Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide.

Gordon, Robert D.; Fieles, William; Schotland, Donald L.; Hogue-Angeletti, Ruth A.; Barchi, Robert L.

doi:10.1073/pnas.84.1.308

Cited by 41 publications

(10 citation statements)

References 40 publications

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“…USA 86 (1989) support for this orientation has come from localization (Fig. SB) of antibody-binding sites (25,26), cAMP-dependent phosphorylation sites (27), and sites of channel inactivation (23). These models all propose that hydrophobic segments S1-S6 are transmembrane segments, although no direct experimental evidence supports these proposals.…”

Section: Discussionmentioning

confidence: 94%

Localization of the receptor site for alpha-scorpion toxins by antibody mapping: implications for sodium channel topology.

Thomsen

Catterall

1989

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

153

107

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Site-directed and monoclonal antibodies recognizing different extracellular regions of the RI, sodium channel a subunit have been used to determine the sequences that comprise the receptor for a-scorpion toxins by evaluating the effect of antibody on voltage-dependent binding of radiolabeled toxin isolated from Leiurus quinquestriatus to both reconstituted rat brain sodium channel and rat brain synaptosomes. Kinetic studies of the antibody effect are consistent with a slowly reversible competition for the toxin receptor site. Our results suggest that the extracellular loops between segments S5 and S6 of domains I and IV comprise at least part of the a-scorpion toxin receptor site and support the membrane topology models in which domains I and IV are adjacent in the tertiary structure of the channel protein and six transmembrane sequences are contained in each of the four homologous domains.The voltage-dependent sodium channel is an integral plasma membrane protein responsible for the increase in sodium ion permeability during the rapidly rising phase of the action potential of excitable cells (1). The sodium channel isolated from rat brain consists of three nonidentical glycoprotein subunits, a 260-kDa a subunit that is covalently associated with a 33-kDaP2 subunit and noncovalently associated with a 36-kDa ,1 subunit (2, 3). Messenger RNA encoding the a subunit alone can direct the synthesis of functional sodium channels in Xenopus oocytes (4, 5) and mammalian somatic cells (6). Computer analysis and similarity comparisons ofthe primary amino acid sequence of sodium channel a subunits (3) has been used to construct topological models of the a subunit within the plasma membrane having four homologous domains containing six (3) or eight (7, 8) transmembrane segments per domain. a-Scorpion toxin V isolated from the venom of the North African scorpion Leiurus quinquestriatus (LqTx) binds to neurotoxin receptor site 3 on the sodium channel in a voltage-dependent manner (9, 10) and slows or blocks sodium channel inactivation at an extracellular locus (11,12). Photoreactive derivatives of LqTx covalently label the a subunits in neurons. synaptosomes, and purified and reconstituted sodium channels (13-16), and sodium channels expressed from a-subunit mRNA in somatic cells have a functional receptor site for LqTx (6). The site of covalent attachment of photoreactive derivatives to the a subunit has been localized by immunoprecipitation with site-directed antipeptide antibodies to the extracellular loop between proposed transmembrane segments S5 and S6 of domain I (16). In the present experiments, we have analyzed the effects of a series of site-directed antipeptide and monoclonal antibodies (mAbs) on the voltage-dependent binding of LqTx to sodium channels to define regions of the channel structure that are located on its extracellular surface and are required for toxin binding at higher resolution.

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Section: Discussionmentioning

confidence: 94%

Localization of the receptor site for alpha-scorpion toxins by antibody mapping: implications for sodium channel topology.

