Properties of single Na+ channels in cut-open Myxicola giant axons

Time- and voltage-dependent behavior of the Na+ conductance in dialyzed intact Myxicola axons was compared with cut-open axons subjected to loose-patch clamp of the interior and to axons where Gigaseals were formed after brief enzyme digestion. Voltage and time dependence of activation, inactivation, and reactivation were identical in whole-axons and loose-patch preparations. Single channels observed in patch-clamp axons had a conductance of 18.3 +/- 2.3 pS and a mean open time of 0.84 +/- 0.12 ms. The time-dependence of Na+ currents found by averaging patch-clamp records was similar to intact axons, as was the voltage dependence of activation. Steady-state inactivation in patch-clamped axons was shifted by an average of 15 mV from that seen in loose-patch or intact axons. Substitution of D2O for H2O decreased single channel conductance by 24 +/- 6% in patch-clamped axons compared with 28 +/- 4% in intact axons, slowed inactivation by 58 +/- 8% compared with 49 +/- 6%, and increased mean open time by 52 +/- 7%. The results confirm observations on macroscopic channel behavior in Myxicola and resemble that seen in other excitable tissues.     

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.     

Inactivation of the Na permeability in squid giant axons

Inactivation of the Na permeability has been studied in intact and perfused squid giant axons with the voltage clamp method. The main results are: 1. Upon depolarization inactivation develops along an exponential time course; the upper limit for an initial delay in the development of inactivation is 50-100 musec. 2. Adding 20-40 mM KCl to K-free external solution accelerates the development of inactivation and slows its removal. 3. Scorpion venoms increase the maintained conductance, i.e. make inactivation less complete; the voltage dependence of the maintained conductance is different from that of the peak conductance.     

Removal of sodium inactivation and block of sodium channels by chloramine-T in crayfish and squid giant axons.

Modification of sodium channels by chloramine-T was examined in voltage clamped internally perfused crayfish and squid giant axons using the double sucrose gap and axial wire technique, respectively. Freshly prepared chloramine-T solution exerted two major actions on sodium channels: (a) an irreversible removal of the fast Na inactivation, and (b) a reversible block of the Na current. Both effects were observed when chloramine-T was applied internally or externally (5-10 mM) to axons. The first effect was studied in crayfish axons. We found that the removal of the fast Na inactivation did not depend on the states of the channel since the channel could be modified by chloramine-T at holding potential (from -80 to -100 mV) or at depolarized potential of -30 mV. After removal of fast Na inactivation, the slow inactivation mechanism was still present, and more channels could undergo slow inactivation. This result indicates that in crayfish axons the transition through the fast inactivated state is not a prerequisite for the slow inactivation to occur. During chloramine-T treatment, a distinct blocking phase occurred, which recovered upon washing out the drug. This second effect of chloramine-T was studied in detail in squid axons. After 24 h, chloramine-T solution lost its ability to remove fast inactivation but retained its blocking action. After removal of the fast Na inactivation, both fresh and aged chloramine-T solutions blocked the Na currents with a similar potency and in a voltage-dependent manner, being more pronounced at lower depolarizing potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Alteration of birefringence signals from squid giant axons by intracellular perfusion with protease solution

The optical signal, arising from a transient birefringence change associated with excitation, was recorded from a squid giant axon together with the membrane potential change, and the effect of removal of the axoplasm on the optical signal was examined. In an unperfused axon, repetitive stimulation at a frequency of about 100 Hz produced two kinds of optical response. The initial response had a brief, spike-like time course and was elicited by each stimulating pulse. The delayed response had a slow time course and the sign of decreased light intensity, and summated with repetitive stimulation. Most of the axoplasm was removed from interior of the axon by intracellular perfusion with solutions containing pronase at a concentration of 0.1 mg/ml. The delayed response could selectively be eliminated by perfusion with a pronase-containing solution for 2–8 min. The result was interpreted as showing that the delayed birefringence signal originates from axoplasm when its gel structure was transiently disturbed by an increased Ca²⁺ influx associated with excitation. When perfusion was further continued the duration of the action potential started increasing and often a prominent after-depolarization appeared. At this stage the initial optical response was again followed by a large show signal with the sign of increased light intensity. This reversed delayed response was tentatively assumed to originate from the membrane with some remaining axoplasm, but its cause is still not understood.     

