Interactions of aminopyridines with potassium channels of squid axon membranes.

The effects of aminopyridines on ionic conductances of the squid giant axon membrane were examined using voltage clamp and internal perfusion techniques. 4-Aminopyridine (4-AP) reduced potassium currents, but had no effect upon transient sodium currents. The block of potassium channels by 4-AP was substantially less with (a) strong depolarization to positive membrane potentials, (b) increasing the duration of a given depolarizing step, and (c) increasing the frequency of step depolarizations. Experiments with high external potassium concentrations revealed that the effect of 4-AP was independent of the direction of potassium ion movement. Both 3- and 2-aminopyridine were indistinguishable from 4-AP except in potency. It is concluded that aminopyrimidines may be used as tools to block the potassium conductance in excitable membranes, but only within certain specific voltage and frequency limits.     

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.     

Effects of batrachotoxin on membrane potential and conductance of squid giant axons

The effects of batrachotoxin (BTX) on the membrane potential and conductances of squid giant axons have been studied by means of intracellular microelectrode recording, internal perfusion, and voltage clamp techniques. BTX (550–1100 nM) caused a marked and irreversible depolarization of the nerve membrane, the membrane potential being eventually reversed in polarity by as much as 15 mv. The depolarization progressed more rapidly with internal application than with external application of BTX to the axon. External application of tetrodotoxin (1000 nM) completely restored the BTX depolarization. Removal or drastic reduction of external sodium caused a hyperpolarization of the BTX-poisoned membrane. However, no change in the resting membrane potential occurred when BTX was applied in the absence of sodium ions in both external and internal phases. These observations demonstrate that BTX specifically increases the resting sodium permeability of the squid axon membrane. Despite such an increase in resting sodium permeability, the BTX-poisoned membrane was still capable of undergoing a large sodium permeability increase of normal magnitude upon depolarizing stimulation provided that the membrane potential was brought back to the original or higher level. The possibility that a single sodium channel is operative for both the resting sodium, permeability and the sodium permeability increase upon stimulation is discussed.     

Effect of phloretin on the permeability of thin lipid membranes

Phloretin dramatically increases cation conductances and decreases anion conductances of membranes treated with ion carriers (nonactin, valinomycin, carbonyl-cyanide-m-chlorophenylhydrazone [CCCP], and Hg(C6F5)2) or lipophilic ions (tetraphenylarsonium [tphAs+] and tetraphenylborate [TPhB-]). For example, on phosphatidylethanolamine membranes, 10(-4) M phloretin increases K+ -nonactin and TPhAs+ conductances and decreases CCCP- and TPhB- conductances 10(3)-fold; on lecithin: cholesterol membranes, it increases K+-nonactin conductance 10(5)-fold and decreases CCCP- conductance 10(3)-fold. Similar effects are obtained with p- and m-nitrophenol at 10(-2) M. These effects are produced by the un-ionized form of phloretin and the nitrophenols. We believe that phloretin, which possesses a large dipole moment, adsorbs and orients at the membrane surface to introduce a dipole potential of opposite polarity to the preexisting positive one, thus increasing the partition coefficient of cations into the membrane interior and decreasing the partition coefficient of anions. (Phloretin may also increase the fluidity of cholesterol-containing membranes; this is manifested by its two- to three-fold increase in nonelectrolyte permeability and its asymmetrical effect on cation and anion conductances in cholesterol-containing membranes.) It is possible that pholoretin's inhibition of chloride, urea, and glucose transport in biological membranes results from the effects of these intense intrafacial dipole fields on the translocator(s) of these molecules.     

Anion conductances of the giant axon of squid Sepioteuthis.

Anion conductances of giant axons of squid, Sepioteuthis, were measured. The axons were internally perfused with a 100-mM tetraethylammonium-phosphate solution and immersed in a 100-mM Ca-salt solution (or Mg-salt solution) containing 0.3 microns tetrodotoxin. The external anion composition was changed. The membrane currents had a large amount of outward rectification due to anion influx across Cl- channels of the membrane (Inoue, 1985). The amount of outward rectification depended on the species of anion used and was strongly influenced by temperature and internal pH. In contrast to the anion conductances themselves, the conductance relative to Cl- (gA/gCl) was found to be quite stable against changes in the membrane potential, temperature, and pH. It is therefore suggested that each gA/gCl is an intrinsic quantity of the Cl- channel of the squid axon membrane. The sequence and values of gA/gCl obtained in this study were NO3- (1.80) greater than I- (1.40) greater than Br- (1.07) greater than Cl- (1.00) greater than MeSO3- (0.46) greater than H2PO2- (0.33) greater than CH3COO- (0.29) greater than SO4(2-) (0.06).     

