scholarly journals Conotoxins as Sensors of Local pH and Electrostatic Potential in the Outer Vestibule of the Sodium Channel

2003 ◽  
Vol 122 (1) ◽  
pp. 63-79 ◽  
Author(s):  
Kwokyin Hui ◽  
Deane McIntyre ◽  
Robert J. French
Keyword(s):  
Lipid Bilayers ◽  
Sodium Channels ◽  
Electrostatic Potential ◽  
Single Channel ◽  
Local Environment ◽  
Bound State ◽  
Channel Changes ◽  
Voltage Dependent ◽  
Charged Ring ◽  
Derivatives Of

We examined the block of voltage-dependent rat skeletal muscle sodium channels by derivatives of μ-conotoxin GIIIA (μCTX) having either histidine, glutamate, or alanine residues substituted for arginine-13. Toxin binding and dissociation were observed as current fluctuations from single, batrachotoxin-treated sodium channels in planar lipid bilayers. R13X derivatives of μCTX only partially block the single-channel current, enabling us to directly monitor properties of both μCTX-bound and -unbound states under different conditions. The fractional residual current through the bound channel changes with pH according to a single-site titration curve for toxin derivatives R13E and R13H, reflecting the effect of changing the charge on residue 13, in the bound state. Experiments with R13A provided a control reflecting the effects of titration of all residues on toxin and channel other than toxin residue 13. The apparent pKs for the titration of residual conductance are shifted 2–3 pH units positive from the nominal pK values for histidine and glutamate, respectively, and from the values for these specific residues, determined in the toxin molecule in free solution by NMR measurements. Toxin affinity also changes dramatically as a function of pH, almost entirely due to changes in the association rate constant, kon. Interpreted electrostatically, our results suggest that, even in the presence of the bound cationic toxin, the channel vestibule strongly favors cation entry with an equivalent local electrostatic potential more negative than −100 mV at the level of the “outer charged ring” formed by channel residues E403, E758, D1241, and D1532. Association rates are apparently limited at a transition state where the pK of toxin residue 13 is closer to the solution value than in the bound state. The action of these unique peptides can thus be used to sense the local environment in the ligand-–receptor complex during individual molecular transitions and defined conformational states.

scholarly journals Grayanotoxin-I-modified eel electroplax sodium channels. Correlation with batrachotoxin and veratridine modifications.

1992 ◽  
Vol 100 (4) ◽  
pp. 623-645 ◽  
Author(s):  
D S Duch ◽  
A Hernandez ◽  
S R Levinson ◽  
B W Urban
Keyword(s):  
Lipid Bilayers ◽  
Sodium Channels ◽  
Single Channel ◽  
Electric Organ ◽  
Open Time ◽  
Activation Gating ◽  
Electric Eel ◽  
Voltage Dependent ◽  
Parallel Voltage ◽  
Dependent Processes

To probe the structure-function relationships of voltage-dependent sodium channels, we have been examining the mechanisms of channel modification by batrachotoxin (BTX), veratridine (VTD), and grayanotoxin-I (GTX), investigating the unifying mechanisms that underlie the diverse modifications of this class of neurotoxins. In this paper, highly purified sodium channel polypeptides from the electric organ of the electric eel were incorporated into planar lipid bilayers in the presence of GTX for comparison with our previous studies of BTX (Recio-Pinto, E., D. S. Duch, S. R. Levinson, and B. W. Urban. 1987. J. Gen. Physiol. 90:375-395) and VTD (Duch, D. S., E. Recio-Pinto, C. Frenkel, S. R. Levinson, and B. W. Urban. 1989. J. Gen. Physiol. 94:813-831) modifications. GTX-modified channels had a single channel conductance of 16 pS. An additional large GTX-modified open state (40-55 pS) was found which occurred in bursts correlated with channel openings and closings. Two voltage-dependent processes controlling the open time of these modified channels were characterized: (a) a concentration-dependent removal of inactivation analogous to VTD-modified channels, and (b) activation gating similar to BTX-modified channels, but occurring at more hyperpolarized potentials. The voltage dependence of removal of inactivation correlated with parallel voltage-dependent changes in the estimated K1/2 of VTD and GTX modifications. Ranking either the single channel conductances or the depolarization required for 50% activation, the same sequence is obtained: unmodified > BTX > GTX > VTD. The efficacy of the toxins as activators follows the same ranking (Catterall, W. A. 1977. J. Biol. Chem. 252:8669-8676).

scholarly journals Purified and unpurified sodium channels from eel electroplax in planar lipid bilayers.

