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.     

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.     

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.     

Activation-inactivation of potassium channels and development of the potassium-channel spike in internally perfused squid giant axons

A spike that is the result of calcium permeability through potassium channels was separated from the action potential is squid giant axons internally perfused with a 30 mM NaF solution and bathed in a 100 mM CaCl2 solution by blocking sodium channels with tetrodotoxin. Currents through potassium channels were studied under voltage clamp. The records showed a clear voltage-dependent inactivation of the currents. The inactivation was composed of at least two components; one relatively fast, having a time constant of 20--30 ms, and the other very slow, having a time constant of 5--10 s. Voltage clamp was carried out with a variety of salt compositions in both the internal and external solutions. A similar voltage-dependent inactivation, also composed of the two components, was recognized in all the current through potassium channels. Although the direction and intensity of current strongly depended on the salt composition of the solutions, the time-courses of these currents at corresponding voltages were very similar. These results strongly suggest that the inactivation of the currents in attributable to an essential, dynamic property of potassium channels themselves. Thus, the generation of a potassium-channel spike can be understood as an event that occurs when the equilibrium potential across the potassium channel becomes positive.     

Effects of N-alcohols on potassium conductance in squid giant axons

The effect of bath application of several short chain N-alcohols on voltage-dependent potassium conductance has been studied in intact giant axons of Loligo forbesi under voltage-clamp conditions. All tested alcohols (methanol, ethanol, propanol, butanol, heptanol and octanol) were found to depress potassium conductance only at concentrations much larger than those necessary to reduce sodium conductance. The efficacy of the different molecules was correlated with the carbon-chain length. In all cases the effects were found to be at least partly reversible. Low concentrations of propanol (100 mM) or heptanol (1 mM) were found to increase potassium conductance whereas higher concentrations had the usual depressing effect. The two alcohols were found to induce a slow inactivation of the potassium conductance. A detailed analysis of the time course of the turning-on of the potassium current for various pulse potentials in the presence of TTX revealed that, for membrane potential values more positive than -20 mV, the time constant of activation was reduced in the presence of propanol or heptanol. The delay which separates the change in potential and the turning-on of the potassium current, which was systematically analysed for different pulse and prepulse potential values, was increased by the two alcohols, the curve relating this delay to prepulse potential being shifted towards larger (positive) delays. This high degree of complexity in the effects on potassium conductance suggests that the alcohol molecules modify several more or less independent mechanisms associated with the turning-on of the potassium current.     

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.     

Modified Hodgkin-Huxley gating kinetics of sodium activation in giant axons of squid (Doryteuthis bleekeri)

The probabilities m of the sodium activation gate being open are shown to fit experimentally-determined running integrals Qg of recordings of the colchicine-sensitive fraction of the asymmetry current, within the Hodgkin-Huxley framework that the gate could have only two conformations, open and closed. Using the Hodgkin-Huxley framework, we are obliged to assume that the transition velocities, alpha m and beta m, between the open and closed gates depend not only on membrane potentials V but also on the time after a potential step was externally applied. We introduce the following functions of alpha m and beta m. (sequence in text) where VH, td and tau p stand for holding potential, constant delay time of 10 microseconds, and transit time of the transition velocity of alpha m (or beta m) from its initial value alpha om (or beta om) to its final steady value alpha infinity m (or beta infinity m), respectively. The transit time tau p was found to be potential-dependent; typically it was 30 microseconds at -20 mV, and 100 microseconds at 20-40 mV. The values of alpha infinity m, alpha om, beta infinity m and beta om were found to be in reasonable agreement with those obtained by others, under the Hodgkin-Huxley assumption that the gate followed first-order kinetics. The requirement of new parameters, tau p and td, in the transition velocities was discussed in a relation to a membrane model where a voltage-receptor and a sodium channel macromolecule are spatially separated but functionally connected through underlying cytoskeletons (Matsumoto, 1984).     

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.     

Gating current and potassium channels in the giant axon of the squid.

Gating current (Ig) underlying Na-channel activation is large enough to enable resolution of components both preceding and paralleling Na conductance (gNa) turn-on. For large depolarizations (beyond +20 mV), an additional "slow phase" of Ig is observed during a time when Na activation is already complete, but when K-channel opening is just becoming detectable. If Na- and K-channel gating are similar, the slow kinetics and long delay for K activation predict that K channel Ig must be relatively small and slow. Externally applied dibucaine almost totally blocks gNa and greatly reduces the fast (Na channel) Ig without altering gK or the Ig slow phase. The slow phase of Ig depends in part of the presence of functional K channels. Selective diminution in amplitude of the slow phase is consistently observed after a 30-min perfusion with both external and internal K-free media, a procedure which destroys nearly all K channels. This decrease of Ig amounts to approximately 10% of the total charge movements at +40 to +80 mV, with gating charge and K channels disappearing in a ratio of less than 1 e- per picosiemens of gK. These findings are consistent with the idea that part of the Ig slow phase represents gating current generated by the early steps in K-channel activation.     

Asymmetric modulation and blockade of the delayed rectifier in squid giant axons by divalent cations.

