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

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.     

Molecular motion underlying activation and inactivation of sodium channels in squid giant axons

Summary Measurements of the changes in birefringence associated with changes in membrane potential were made with internally perfused squid giant axons in low sodium solutions at 0–8°C. The time course of the birefringence changes share many properties of the gating (polarization) currents previously studied in this nerve. Both can be demonstrated as an asymmetry in the response to voltage pulses symmetrical about the resting potential which is not present about a hyperpolarized holding potential. Both have a rapid relaxation, which precedes the sodium permeability change. Both exhibit an initial delay or rising phase. Both are reversibly blocked by perfusion with 30mm colchicine; neither are altered by changes on sodium concentrations or 300nm tetrodotoxin. The birefringence response has a decrease in the amplitude of the rapid relaxation associated with the appearance of a slow relaxation. This is similar to the immobilization of fast gating charges which parallels sodium current inactivation.The amplitude of the birefringence and the gating current responses is consistent with a change in the alignment of several hundred peptide bonds per sodium channel.     

Sodium inactivation mechanism modulates QX-314 block of sodium channels in squid axons.

Blocking action of Na channels by QX-314, a quaternary derivative of lidocaine, was studied in internally perfused and voltage-clamped axons of squid. In axons with intact Na inactivation, QX-314 exhibited both a frequency- and a voltage-dependent block of Na channels. Repetitive pulsing to more positive potentials enhanced the degree of block. Both frequency- and voltage-dependent blocks disappeared in axons in which Na inactivation had been destroyed by either pronase or N-bromoacetamide treatment. These results support the notion that Na inactivation not only modulates the frequency-dependent block but also involves the voltage-dependent binding reaction between QX-314 and Na channels.     

Local anesthetic block of sodium channels in normal and pronase-treated squid giant axons

The inhibition of sodium currents by local anesthetics and other blocking compounds was studied in perfused, voltage-clamped segments of squid giant axon. When applied internally, each of the eight compounds studied results in accumulating "use-depnedent" block of sodium currents upon repetitive pulsing. Recovery from block occurs over a time scale of many seconds. In axons treated with pronase to completely eliminate sodium inactivation, six of the compounds induce a time- and voltage-dependent decline of sodium currents after activation during a maintained depolarization. Four of the time-dependent blocking compounds--procaine, 9-aminoacridine, N-methylstrychnine, and QX572--also induce altered sodium tail currents by hindering closure of the activation gating mechanism. Treatment of the axon with pronase abolishes use-dependent block completely by QX222, QX314, 9-aminoacridine, and N-methylstrychnine, but only partially be tetracaine and etidocaine. Two pulse experiments reveal that recovery from block by 9-aminoacridine or N-methyl-strychnine is greatly accelerated after pronase treatment. Pronase treatment abolishes both use-dependent and voltage-dependent block by QX222 and QX314. These results provide support for a direct role of the inactivation gating mechanism in producing the long-lasting use-dependent inhibition brought about by local anesthetic compounds.     

n-Alkanols potentiate sodium channel inactivation in squid giant axons.

The effects of n-octanol and n-decanol on nerve membrane sodium channels were examined in internally perfused, voltage-clamped squid giant axons. Both n-octanol and n-decanol almost completely eliminated the residual sodium conductance at the end of 8-ms voltage steps. In contrast, peak sodium conductance was only partially reduced. This block of peak and residual sodium conductance was very reversible and seen with both internal and external alkanol application. The differential sensitivity of peak and residual conductance to alkanol treatment was eliminated after internal pronase treatment, suggesting that n-octanol and n-decanol enhance the normal inactivation mechanism rather than directly blocking channels in a time-dependent manner.     

Removal of sodium channel inactivation in squid axon by the oxidant chloramine-T

We have investigated the effects of a mild oxidant, chloramine-T(CT), on the sodium and potassium currents of squid axons under voltage-clamp conditions. Sodium channel inactivation of squid giant axons can be completely removed by CT at neutral pH. Internal and external CT treatment are both effective. CT apparently removes inactivation in an irreversible, all-or-none manner. The activation process of sodium channels is little affected, as judged from the voltage dependence of peak sodium currents, the rising phase of sodium currents, and the time course of tail currents following the repolarization. The removal of inactivation by CT is pH-dependent; higher pH decreases the removal rate, whereas lower pH increases it. Internal metabisulfite, a strong reductant, does not protect inactivation from the action of external CT, nor does external metabisulfite protect from internal CT application. CT slightly depresses the peak potassium currents at comparable concentrations but has no apparent effects on their kinetics. Our results suggest that the neutral form of CT modifies an embedded methionine residue that is involved in sodium channel inactivation.     