1987 ◽  
Vol 90 (3) ◽  
pp. 375-395 ◽  
Author(s):  
E Recio-Pinto ◽  
D S Duch ◽  
S R Levinson ◽  
B W Urban
Keyword(s):  
Sodium Channel ◽  
Lipid Bilayers ◽  
Sodium Channels ◽  
Single Channel ◽  
Planar Bilayers ◽  
Electric Eel ◽  
Voltage Dependent ◽  
Single Channel Activity ◽  
Single Channel Currents ◽  
Channel Currents

Highly purified sodium channel protein from the electric eel, Electrophorus electricus, was reconstituted into liposomes and incorporated into planar bilayers made from neutral phospholipids dissolved in decane. The purest sodium channel preparations consisted of only the large, 260-kD tetrodotoxin (TTX)-binding polypeptide. For all preparations, batrachotoxin (BTX) induced long-lived single-channel currents (25 pS at 500 mM NaCl) that showed voltage-dependent activation and were blocked by TTX. This block was also voltage dependent, with negative potentials increasing block. The permeability ratios were 4.7 for Na+:K+ and 1.6 for Na+:Li+. The midpoint for steady state activation occurred around -70 mV and did not shift significantly when the NaCl concentration was increased from 50 to 1,000 mM. Veratridine-induced single-channel currents were about half the size of those activated by BTX. Unpurified, nonsolubilized sodium channels from E. electricus membrane fragments were also incorporated into planar bilayers. There were no detectable differences in the characteristics of unpurified and purified sodium channels, although membrane stability was considerably higher when purified material was used. Thus, in the eel, the large, 260-kD polypeptide alone is sufficient to demonstrate single-channel activity like that observed for mammalian sodium channel preparations in which smaller subunits have been found.

scholarly journals Batrachotoxin-modified sodium channels in planar lipid bilayers. Characterization of saxitoxin- and tetrodotoxin-induced channel closures.

1987 ◽  
Vol 89 (6) ◽  
pp. 873-903 ◽  
Author(s):  
W N Green ◽  
L B Weiss ◽  
O S Andersen
Keyword(s):  
Binding Site ◽  
Lipid Bilayers ◽  
Sodium Channels ◽  
Conformational Changes ◽  
Single Channel ◽  
Planar Lipid Bilayers ◽  
Net Charge ◽  
Toxin Binding ◽  
Wide Range ◽  
Voltage Dependent

The guanidinium toxin-induced inhibition of the current through voltage-dependent sodium channels was examined for batrachotoxin-modified channels incorporated into planar lipid bilayers that carry no net charge. To ascertain whether a net negative charge exists in the vicinity of the toxin-binding site, we studied the channel closures induced by tetrodotoxin (TTX) and saxitoxin (STX) over a wide range of [Na+]. These toxins carry charges of +1 and +2, respectively. The frequency and duration of the toxin-induced closures are voltage dependent. The voltage dependence was similar for STX and TTX, independent of [Na+], which indicates that the binding site is located superficially at the extracellular surface of the sodium channel. The toxin dissociation constant, KD, and the rate constant for the toxin-induced closures, kc, varied as a function of [Na+]. The Na+ dependence was larger for STX than for TTX. Similarly, the addition of tetraethylammonium (TEA+) or Zn++ increased KD and decreased kc more for STX than for TTX. These differential effects are interpreted to arise from changes in the electrostatic potential near the toxin-binding site. The charges giving rise to this potential must reside on the channel since the bilayers had no net charge. The Na+ dependence of the ratios KDSTX/KDTTX and kcSTX/kcTTX was used to estimate an apparent charge density near the toxin-binding site of about -0.33 e X nm-2. Zn++ causes a voltage-dependent block of the single-channel current, as if Zn++ bound at a site within the permeation path, thereby blocking Na+ movement. There was no measurable interaction between Zn++ at its blocking site and STX or TTX at their binding site, which suggests that the toxin-binding site is separate from the channel entrance. The separation between the toxin-binding site and the Zn++ blocking site was estimated to be at least 1.5 nm. A model for toxin-induced channel closures is proposed, based on conformational changes in the channel subsequent to toxin binding.

scholarly journals A molecular link between activation and inactivation of sodium channels.

