+ channels exist during the first two postnatal weeks. Thus, although expression of these two Na+ channel α subunits in heterologous systems yields currents with very similar electrophysiological and pharmacological properties, their distinct spatial and temporal patterning, and association with auxiliary subunits in brain, suggest that they perform distinct, nonoverlapping functions in situ. J. Comp. Neurol. 412:342–352, 1999. © 1999 Wiley‐Liss, Inc.

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Purification of the tetrodotoxin-binding component associated with the voltage-sensitive sodium channel from Electrophorus electricus electroplax membranes.

Cited by 248 publications

References 22 publications

Electron microscopic visualization of the tetrodotoxin-binding protein from Electrophorus electricus.

Electron microscopic visualization of the tetrodotoxin-binding protein from Electrophorus electricus.

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

Type I and type II Na+ channel ?-subunit polypeptides exhibit distinct spatial and temporal patterning, and association with auxiliary subunits in rat brain

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