Competitive Action of Calcium and Procaine on Lobster Axon : A study of the mechanism of action of certain local anesthetics

Voltage clamp studies with the squid giant axon have shown that changes in the external calcium concentration (Frankenhaeuser and Hodgkin, 1957) shift the sodium and potassium conductance versus membrane potential curves along the potential axis. Taylor (1959) found that procaine acts primarily by reducing the sodium and, to a lesser extent, the potassium conductances. Both procaine and increased calcium also delay the turning on of the sodium conductance mechanism. Calcium and procaine have similar effects on lobster giant axon. In addition, we have observed that the magnitude of the response to procaine is influenced by the external calcium concentration. Increasing external calcium tends to reduce the effectiveness of procaine in decreasing sodium conductance. Conversely, procaine is more effective in reducing the membrane conductance if external calcium is decreased. The amplitude of the nerve action potential reflects these conductance changes in that, for example, reductions in amplitude resulting from the addition of procaine to the medium are partially restored by increasing external calcium, as was first noted by Aceves and Machne (1963). These phenomena suggest that calcium and procaine compete with one another with respect to their actions on the membrane conductance mechanism. The fact that procaine and its analogues compete with calcium for binding to phospholipids in vitro (Feinstein, 1964) suggests that the concept of competitive binding to phospholipids may provide a useful model for interpreting these data.     

On the voltage-dependent action of tetrodotoxin.

The use of the maximum rate-of-rise of the action potential (Vmax) as a measure of the sodium conductance in excitable membranes is invalid. In the case of membrane action potentials, Vmax depends on the total ionic current across the membrane; drugs or conditions that alter the potassium or leak conductances will also affect Vmax. Likewise, long-term depolarization of the membrane lessens the fraction of total ionic current that passes through the sodium channels by increasing potassium conductance and inactivating the sodium conductance, and thereby reduces the effect of Vmax of drugs that specifically block sodium channels. The resultant artifact, an apparent voltage-dependent potency of such drugs, is theoretically simulated for the effects of tetrodotoxin on the Hodgkin-Huxley squid axon.     

Sodium channel selectivity. Dependence on internal permeant ion concentration

The selectivity of sodium channels in squid axon membranes was investigated with widely varying concentrations of internal ions. The selectivity ratio, PNa/PK, determined from reversal potentials decreases from 12.8 to 5.7 to 3.5 as the concentration of internal potassium is reduced from 530 to 180 to 50 mM, respectively. The internal KF perfusion medium can be diluted by tetramethylammonium (TMA), Tris, or sucrose solutions with the same decrease in PNa/PK. The changes in the selectivity ratio depend upon internal permeant ion concentration rather than ionic strength, membrane potential, or chloride permeability. Lowering the internal concentration of cesium, rubidium, guanidnium, or ammonium also reduces PNa/Pion. The selective sequence of the sodium channel is: Na greater than guanidinium greater than ammonium greater than K greater than Rb greater than Cs.     

Potassium ion accumulation in a periaxonal space and its effect on the measurement of membrane potassium ion conductance

Summary Potassium currents of various durations were obtained from squid giant axons voltage-clamped in artificial seawater solutions containing sufficient tetrodotoxin to block the sodium conductance completely. From instantaneous potassium current-voltage relations, the reversal potentials immediately at the end of these currents were determined. On the basis of these reversal potential measurements, the potassium ion concentration gradient across the membrane was shown to decrease as the potassium current duration increased. The kinetics of this change was shown to vary monotonically with the potassium ion efflux across the membrane estimated from the integral over time of the potassium current divided by the Faraday, and to be independent of both the external sodium ion concentration and the presence or absence of membrane series resistance compensation. It was assumed that during outward potassium current flow, potassium ions accumulated in a periaxonal space bounded by the membrane and an external diffusion barrier. A model system was used to describe this accumulation as a continuous function of the membrane currents. On this basis, the mean periaxonal space thickness and the permeability of the external barrier to K⁺ were found to be 357 Å and 3.21×10^–4 cm/sec, respectively. In hyperosmotic seawater, the value of the space thickness increased significantly even though the potassium currents were not changed significantly. Values of the resistance in series with the membrane were calculated from the values of the permeability of the external barrier and these values were shown to be roughly equivalent to series resistance values determined by current clamp measurements. Membrane potassium ion conductances were determined as a function of time and voltage. When these were determined from data corrected for the potassium current reversal potential changes, larger maximal potassium conductances were obtained than were obtained using a constant reversal potential. In addition, the potassium conductance turn-on with time at a variety of membrane potentials was shown to be slower when potassium conductance values were obtained using a variable reversal potential than when using a constant reversal potential.     

Effects of proteolytic enzymes on ionic conductances of squid axon membranes.