1995 ◽  
Vol 106 (4) ◽  
pp. 641-658 ◽  
Author(s):  
M E O'Leary ◽  
L Q Chen ◽  
R G Kallen ◽  
R Horn
Keyword(s):  
Sodium Channels ◽  
Voltage Dependence ◽  
Single Channel ◽  
Critical Role ◽  
Channel Measurements ◽  
Wild Type ◽  
Open Probability ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Normal Coupling

A pair of tyrosine residues, located on the cytoplasmic linker between the third and fourth domains of human heart sodium channels, plays a critical role in the kinetics and voltage dependence of inactivation. Substitution of these residues by glutamine (Y1494Y1495/QQ), but not phenylalanine, nearly eliminates the voltage dependence of the inactivation time constant measured from the decay of macroscopic current after a depolarization. The voltage dependence of steady state inactivation and recovery from inactivation is also decreased in YY/QQ channels. A characteristic feature of the coupling between activation and inactivation in sodium channels is a delay in development of inactivation after a depolarization. Such a delay is seen in wild-type but is abbreviated in YY/QQ channels at -30 mV. The macroscopic kinetics of activation are faster and less voltage dependent in the mutant at voltages more negative than -20 mV. Deactivation kinetics, by contrast, are not significantly different between mutant and wild-type channels at voltages more negative than -70 mV. Single-channel measurements show that the latencies for a channel to open after a depolarization are shorter and less voltage dependent in YY/QQ than in wild-type channels; however the peak open probability is not significantly affected in YY/QQ channels. These data demonstrate that rate constants involved in both activation and inactivation are altered in YY/QQ channels. These tyrosines are required for a normal coupling between activation voltage sensors and the inactivation gate. This coupling insures that the macroscopic inactivation rate is slow at negative voltages and accelerated at more positive voltages. Disruption of the coupling in YY/QQ alters the microscopic rates of both activation and inactivation.

scholarly journals Batrachotoxin-modified sodium channels from squid optic nerve in planar bilayers. Ion conduction and gating properties.

1989 ◽  
Vol 93 (1) ◽  
pp. 23-41 ◽  
Author(s):  
M I Behrens ◽  
A Oberhauser ◽  
F Bezanilla ◽  
R Latorre
Keyword(s):  
Optic Nerve ◽  
Dissociation Constant ◽  
Sodium Channels ◽  
Single Channel ◽  
Dependent Manner ◽  
Channel Conductance ◽  
Apparent Dissociation Constant ◽  
Planar Bilayers ◽  
Voltage Range ◽  
Voltage Dependent

Squid optic nerve sodium channels were characterized in planar bilayers in the presence of batrachotoxin (BTX). The channel exhibits a conductance of 20 pS in symmetrical 200 mM NaCl and behaves as a sodium electrode. The single-channel conductance saturates with increasing the concentration of sodium and the channel conductance vs. sodium concentration relation is well described by a simple rectangular hyperbola. The apparent dissociation constant of the channel for sodium is 11 mM and the maximal conductance is 23 pS. The selectivity determined from reversal potentials obtained in mixed ionic conditions is Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+. Calcium blocks the channel in a voltage-dependent manner. Analysis of single-channel membranes showed that the probability of being open (Po) vs. voltage relation is sigmoidal with a value of 0.5 between -90 and -100 mV. The fitting of Po requires at least two closed and one open state. The apparent gating charge required to move through the whole transmembrane voltage during the closed-open transition is four to five electronic charges per channel. Distribution of open and closed times are well described by single exponentials in most of the voltage range tested and mean open and mean closed times are voltage dependent. The number of charges associated with channel closing is 1.6 electronic charges per channel. Tetrodotoxin blocked the BTX-modified channel being the blockade favored by negative voltages. The apparent dissociation constant at zero potential is 16 nM. We concluded that sodium channels from the squid optic nerve are similar to other BTX-modified channels reconstituted in bilayers and to the BTX-modified sodium channel detected in the squid giant axon.