Ion permeation in normal and batrachotoxin-modified Na+ channels in the squid giant axon

Na+ permeation through normal and batrachotoxin (BTX)-modified squid axon Na+ channels was characterized. Unmodified and toxin-modified Na+ channels were studied simultaneously in outside-out membrane patches using the cut-open axon technique. Current-voltage relations for both normal and BTX-modified channels were measured over a wide range of Na+ concentrations and voltages. Channel conductance as a function of Na+ concentration curves showed that within the range 0.015-1 M Na+ the normal channel conductance is 1.7-2.5-fold larger than the BTX-modified conductance. These relations cannot be fitted by a simple Langmuir isotherm. Channel conductance at low concentrations was larger than expected from a Michaelis-Menten behavior. The deviations from the simple case were accounted for by fixed negative charges located in the vicinity of the channel entrances. Fixed negative charges near the pore mouths would have the effect of increasing the local Na+ concentration. The results are discussed in terms of energy profiles with three barriers and two sites, taking into consideration the effect of the fixed negative charges. Either single- or multi-ion pore models can account for all the permeation data obtained in both symmetric and asymmetric conditions. In a temperature range of 5-15 degrees C, the estimated Q10 for the conductance of the BTX-modified Na+ channel was 1.53. BTX appears not to change the Na+ channel ion selectively (for the conditions used) or the surface charge located near the channel entrances.     

A new internal perfusion method for transport studies in squid giant axons

A new internal perfusion method has been developed which allows control of the internal solute composition in squid axons. The superiority of this technique compared to the old perfusion methods is shown by the experiments performed which have reproduced, both qualitatively and quantitatively, the Na⁺ and Ca²⁺ fluxes observed in intact and dialyzed axons. Compared with the internal dialysis, the perfusion method has the advantage that the permeability barrier given by the porous capillary has been eliminated. This allows the introduction into the axon of solutes with very high molecular weight, at the same time that a fast and reliable internal control can be achieved.     

A new internal perfusion method for transport studies in squid giant axons

A new internal perfusion method has been developed which allows control of the internal solute composition in squid axons. The superiority of this technique compared to the old perfusion methods is shown by the experiments performed which have reproduced, both qualitatively and quantitatively, the Na+ and Ca2+ fluxes observed in intact and dialyzed axons. Compared with the internal dialysis, the perfusion method has the advantage that the permeability barrier give by the porous capillary has been eliminated. This allows the introduction into the axon of solutes with very high molecular weight, at the same time that a fast and reliable internal control can be achieved.     

Sidedness of the ATP-Na+-K+ interactions with the Na+ pump in squid axons

Using dialysed squid axons we have been able to control internal and external ionic compositions under conditions in which most of the Na⁺ efflux goes through the Na⁺ pump. We found that (i) internal K⁺ had a strong inhibitory effect on Na⁺ efflux; this effect was antagonized by ATP, with low affinity, and by internal Na⁺, (ii) a reduction in ATP levels from 3 mM to 50 μM greatly increased the apparent affinity for external K⁺, but reduced its effectiveness compared with other monovalent cations, as an activator of Na⁺ efflux, and (iii) the relative effectiveness of different K⁺ congeners as external activator of the Na⁺ efflux, though affected by the ATP concentration, was not affected by the Na⁺/⁺ ratio inside the cells. These results are consistent with the idea that the same conformation of the (Na⁺ + K⁺)-ATPase can be reached by interaction with external K⁺ after phosphorylation and with internal K⁺ before rephosphorylation. They also stress a nonphosphorylating regulatory role of ATP.     

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.     

Divalent cations and the activation kinetics of potassium channels in squid giant axons

The effects of external Zn+2 and other divalent cations on K channels in squid giant axons were studied. At low concentration (2 mM) Zn+2 slows opening kinetics without affecting closing kinetics. Higher concentrations (5-40 mM) progressively slow opening and speed channel closing to a lesser degree. In terms of "shifts," opening kinetics are strongly shifted to the right on the voltage axis, and off kinetics much less so. The shift of the conductance-voltage relation along the axis is intermediate. Zinc's kinetic effects show little sign of saturation at the highest concentration attainable. Zn does not alter the shape of the instantaneous current-voltage relation of open channels. Some other divalent cations have effects similar to Zn+2, Hg2+ being the most potent and Ca+2 the least. After treatment with Hg+2, which is irreversible, Zn+2 still slows opening kinetics, which suggests that each channel has at least two sites for divalent cation action. The results are not compatible with a simple theory of fixed, uniform surface charges. They suggest that external cations interact directly with a negatively charged element of the gating apparatus that moves inward from the membrane's outer surface during activation. Examination of normal kinetics shows that there is a slow step somewhere in the chain leading to channel opening. But the slowest step must not be the last one.     