Removal of Na+ channels in squid giant axons by perfusion with trypsin

The irreversible effects of the proteolytic enzyme trypsin on ionic and gating currents of voltage-clamped squid axon membranes have been studied. At physiological pH, internal perfusion of the fibre with trypsin was found to be very effective in removing Na+ channels leaving the potassium system almost unaltered. At T = 13 degrees C the rates of channel-cleavage averaged 1/10 min-1 for the Na+ and 1/128 min-1 for the K+ channel, respectively. As estimated by the decrement of peak sodium conductance, the rate of loss of Na+ channels correlates well with the rate of decrease of the total charge associated with the ON component of gating currents, indicating that trypsin probably interacts with an essential proteic portion of the channel whose removal might prevent both the displacement of gating charges and the subsequent opening of the channel. Intracellular pH remarkably influences the action of the enzyme. A plot of the pH-dependence of the rate of cleavage of Na+ channels suggests the involvement of a positively charged group (either lysine or arginine) in the substrate region of the trypsin catalytic reaction.     

Hydrogen ion block of the sodium pore in squid giant axons

The block of squid axon sodium channels by H ions was studied using voltage-clamp and internal perfusion techniques. An increase in the concentration of internal permeant ions decreased the block produced by external H ions. The voltage dependence of the block was found to be nonmonotonic: it was reduced by both large positive and large negative potentials. The ability of internal ions to modify the block by external H+ is explained by a competition among these ions for a binding site within the pore. The nonmonotonic voltage dependence is consistent with this picture if the hydrogen ions are allowed to be permeant.     

Dynamics of 9-aminoacridine block of sodium channels in squid axons

The interactions of 9-aminoacridine with ionic channels were studied in internally perfused squid axons. The kinetics of block of Na channels with 9-aminoacridine varies depending on the voltage-clamp pulses and the state of gating machinery of Na channels. In an axon with intact h gate, the block exhibits frequency- and voltage-dependent characteristics. However, in the pronase-perfused axon, the frequency- dependent block disappears, whereas the voltage-dependent block remains unchanged. A time-dependent decrease in Na currents indicative of direct block of Na channel by drug molecule follows a single exponential function with a time constant of 2.0 +/- 0.18 and 1.0 +/- 0.19 ms (at 10 degrees C and 80 m V) for 30 and 100 microM 9- aminoacridine, respectively. A steady-state block can be achieved during a single 8-ms depolarizing pulse when the h gate has been removed. The block in the h-gate intact axon can be achieved only with multiple conditioning pulses. The voltage-dependent block suggests that 9-aminoacridine binds to a site located halfway across the membrane with a dissociation constant of 62 microM at 0 m V. 9-Aminoacridine also blocks K channels, and the block is time- and voltage-dependent.     

Anticalmodulin drugs block the sodium gating current of squid giant axons

Summary The effects of calmodulin (CaM) antagonists (W-7, W-5, trifluoperazine, chlorpromazine, quinacrine, diazepam, propericyazine and carmidazolium) on the sodium and potassium channels were studied on the intracellularly perfused and voltage-clamped giant axon of the squid. It was found that the drugs are more potent blockers of the sodium current than of the potassium current. The drugs also reduce the sodium gating current. The blockage of the sodium and gating current can be explained by assuming that the drugs interact with the sodium gating subunit in one of its closed states. The site of action is probably the intracellular surface of the axolemma where presumably a Ca²⁺-calmodulin complex can be formed.     

QX-314 restores gating charge immobilization abolished by chloramine-T treatment in squid giant axons.

The gating status of the QX-314 bound Na channels before and after suppressing the fast inactivation by chloramine-T (CT) was investigated by studying the gating charge immobilization using the OFF gating current (Ig,OFF). CT treatment, which abolishes the charge immobilization induced by a prolonged depolarization, altered the kinetics of Ig,OFF: the fast phase became insensitive to the pulse duration and the slow phase became three times faster than the control one. However, internally applied QX-314 (in the presence of external TTX) caused an immediate charge immobilization similar to that observed in the absence of CT treatment. The Ig,OFF exhibited kinetics similar to the inactivated channels, decaying with a very fast time course. We conclude that the charge immobilization is restored by QX-314 in the chloramine-T-treated axon and that the gating state of the QX-314-bound channel is similar to the inactivated one. The role of the gating charge immobilization in the use-dependent block mechanism is discussed.     

Inactivation of the sodium current in squid giant axons by hydrocarbons.