Cation channels from Tetrahymena cilia incorporated into planar lipid bilayers

1985 ◽  
Vol 249 (1) ◽  
pp. C177-C179 ◽  
Author(s):  
Y. Oosawa ◽  
M. Sokabe
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Cation Channel ◽  
Cation Channels ◽  
Planar Lipid Bilayers ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Ciliary Membrane ◽  
Voltage Dependent

A single cation channel from Tetrahymena cilia was incorporated into planar lipid bilayers. This channel selected for K+, Na+, and Li+ over Cl- and gluconate-, and its single channel conductance (at +25 mV) was 211 +/- 8 pS (mean +/- SE) in 100 mM K+-gluconate. The channel was not voltage dependent and may contribute to the resting K+ conductance of ciliary membrane.

Calcium-dependent chloride channels in endosomes from rabbit kidney cortex

1994 ◽  
Vol 266 (3) ◽  
pp. C741-C750 ◽  
Author(s):  
W. B. Reeves ◽  
R. W. Gurich
Keyword(s):  
Lipid Bilayers ◽  
Membrane Trafficking ◽  
Single Channel ◽  
Cell Volume Regulation ◽  
Rabbit Kidney ◽  
Disulfonic Acid ◽  
Kidney Cortex ◽  
Open Probability ◽  
Endosomal Acidification ◽  
Voltage Dependent

Ion channels in endosomal membranes from rabbit kidney cortex were studied after reconstitution into planar lipid bilayers. The most frequently observed ion channel was anion selective (PCl/PK = 13) and had a single-channel conductance of 116 pS when the cis and trans solutions contained 410 and 150 mM KCl, respectively, and a conductance of 90 pS in symmetrical 150 mM KCl solutions. The anion selectivity sequence of the channel was NO3- > F- > Br- > Cl- >> I-. The activity of the channel was voltage dependent such that hyperpolarization of the cis, or cytoplasmic, surface of the channel increased the open probability (Po). The activity of the channel was also highly dependent on the calcium activity of the cis but not the trans solution. Channels were fully active (Po > 0.7) at Ca2+ concentration > 1 microM, but channel activity was completely absent (Po < 0.001) at Ca2+ concentration < 250 nM. The effects of calcium on Po were not voltage dependent. The Cl(-)-channel blocker 2-[(2-cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H-inden -5- yl)oxy]-acetic acid (IAA-94/95) produced a concentration-dependent reversible flickering block of the endosomal channel with a Ki of 15 microM. 4,4'-Dinitrostilbene-2,2'-disulfonic acid, a disulfonic stilbene, also produced a flickering block of the channel with a Ki of approximately 5 microM. Because endosomal Cl- channels are believed to facilitate endosomal acidification, we tested the effects of IAA-94/95 and deletion of Ca2+ on the rate of acidification of intact endosomes. Because neither maneuver affected acidification, we conclude that the 116-pS channel does not participate in endosomal acidification. This channel may be involved in other endosomal processes, e.g., cell volume regulation and control of membrane trafficking.

Effects of dihydropyridine calcium channel modulators on cardiac sodium channels

1988 ◽  
Vol 254 (1) ◽  
pp. H140-H147 ◽  
Author(s):  
A. Yatani ◽  
D. L. Kunze ◽  
A. M. Brown
Keyword(s):  
Calcium Channels ◽  
Sodium Channels ◽  
Single Channel ◽  
Bay K 8644 ◽  
Sodium Currents ◽  
Whole Cell ◽  
Blocking Effects ◽  
Voltage Dependent ◽  
Cardiac Sodium Channels ◽  
Single Channel Currents