The effects of proteolytic enzymes on ionic conductances of squid axon membranes have been studied by means of the voltage clamp technique. When perfused internally alpha-chymotrypsin (1 mg/ml) increased and prolonged the depolarizing after-potential. Sodium inactivation was partially inhibited causing a prolonged sodium current, and peak sodium and steady-state potassium currents were suppressed. The time for sodium current to reach its peak was not affected. Leakage conductance increased later. On the other hand, carboxypeptidases A and B, both at 1mg/ml, suppressed the sodium and potassium conductance increases with little or no change in sodium inactivation. The mechanism that controls sodium inactivation appears to be associated with the structure of membrane proteins which is modified by alpha-chymotrypsin but not by carboxypeptidases and is located in a position accessible to alpha-chymotrypsin only from inside the membrane.     

Dynamics of potassium ion currents in squid axon membrane. A re-examination.

The original experiments of Cole and Moore (1960. Biophys. J. 1:161-202.), using conditioning and test membrane potentials to examine the dynamics of the potassium channel conductance in the squid axon, have been extended to test voltage levels by the use of tetrodotoxin to block the sodium conductance. The potassium currents for test voltage levels from -20 to +85 mV were superposable by translation along the time axis for all conditions tested: (a) with depolarizing conditioning voltages; (b) with hyperpolarizing conditioning voltages; and (c) in normal and in high potassium external media. The only deviations from superposition seen were when the internal sodium concentration was abnormally high and the potassium currents showed saturation at high levels of depolarization. Some restoration toward normal kinetics could be obtained by rapidly repeated depolarizations.     

Kinetic analysis of pancuronium interaction with sodium channels in squid axon membranes

The interaction of pancuronium with sodium channels was investigated in squid axons. Sodium current turns on normally but turns off more quickly than the control with pancuronium 0.1-1mM present internally; The sodium tail current associated with repolarization exhibits an initial hook and then decays more slowly than the control. Pancuronium induces inactivation after the sodium inactivation has been removed by internal perfusion of pronase. Such pancuronium-induced sodium inactivation follows a single exponential time course, suggesting first order kinetics which represents the interaction of the pancuronium molecule with the open sodium channel. The rate constant of association k with the binding site is independent of the membrane potential ranging from 0 to 80 mV, but increases with increasing internal concentration of pancuronium. However, the rate constant of dissociation l is independent of internal concentration of pancuronium but decreases with increasing the membrane potential. The voltage dependence of l is not affected by changine external sodium concentration, suggesting a current-independent conductance block, The steady-state block depends on the membrane potential, being more pronounced with increasing depolarization, and is accounted for in terms of the voltage dependence of l. A kinetic model, based on the experimental observations and the assumption on binding kinetics of pancuronium with the open sodium channel, successfully simulates many features of sodium current in the presence of pancuronium.     

Simulation of the electrical activity of an excitable membrane with an electronic analog (author's transl)

1. The sodium and potassium conductances of the HODGKIN-HUXLEY model are simulated by a field effect transistor with a series resistor. This arrangement leads to a simple analog model of the excitable membrane (fig. 1 and 2). 2. Normally, the model is silent (fig. 3), but it becomes automatic (fig. 4) when the decay time (de-activation) of the potassium conductance is at least twice the recovery from inactivation time of the sodium conductance (taud greater than 2 tauri). 3. The effects of changes in sodium (fig. 5 and 6) and potassium (fig. 7, 8 and 9) concentration gradients upon the membrane potential and the ionic currents are easily studied when the model is silent or automatic. 4. When automatic, an increase in the potassium concentration gradient induces a lengthening of the period and ultimately, when the gradient is very high, spontaneous activity is blocked (fig. 9). On the other hand, increases of sodium gradient over 30% of normal value do not modify the period (fig 6). 5. The potassium concentration gradient modifies the excitability solely through membrane polarization (fig. 8), while sodium concentration has no effect on it (fig. 5). 6. Results with the model strengthen the hypothesis that tetraethylammonium (TEA) acts on both the maximum potassium conductance (gK) and the mechanism of sodium conductance inactivation (Tauh) to lengthen the action potential as observed on the Ranvier node (fig. 10). Effects of TEA on potassium conductance activation are also discussed. 7. Because of its simplicity and accuracy, this model lends itself easily to many other simulations.     