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.     

Sidedness of the ATP-Na+-K+ interactions with the Na+ pump in squid axons.

Using dialysed squid axons we have been able to control internal and external ionic compositions under conditions in which most of the Na+ efflux goes through the Na+ pump. We found that (i) internal K+ had a strong inhibitory effect on Na+ efflux; this effect was antagonized by ATP, with low affinity, and by internal Na+, (ii) a reduction in ATP levels from 3 mM to 50 microM greatly increased the apparent affinity for external K+, but reduced its effectiveness compared with other monovalent cations, as an activator of Na+ efflux, and (iii) the relative effectiveness of different K+ congeners as external activator of the Na+ efflux, though affected by the ATP concentration, was not affected by the Na+/K+ ratio inside the cells. These results are consistent with the idea that the same conformation of the (Na+ + K+)-ATPase can be reached by interaction with external K+ after phosphorylation and with internal K+ before rephosphorylation. They also stress a nonphosphorylating regulatory role of ATP.     

Transport of Na+ inside the giant axon of squid

The transport mechanism of Na ions within the nerve cell was studied by measuring the radioactivity distribution profile of 22Na that had been intracellularly injected into the giant axon. Specifically, we tested whether or not the movement of Na ions is coupled with the process of "fast axonal transport." Results of our measurements indicate that the intracellular transport of Na+ and the fast axonal transport are two independent processes. Very few Na ions are irreversibly sequestered into the axoplasmic vesicles involved in axonal transport. The movement of Na+ inside the axon can be modeled by a one-dimension diffusion. The effective diffusion coefficient of the intracellular Na+ was determined in this study.     

Transport of Na+ inside the giant axon of squid

The transport mechanism of Na ions within the nerve cell was studied by measuring the radioactivity distribution profile of²²Na that had been intracellularly injected into the giant axon. Specifically, we tested whether or not the movement of Na ions is coupled with the process of “fast axonal transport.” Results of our measurements indicate that the intracellular transport of Na⁺ and the fast axonal transport are two independent processes. Very few Na ions are irreversibly sequestered into the axoplasmic vesicles involved in axonal transport. The movement of Na⁺ inside the axon can be modeled by a one-dimension diffusion. The effective diffusion coefficient of the intracellular Na⁺ was determined in this study.     

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.     

Analysis of potassium conductance in squid giant axons

Assuming a model of facilitated ionic transport across axonal membranes proposed by McIlroy (1975) and extended by McIlroy and Hahn (1978), it is shown that if the selectivity coefficient, π_K, of the potassium conducting system ?59 the permeabilityP _Ks, of the periaxonal barrier of the squid giant axon for K⁺ ions?(1.2±0.44)×10^?4 cm sec^?1 and the thickness of the periaxonal space ?477±168 Å. Using a value (10^?4 cm sec^?1) ofP _Ks in the foregoing range the experimental curves for the steady state membrane ionic conductance versus measured membrane potential difference (p.d.), ?, of Gilbert and Ehrenstein (1969) are corrected for the effect of accumulation of K⁺ in the periaxonal space. This correction is most marked for the axon immersed in a natural ionic environment, whose conductance curve is shifted ?70mV along the voltage axis in the hyperpolarization direction. By assuming that the physico-chemical connection between a depolarization of the axonal membrane and the consequent membrane conductance changes is a Wien dissociative effect of the membrane's electric field on a weak electrolyte situated in the axolemma, the position of the peaks of the corrected conductance versus ? curves can be identified with zero membrane electric field and hence with zero p.d.across the axolemma. A set of values for the double-layer p.d.s at the axonal membrane interfaces with the external electrolytes in the vicinity of the K⁺ conducting pores can therefore be deduced for the various external electrolytes employed by Gilbert and Ehrenstein. A model of these double-layer p.d.s in which the membrane interfaces are assumed to possess fixed monovalent negatively charged sites, at least in the neighbourhood of the K⁺ conducting pores, is constructed. It is shown that, using the previously deduced values for the doublelayer p.d.s, such a model has a consistent, physically realistic solution for the distance between the fixed charged sites and for the dissociation constants of these sites in their interaction with the ions of the extramembrane electrolytes.