The effects of intracellular magnesium ions and extracellular calcium and magnesium ions on the delayed rectifier potassium ion channel, IK, were investigated from intracellularly perfused squid giant axons. Cao+2 and Mgo+2 both blocked IK in a voltage-independent manner with a KD of approximately 100 and 500 mM, respectively. This effect was obscured at potentials in the vicinity of the resting potential (approximately -60 mV) by a rightward shift of the steady-state IK inactivation curve along the voltage axis. The addition of either calcium or magnesium ions to the extracellular solution also produced the well known shift of the IK activation curve along the voltage axis. Cao+2 was approximately twice as effective in this regard as Mgo+2. The IK activation kinetics were slowed by Cao+2, but deactivation kinetics were not altered, as shown previously. Similar results were obtained with Mgo+2. The addition of magnesium ions to the intracellular perfusate shifted the activation curve along the voltage axis in the negative direction (without producing block) by approximately the same among as the Mgo+2 shift of this curve in the positive direction. Moreover, Mgi+2 substantially slowed the deactivation kinetics, whereas the effects of Mgi+2 on activation kinetics at strongly depolarized potentials were relatively minor. At modest depolarizations, Mgi+2 significantly reduced the delay before IK activation. These results are essentially the mirror image of the effects on gating of extracellular divalent cations.     

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.     

Phosphorylation modulates potassium conductance and gating current of perfused giant axons of squid

The presence of internal Mg-ATP produced a number of changes in the K conductance of perfused giant axons of squid. For holding potentials between -40 and -50 mV, steady-state K conductance increased for depolarizations to potentials more positive than approximately -15 mV and decreased for smaller depolarizations. The voltage dependencies of both steady-state activation and inactivation also appears shifted toward more positive potentials. Gating kinetics were affected by internal ATP, with the activation time constant slowed and the characteristic delay in K conductance markedly enhanced. The rate of deactivation also was hastened during perfusion with ATP. Internal ATP affected potassium channel gating currents in similar ways. The voltage dependence of gating charge movement was shifted toward more positive potentials and the time constants of ON and OFF gating current also were slowed and hastened, respectively, in the presence of ATP. These effects of ATP on the K conductance occurred when no exogenous protein kinases were added to the internal solution and persisted even after removing ATP from the internal perfusate. Perfusion with a solution containing exogenous alkaline phosphatase reversed the effects of ATP. These results provide further evidence that the effects of ATP on the K conductance are a consequence of a phosphorylation reaction mediated by a kinase present and active in perfused axons. Phosphorylation appears to alter the K conductance of squid giant axons via a minimum of two mechanisms. First, the voltage dependence of gating parameters are shifted toward positive potentials. Second, there is an increase in the number of functional closed states and/or a decrease in the rates of transition between these states of the K channels.     

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.     

Kinetics of activation of the potassium conductance in the squid giant axon

A quantitative re-investigation of the time course of the initial rise of the potassium current in voltage-clamped squid giant axons is described. The n4 law of the Hodgkin-Huxley equations was found to be well obeyed only for the smallest test pulses, and for larger ones a good fit of the inflected rise required use of the expression (1-exp[-t/tau n1])X-1(1-exp[-t/tau n2]), where both of the time constants and the power X varied with the size of the test pulse. Application of a negative prepulse produced a delay in the rise resulting mainly from an increase of X from a value of about 3 at -70 mV to 8 at -250 mV, while tau n1 remained constant and tau n2 was nearly doubled. The process responsible for generating this delay was switched on with a time constant of 8 ms at 4 degrees C, which fell to about 1 ms at 15 degrees C. Analysis of the inward tail currents at the end of a voltage-clamp pulse showed that there was a substantial external accumulation of potassium owing to the restriction of its diffusion out of the Schwann cell space, which, when duly allowed for, roughly doubled the calculated value of the potassium conductance. Computations suggested that the principal effect of such a build-up of [K]o would be to reduce the fitted values of tau n1 and tau n2 to two-thirds or even half their true sizes, while the power X would generally be little changed; but it would not affect the necessity to introduce a second time constant, nor would it invalidate our findings on the effect of negative prepulses.     

Potassium ion accumulation slows the closing rate of potassium channels in squid axons.

Potassium ion accumulation in the periaxonal space between squid axonal membrane and the Schwann cell surrounding the axon slows the rate of potassium channel closing to a degree that is consistent with the effect on channel closing of an equivalent change in the bulk external potassium concentration. The alteration of channel gating is independent of membrane potential, V, for V less than or equal to -60 mV, which suggests that the effect is mediated at a site on the outer surface of the membrane, rather than a site within the channel.     

Interaction of internal anions with potassium channels of the squid giant axon

The interaction of internal anions with the delayed rectifier potassium channel was studied in perfused squid axons. Changing the internal potassium salt from K+ glutamate- to KF produced a reversible decline of outward K currents and a marked slowing of the activation of K channels at all voltages. Fluoride ions exert a differential effect upon K channel gating kinetics whereby activation of IK during depolarizing steps is slowed dramatically, but the rate of closing after the step is not much altered. These effects develop with a slow time course (30-60 min) and are specific for K channels over Na channels. Both the amplitude and activation rate of IK were restored within seconds upon return to internal glutamate solutions. The fluoride effect is independent of the external K+ concentration and test membrane potential, and does not recover with repetitive application of depolarizing voltage steps. Of 11 different anions tested, all inorganic species induced similar decreases and slowing of IK, while K currents were maintained during extended perfusion with several organic anions. Anions do not alter the reversal potential or shape of the instantaneous current-voltage relation of open K channels. The effect of prolonged exposure to internal fluoride could be partially reversed by the addition of cationic K channel blocking agents such as TEA+, 4-AP+, and Cs+. The competitive antagonism between inorganic anions and internal cationic K channel blockers suggests that they may interact at a related site(s). These results indicate that inorganic anions modify part of the K channel gating mechanism (activation) at a locus near the inner channel surface.