The voltage dependence of the steady state inactivation parameter (h infinity) of the sodium current in the squid giant axon is known to be shifted in the hyperpolarizing direction by hydrocarbons and it has been suggested that the shifts arise from thickness changes in the axon membrane, analogous to those produced in lipid bilayers (Haydon, D. A., and J. E. Kimura, 1981, J. Physiol. [Lond.], 312:57-70; Haydon, D. A., and B. W. Urban, 1983, J. Physiol. [Lond.], 338:435-450; Haydon, D. A., J. R. Elliott, and B. M. Hendry, 1984, Curr. Top. Membr. Transp., 22:445-482). This hypothesis has been tested systematically by examining the effects of a range of concentrations of cyclopentane on the high-frequency capacitance per unit area both of the axonal membrane and of lipid bilayers formed from monoolein plus squalene. A similar comparison has been made for cyclopropane and n-butane, both at a pressure of 1 atm. The results are consistent with the notion that thickness increases in the axolemma produce the shifts in h infinity. Except at very high concentrations, however, the thickness changes in the lipid bilayer were too small to account for the h infinity shifts. A possible explanation of this finding is discussed.     

Kinetic analysis of sodium channel block by internal methylene blue in pronased crayfish giant axons

The cationic dye methylene blue (MB+) blocks INa in a voltage and time-dependent manner and exhibits no frequency dependent block at 1 Hz when internally perfused in normal or pronase-treated crayfish axons. Peak INa decreases with increasing MB+ concentrations in the range 50 microM to 5 mM, but the blocking time constant approaches an asymptote at concentrations above 500 microM. IgON is not noticeably affected by internal MB+ at concentrations of 500 microM or below, in the absence of external tetrodotoxin (TTX). However, 5 mM MB+ produces a visible suppression of IgON that is reversible following washout. A pseudo-first-order analysis of MB+ blocking kinetics suggests a drug binding site deep in the transmembrane voltage field (dz = 0.85, KD = 11 microM at 0 mV). The voltage sensitivity of the individual rate constants is highly asymmetric, suggesting that the major energy barrier for MB+ is very close to the axoplasmic margin of the voltage field. Reversing the Na+ gradient and direction of INa has little effect on the kinetics of MB+ block. The kinetic properties of state-dependent vs. state-independent blocking schemes are investigated and compared with our observations of MB+ block. Analysis of hooked sodium tail currents following depolarization to various test potentials demonstrates quantitatively that MB+ binds in a state-dependent manner to open sodium channels. The appropriateness of first-order kinetic analysis of drug block is then considered in light of these observations.     

Block of sodium conductance by n-octanol in crayfish giant axons

The block of the Na+ current by n-octanol was studied in crayfish giant axons under axial wire voltage-clamp conditions. Standard kinetic analysis of the Na+ currents was undertaken to test the hypothesis tha the n-octanol-induced block of the Na+ current could be accounted for on the basis of changes in the voltage dependence of the kinetic parameters. Alterations in the membrane dipolar potential arising from rearrangement of membrane lipids would be the anticipated source of changes in the voltage dependence. Although some changes in voltage dependence did evolve with the block by n-octanol, the changes were not of sufficient magnitude to account for the block. In conclusion, although higher concentrations of n-octanol produced shifts along the voltage axis of the kinetic parameters, direct blocking action of n-octanol on the channel appears to be the most important mechanism of the block.     

Interaction of barium ions with potassium channels in squid giant axons.

Blocking of potassium channels by internally and externally applied barium ions has been studied in squid giant axons. Internal Ba (3-5 mM) causes rapid decay or "inactivation" of potassium current (IK). The kinetics and degree of block are strongly voltage-dependent. Large positive voltages speed blocking and make it more profound. Raising the external potassium concentration (Ko) from 0 to 250 mM has the opposite effect: block is made slower and less severe. In contrast, for positive voltages block by the tetraethylammonium derivative 3-phenylpropyltriethylammonium ion is almost independent of Ko and voltage. Recovery from block by internal Ba has a rapid phase lasting a few milliseconds and a slow phase lasting approximately 5 min. Internal Ba causes a "hook" in the IK tails recorded on repolarizing the fiber in high potassium external medium. External Ba, on the other hand, blocks without much altering IK time-course. KD (the dissociation constant) for block by external Ba is a few millimolar, and depends on the internal potassium concentration, the holding potential, and other factors. A reaction scheme for Ba and K channels is presented, postulating that internal and external Ba reach the same point in the channel. Once there, Ba blocks and also stabilizes the closed conformation of the channel. The extreme stability of the Ba channel complex implies the existence of negative charge within the channel.     

