Effects of some chemical reagents on sodium current inactivation in myelinated nerve fibers of the frog

Rack, Michael; Rubly, N.; Waschow, Christel

doi:10.1016/s0006-3495(86)83495-6

Cited by 39 publications

(19 citation statements)

References 34 publications

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“…Catterall (22) suggests a helical displacement for an a-helical Cl' domain, a structure that should be less mobile than a 310-helix. Chemical modification of groups in nerves can sometimes lead to changes in inactivation curves (28) but cannot be interpreted on the molecular level until the sequence positions of specific amino acid changes are delineated.…”

Section: Discussionmentioning

confidence: 99%

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Mapping a region associated with Na channel inactivation using antibodies to a synthetic peptide corresponding to a part of the channel.

Meiri¹,

Spira²,

Sammar³

et al. 1987

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

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Antibodies to the synthetic peptide (carriercoupled) corresponding to amino acids 210-223 of the primary sequence of eel Na channel (Cl+ peptide) were generated. The antipeptide antibodies were used to identify functional roles as well as the accessibility from the external membrane surface of the C1 domains. Rabbit antipeptide antibodies bound specifically to the Cl' synthetic peptide and to an eel membrane fraction bearing a high density of Na channels. When applied to the external surface of cultured dorsal root ganglion cells obtained from newborn rats, the antibodies modify Na channel inactivation by shifting the steady-state Na current-inactivation parameter, h., curve to more negative potentials in fast and slow Na currents. The rate of inactivation of the slow channel is shown to be increased. The antibodies do not have a significant effect on activation of the channels. Part of the amino acid sequence corresponding to Cl+ peptide is therefore accessible, in the mammalian Na channel, from the external membrane surface and is associated with the inactivation gate. Na channels are transmembrane protein macromolecules essential for the generation of action potentials in excitable tissues (1, 2). In species studied thus far, the channel molecules consist of a large single polypeptide (a subunit) of relative Mr = 260,000 (3-6). Those purified from mammalian brain (4) and rabbit skeletal muscle (6) contain the a subunit accompanied by two smaller polypeptides (f3 subunits) of Mr = 33,000-43,000. The primary structures of the Electrophorus Na channel and of two distinct Na channel a polypeptides from rat brain (rat Na channels I and II) have been elucidated by cloning and sequencing of the DNAs complementary to the mRNA (7,8). Functional tests of rat a subunit expressed in Xenopus oocytes have shown the presence of most of the structural features required for channel voltage sensitivity and ion selectivity (9-11).All three a subunits of the Na channel molecules that have been already cloned contain four homologous internal repeats. The four repeats are highly conserved among all Na channels, and it was postulated that these repeats form the channel (7,8,12). The remaining regions, much of which may be exposed to either the cytoplasm or the extracellular medium, are less well conserved. Each of the four internal repeats has hydrophobic segments (in one model, three or four; in others, five) and one positively charged amphiphilic segment (S4). The S4 segments ofthe four internal repeats contain four to six positively charged amino acids (arginine or lysine) residues located at every third position with neutral, mostly hydrophobic, residues intervening between the basic residues. The homology of this region is almost complete in the three Na channels for which the sequences are known (7,8).There have been a number of attempts to model the tertiary structure of the Na channel, beginning with the primary amino acid sequence (12-15). Although these models differ substantially, all agree that the S4 segment pl...

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Section: Discussionmentioning

confidence: 99%

“…Cultures were maintained in 6% CO2 in air at 37°C. After 6-7 hr in culture, the neurons (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) ,Am in diameter) could be distinguished from the fibroblasts, Schwann cells, and other cells by their round shape, bright hillarium, and short, thin neurites.…”

mentioning

confidence: 99%

Mapping a region associated with Na channel inactivation using antibodies to a synthetic peptide corresponding to a part of the channel.

Meiri¹,

Spira²,

Sammar³

et al. 1987

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

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“…It is well known that chemical modifications can change TTX sensitivity, and that these modifications are often associated with slowing current kinetics [54,61]. NGF cannot be included among these factors, since it does not change the characteristics of the currents, and has no acute effect on current properties.…”

Section: Post-translational Modulationmentioning

confidence: 99%

Characterization of sodium currents in mammalian sensory neurons cultured in serum-free defined medium with and without nerve growth factor