Thomsen

Catterall

1989

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

153

107

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“…The site for alpha-scorpion toxins (ScTx) between $5 and $6 of repeat I [6]. CT: C terminal region [13]; R6: cytoplasmic region [14]. The C~ region is pointed.…”

Section: Structural Basis For the Differences Between The Na Channelsmentioning

confidence: 99%

Structural and developmental differences between three types of Na channels in dorsal root ganglion cells of newborn rats

1990

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The changes in Na current during development were studied in the dorsal root ganglion (DRG) cells using the whole-cell patch-clamp technique. Cells obtained from rats 1-3 and 5-8 days after birth were cultured and their Na currents were compared. On top of the two types of Na currents reported in these cells (fast-FA current and slow-S current) a new fast current was found (FN). The main characteristics of the three currents are: (i) The voltages of activation are -37, -36 and -23 mV for the FN, FA and S currents, respectively. (ii) The activation and inactivation kinetics of FN and FA currents are about five times faster than those of the S current. (iii) The voltages at which inactivation reaches 50% are -139, -75 and -23 mV for the FN, FA and S currents, respectively. The kinetics and voltage-dependent parameters of the three currents and their density do not change during the first eight days after birth. However, their relative frequency in the cells changes. In the 1-3 day-old rats the percent of cells with S, FA, and mixed S + FN currents is 22, 18, and 60% of the cells, respectively. In the 5-8 day-old, the percent of cells with S, FA, and FN + S is 10, 66 and 22%. The relative increase in the frequency of cells with FA current during development can contribute to the ease of action potential generation compared with cells with FN currents, which are almost completely inactivated under physiological conditions. The predominance of FA cells also results in a significant decrease in the relative frequency of cells with the high-threshold, slow current. Antibodies directed against a part of the S4 region of internal repeat I of the sodium channel (C1+, amino acids 210-223, eel channel numbering) were found to shift the voltage dependence of FA current inactivation (but not of FN or S currents) to more negative potentials. The effect was found only when the antibodies were applied externally. The results suggest that FN, FA and S types of Na currents are generated by channels, which are different in the topography of the C1+ region in the membrane.

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“…FITC (fluorescein isothiocyanate)-conjugated secondary antibodies are then used to visualize the association in the epifluorescence microscope. Similar assays have been used to determine the molecular topography of proteins associated with reconstituted cell membranes (31)(32)(33).…”

Section: Introductionmentioning

confidence: 99%

Lipid bilayer surface association of lung surfactant protein SP-B, amphipathic segment detected by flow immunofluorescence

Longo¹,

Waring²,

Zasadzinski³

1992

Biophysical Journal

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Lung surfactant protein, SP-B, and synthetic amphipathic peptides derived from SP-B were studied in model lung surfactant lipid bilayers by immunofluorescent labeling. Liposomes were formed by hydrating a lipid film on the glass viewing port of a temperature controlled flow chamber. Membrane associated peptides were detected by epifluorescence optical microscopy of the binding of anti-peptide polyclonal monospecific antibodies and FITC-conjugated secondary antibodies added to buffer contained in the flow chamber. Liposomes were bound by antibody to residues 1-25 of SP-B if formed from lipid films containing the 1-25 peptide, (SP-B(1-25)), or if SP-B(1-25) was added to already formed liposomes in buffer solution. The distribution of antigen-antibody complex was temperature dependent with aggregation occurring at greater than or equal to 30 degrees C. Surface association was not detected in liposomes formed from lipid films containing the 49-66 peptides (SP-B(49-66)), using an antibody to the 49-66 peptide, or to a synthetic version of the SP-B protein, (SP-B(1-78)), using both antibodies to the 49-66 peptide and the 1-25 peptide. The detection of SP-B(1-78) with antibody to the 49-66 sequence was only possible after reducing SP-B(1-78) with dithiothreitol, suggesting that the COOH-terminus of the full monomer protein is accessible to the bulk aqueous environment unlike the COOH-terminal peptide. The size, number of layers, and fluidity of the liposomes were not altered by protein or peptides, although they were affected by lipid composition and temperature.

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Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide.

Cited by 41 publications

References 40 publications

Localization of the receptor site for alpha-scorpion toxins by antibody mapping: implications for sodium channel topology.

Localization of the receptor site for alpha-scorpion toxins by antibody mapping: implications for sodium channel topology.

Structural and developmental differences between three types of Na channels in dorsal root ganglion cells of newborn rats

Lipid bilayer surface association of lung surfactant protein SP-B, amphipathic segment detected by flow immunofluorescence

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