Localization of voltage-gated K(+) channels in squid giant axons

We have localized the classical voltage-gated K(+) channel within squid giant axons by immunocytochemistry using the Kv1 antibody of Rosenthal et al. (1996). Widely dispersed patches of intense immunofluorescence were observed in the axonal membrane. Punctate immunofluorescence was also observed in the axoplasm and was localized to approximately 25-50-microm-wide column down the length of the nerve (axon diameter approximately 500 microm). Immunoelectronmicroscopy of the axoplasm revealed a K(+) channel containing vesicles, 30-50 nm in diameter, within this column. These and other vesicles of similar size were isolated from axoplasm using a novel combination of high-speed ultracentrifugation and controlled-pore size, glass bead separation column techniques. Approximately 1% of all isolated vesicles were labeled by K(+) channel immunogold reacted antibody. Incorporation of isolated vesicle fractions within an artificial lipid bilayer revealed K(+) channel electrical activity similar to that recorded directly from the axonal membrane by Llano et al. (1988). These K(+) channel-containing vesicles may be involved in cycling of K(+) channel protein into the axonal membrane. We have also isolated an axoplasmic fraction containing approximately 150-nm-diameter vesicles that may transport K(+) channels back to the cell body.     

Unidirectional sodium and potassium fluxes through the sodium channel of squid giant axons.

Unidirectional 22Na-traced sodium influx or 42K-traced potassium efflux across the membranes of voltage-clamped squid giant axons was measured at various membrane potentials under bi-ionic conditions. Tetrodotoxin almost entirely eliminated the extra K+ efflux induced by short repetitive depolarizations in the presence of tetraethylammonium or 3,4-diaminopyridine. A method of determining the voltage dependence of the unidirectional flux through voltage-gated channels is described. This technique was used to obtain the unidirectional flux-voltage relation for the sodium channel in bi-ionic and single-ion conditions. It allows the determination of the unidirectional flux at the zero-current potential which, for influx, was found to be approximately 20% of the value measured 80 mV negative to the zero-current potential. The unidirectional flux ratio under bi-ionic conditions was also measured and the flux ratio exponent found to average 1.15 with an external sodium and an internal potassium solution. A three-barrier, two-site, multi-occupancy model previously obtained for other conditions was found to predict a similar non-unity average for the flux ratio exponent. It is also shown that some single-occupancy models can predict non-unity values for the flux ratio exponent in bi-ionic conditions.     

Internal cesium and the sodium inactivation gate in Myxicola giant axons.

Internal cesium (CSi), relative to internal potassium (Ki), alters Na current (INa) time course in internally perfused Myxicola giant axons. CSi slows the time to peak INa, slows its decline from peak and increases the steady state to peak current ratio, INainfinity/INapeak. Neither activation nor deactivation kinetics are appreciably affected by CSi. Na current rising phases, times to half maximum and tail current time courses are similar in CSi and Ki. Inactivation time constants determined by both one (tau h) and two (tau c) pulses are also little changed by CSi. The CSi effects are due largely or entirely to an increased INainfinity/INapeak. CSi decreases the steady level of inactivation reached during a step in potential, preventing some fraction of inactivation gates from closing at all, the rest apparently closing normally. Inactivation block in CSi decreases with increasing inward current magnitude and in Ki inactivation block is appreciable only for outward Na channel current, suggesting the site of action is located somewhere in the current pathway. If this site mediates the normal operation of the inactivation gate, then a possible mechanism for gate closure could involve a positively charged structure moving to associate with a negative site near or into the inner channel mouth.     

Batrachotoxin uncouples gating charge immobilization from fast Na inactivation in squid giant axons.

The fast inactivation of sodium currents and the immobolization of sodium gating charge are thought to be closely coupled to each other. This notion was tested in the squid axon in which kinetics and steady-state properties of the gating charge movement were compared before and after removal of the Na inactivation by batrachotoxin (BTX), pronase, or chloramine-T. The immobilization of gating charge was determined by measuring the total charge movement (QON) obtained by integrating the ON gating current (Ig,ON) using a double pulse protocol. After removal of the fast inactivation with pronase or chloramine-T, the gating charge movement was no longer immobilized. In contrast, after BTX modification, the channels still exhibited an immobilization of the gating charge (QON) with an onset time course and voltage dependence similar to that for the activation process. These results show that BTX can uncouple the charge immobilization from the fast Na inactivation mechanism, suggesting that the Na gating charge movement can be immobilized independently of the inactivation of the channel.