To investigate whether cardiac sodium channels have dihydropyridine (DHP) receptors we studied the effects of the optically pure (greater than 95%) enantiomers of the DHPs PN200–110 and BAY-K 8644 and the racemic DHP nitrendipine (NTD). Whole cell and single-channel sodium currents were recorded from cultured ventricular cells of neonatal rats using the patch-clamp method. NTD reduced cardiac sodium currents in a voltage-dependent manner. Inhibitory effects were due to an increase in traces without activity. The unit conductance remained unchanged. At negative holding potentials, NTD transiently increased the probability of channel opening. Both (+) and (-) PN 200–110 blocked sodium channels, although the (-) isomer was about one order of magnitude less effective. The blocking effects were voltage dependent. (+) BAY-K 8644 had similar blocking effects. (-) BAY-K 8644 produced an increase in sodium currents due to an increased frequency of channel openings and a marked prolongation of open time without any significant change in unit conductance. The DHPs have effects on cardiac sodium whole cell and single-channel currents that appear identical to and are as stereospecific as their effects on cardiac calcium currents, although the concentrations required are larger. In contrast the inwardly rectifying potassium channel (IK1) is unaffected by these DHPs. We conclude that functionally equivalent DHP receptors are present in cardiac sodium and calcium channels but not potassium channels and take this as evidence of the homology between sodium and calcium channels.

scholarly journals Removal of sodium channel inactivation in squid giant axons by n-bromoacetamide.

1978 ◽  
Vol 71 (3) ◽  
pp. 227-247 ◽  
Author(s):  
G S Oxford ◽  
C H Wu ◽  
T Narahashi
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Single Channel ◽  
Sodium Current ◽  
Complete Removal ◽  
Specific Protein ◽  
Peptide Bonds ◽  
Voltage Dependent ◽  
Inactivation Gating ◽  
Sodium Inactivation

The group-specific protein reagents, N-bromacetamide (NBA) and N-bromosuccinimide (NBS), modify sodium channel gating when perfused inside squid axons. The normal fast inactivation of sodium channels is irreversibly destroyed by 1 mM NBA or NBS near neutral pH. NBA apparently exhibits an all-or-none destruction of the inactivation process at the single channel level in a manner similar to internal perfusion of Pronase. Despite the complete removal of inactivation by NBA, the voltage-dependent activation of sodium channels remains unaltered as determined by (a) sodium current turn-on kinetics, (b) sodium tail current kinetics, (c) voltage dependence of steady-state activation, and (d) sensitivity of sodium channels to external calcium concentration. NBA and NBS, which can cleave peptide bonds only at tryptophan, tyrosine, or histidine residues and can oxidize sulfur-containing amino acids, were directly compared with regard to effects on sodium inactivation to several other reagents exhibiting overlapping protein reactivity spectra. N-acetylimidazole, a tyrosine-specific reagent, was the only other compound examined capable of partially mimicking NBA. Our results are consistent with recent models of sodium inactivation and support the involvement of a tyrosine residue in the inactivation gating structure of the sodium channel.

scholarly journals Veratridine modification of the purified sodium channel alpha-polypeptide from eel electroplax.

1989 ◽  
Vol 94 (5) ◽  
pp. 813-831 ◽  
Author(s):  
D S Duch ◽  
E Recio-Pinto ◽  
C Frenkel ◽  
S R Levinson ◽  
B W Urban
Keyword(s):  
Sodium Channel ◽  
Lipid Bilayers ◽  
Sodium Channels ◽  
Single Channel ◽  
Reversal Potential ◽  
Channel Structure ◽  
Dependent Manner ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Protein Subunit

In the interest of continuing structure-function studies, highly purified sodium channel preparations from the eel electroplax were incorporated into planar lipid bilayers in the presence of veratridine. This lipoglycoprotein originates from muscle-derived tissue and consists of a single polypeptide. In this study it is shown to have properties analogous to sodium channels from another muscle tissue (Garber, S. S., and C. Miller. 1987. Journal of General Physiology. 89:459-480), which have an additional protein subunit. However, significant qualitative and quantitative differences were noted. Comparison of veratridine-modified with batrachotoxin-modified eel sodium channels revealed common properties. Tetrodotoxin blocked the channels in a voltage-dependent manner indistinguishable from that found for batrachotoxin-modified channels. Veratridine-modified channels exhibited a range of single-channel conductance and subconductance states. The selectivity of the veratridine-modified sodium channels for sodium vs. potassium ranged from 6-8 in reversal potential measurements, while conductance ratios ranged from 12-15. This is similar to BTX-modified eel channels, though the latter show a predominant single-channel conductance twice as large. In contrast to batrachotoxin-modified channels, the fractional open times of these channels had a shallow voltage dependence which, however, was similar to that of the slow interaction between veratridine and sodium channels in voltage-clamped biological membranes. Implications for sodium channel structure are discussed.

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