Omri

Meiri

1990

J. Membrain Biol.

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The influence of nerve growth factor (NGF) on Na currents of rat dorsal root ganglia (DRG) was studied in neurons obtained from newborns and cultured for 2-30 hr in serum-free defined medium (SFM). Cell survival for the period studied was 78-87% both with and without NGF. Na currents were detected in all cells cultured for 6-9 hr. They were also detected after 2 hr in culture in 21.5% of the cells cultured without NGF (-NGF cells), and in 91.5% of the cells cultured with NGF (+NGF cells). Current density of the -NGF cells was 2.3 and 2 pA/microns 2 after growth for 2 and 6-9 hr, respectively, compared to 3.0 and 3.9 pA/microns 2 for the +NGF cells. The +NGF cells were separated into fast (F), Intermediate (I) and slow (S) cells, based on the Na current they expressed, while -NGF cells were all of the I type. F, I and S currents differed in their voltage-dependent inactivation (Vh50 = -79, -28 and -20 mV), kinetics of inactivation (tau h = 0.55, 1.3 and 7.75 msec), and TTX sensitivity (Ki = 60, 550 and 1100 nM). All currents were depressed by [Ca]0 with a KdCa of 22, 17 and 8 mM for F, I and S currents, respectively. Current density of F and S currents was 5.5 and 5 pA/micron 2 for the I current. The concentration-dependent curve of I current vs. TTX indicated that I current has two sites: one with F-like and another with S-like Ki for TTX. Hybridization of F and S currents yielded I-like currents. Thus, the major effect of NGF on Na currents in SFM is the acceleration of Na current acquisition and diversity, reflected in an increase of either the S or F type in a cell.

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“…Our present concepts regarding the molecular mechanisms of inactivation gating in Na+ channels have arisen not only from detailed biophysical analysis of behaviours at the single-and multichannel level, but also from attempts to selectively modify these behaviours with group-specific chemical reagents and enzymes. Clues concerning the membrane orientation and nature of reactive residues involved in inactivation have resulted from this approach (Oxford, Wu & Narahashi, 1978;Eaton, Brodwick, Oxford & Rudy, 1978;Nonner, Spaulding & Hille, 1980;Brodwick & Eaton, 1982;Wang, Brodwick & Eaton, 1985;Rack, Rubly & Waschow, 1986;Gonoi & Hille, 1987). Modification of inactivation by these agents has also been manifest at the single-channel level and has aided in discrimination of gating models (Patlak & Horn, 1982).…”

Section: Introductionmentioning

confidence: 99%

The inactivating K+ current in GH3 pituitary cells and its modification by chemical reagents.

Oxford

Wagoner

1989

The Journal of Physiology

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SUMMARY1. Whole-cell and single-channel recording techniques were applied to the study of the permeability and gating of inactivating K+ channels from clonal pituitary cells.2. The cation selectivity sequence (measured from reversal potentials) for the channels underlying the inactivating K+ current was TlV > K+ > Rb+ > NH4+. The conductance sequence (determined from current amplitudes) was K+ = Tl > Rb+ > NH4t3. The inactivating current (IK(i)) which was blocked by 4-aminopyridine (4-AP), activated at voltages more positive than -40 mV and half-inactivated at that voltage. Inactivation proceeded as the sum of two exponentials with mean time constants of 21 and 82 ms. Deactivation followed a single-exponential time course.4. Recovery from inactivation was slow, voltage dependent and multi-exponential, taking more than 50 s near the cell's resting potential.5. The magnitudes of outward current and of slope conductance increased as the concentration of external K+ was increased.6. On-cell and outside-out membrane patches revealed minicurrents with gating and pharmacological properties identical to whole-cell currents. 'Single channels with inactivating characteristics, while rarely observed, had an average slope conductance of 6-8 pS.7. Intracellular application of the disulphonic stilbene derivative, SITS, and the protein-modifying reagent, N-bromoacetamide (NBA), at concentrations of 0 2-1 mm for several tens of minutes dramatically slowed the decay (inactivation) of K+ currents and caused coincident increases in the magnitude of outward IK(i).8. Extracellular application of NBA at much lower concentrations (1-100 1uM) and much shorter exposure times (1-30 s) also slowed inactivation. This effect was reversible for brief applications at low doses, but became irreversible after longer exposures.9. Both internal and external NBA shifted the steady-state inactivation-voltage relation by+ 10 mV and reduced inactivation at voltages more positive than 0 mV.10. The efficacy of external NBA was independent of holding potential between -80 and 0 mV.

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Effects of some chemical reagents on sodium current inactivation in myelinated nerve fibers of the frog

Cited by 39 publications

References 34 publications

Mapping a region associated with Na channel inactivation using antibodies to a synthetic peptide corresponding to a part of the channel.

Mapping a region associated with Na channel inactivation using antibodies to a synthetic peptide corresponding to a part of the channel.

Characterization of sodium currents in mammalian sensory neurons cultured in serum-free defined medium with and without nerve growth factor

The inactivating K+ current in GH3 pituitary cells and its modification by chemical reagents.

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