Catecholamine-stimulated ion transport in duck red cells. Gradient effects in electrically neutral [Na + K + 2Cl] Co-transport

The transient increase in cation permeability observed in duck red cells incubated with norepinephrine has been shown to be a linked, bidirectional, co-transport of sodium plus potassium. This pathway, sensitive to loop diuretics such as furosemide, was found to have a [Na + K] stoichiometry of 1:1 under all conditions tested. Net sodium efflux was inhibited by increasing external potassium, and net potassium efflux was inhibited by increasing external sodium. Thus, the movement of either cation is coupled to, and can be driven by, the gradient of its co-ion. There is no evidence of trans stimulation of co- transport by either cation. The system also has a specific anion requirement satisfied only by chloride or bromide. Shifting the membrane potential by varying either external chloride (at constant internal chloride) or external potassium (at constant internal potassium in the presence of valinomycin and DIDs [4,4'-diisothiocyano- 2,2'-disulfonic acid stilbene]), has no effect on nor-epinephrine- stimulated net sodium transport. Thus, this co-transport system is unaffected by membrane potential and is therefore electrically neutral. Finally, under the latter conditions-when Em was held constant near EK and chloride was not at equilibrium-net sodium extrusion against a substantial electrochemical gradient could be produced by lowering external chloride at high internal concentrations, thereby demonstrating that the anion gradient can also drive co-transport. We conclude, therefore, that chloride participates directly in the co- transport of [Na + K + 2Cl].     

The Cole-Moore Effect: Still Unexplained?

In the first issue, on the first page of the Biophysical Journal in 1960, Cole and Moore provided the first confirmation of the Hodgkin and Huxley formulation of the sodium and potassium conductances that underlie the action potential. In addition, working with the squid giant axon, Cole and Moore noted that strong hyperpolarization preceding a depolarizing voltage-clamp pulse delayed the rise of the potassium conductance: once started, the time course of the rise was always the same but after significant hyperpolarization there was a long lag before the rise began. This phenomenon has come to be known as the Cole-Moore effect. Their article examines and disproves the hypothesis that the lag reflects the time required to refill the membrane with potassium ions after the ions are swept out of the membrane into the axoplasm by hyperpolarization. The work by Cole and Moore indirectly supports the idea of a membrane channel for potassium conductance. However, the mechanism of the Cole-Moore effect remains a mystery even now, buried in the structure of the potassium channel, which was completely unknown at the time.     

Modulation of aminopyridine block of potassium currents in squid axon.

Aminopyridines are known to block potassium (K) currents in excitable membranes in a manner dependent upon membrane potential, such that the block is relieved by depolarization and restored upon repolarization. In the present study, the effects of aminopyridines on voltage-dependent potassium (K) channels were examined in internally perfused, voltage-clamped squid giant axons. The time course of block restoration after conditioning depolarization was found to be modulated by membrane electric field, K-channel gating, and external cations. Depolarized holding potentials accelerated block restoration without altering steady-state block levels, suggesting that the voltage dependence of block restoration may be related to K channel gating rather than drug binding per se. In support of this notion, low external calcium concentration, which shifts the voltage dependence of K-channel gating to more negative potentials, also accelerated block restoration. Conversely, the relationship between the rate of block restoration and membrane holding potential was shifted in the depolarizing direction by phloretin, an agent that shifts the dependence of K-channel opening on membrane potential in a similar manner. Modification of K-channel gating also was found to alter the rate of block restoration. Addition of internal zinc or internal treatment with glutaraldehyde slowed the time course of both K-channel activation and aminopyridine block restoration. Aminopyridines also were found to interact in the K channel with external Cs+, NH4+, and Rb+, each of which slowed aminopyridine block restoration. Our results suggest that aminopyridines enter and occlude K channels, and that the availability of the binding site may be modulated by channel gating such that access is limited by the probability of the channel reaching an intermediate closed state at the resting potential.     

Probing amphotericin B single channel activity by membrane dipole modifiers

The effects of dipole modifiers and their structural analogs on the single channel activity of amphotericin B in sterol-containing planar phosphocholine membranes are studied. It is shown that the addition of phloretin in solutions bathing membranes containing cholesterol or ergosterol decreases the conductance of single amphotericin B channels. Quercetin decreases the channel conductance in cholesterol-containing bilayers while it does not affect the channel conductance in ergosterol-containing membranes. It is demonstrated that the insertion of styryl dyes, such as RH 421, RH 237 or RH 160, in bilayers with either cholesterol or ergosterol leads to the increase of the current amplitude of amphotericin B pores. Introduction of 5α-androstan-3β-ol into a membrane-forming solution increases the amphotericin B channel conductance in a concentration-dependent manner. All the effects are likely to be attributed to the influence of the membrane dipole potential on the conductance of single amphotericin B channels. However, specific interactions of some dipole modifiers with polyene-sterol complexes might also contribute to the activity of single amphotericin B pores. It has been shown that the channel dwell time increases with increasing sterol concentration, and it is higher for cholesterol-containing membranes than for bilayers including ergosterol, 6-ketocholestanol, 7-ketocholestanol or 5α-androstan-3β-ol. These findings suggest that the processes of association/dissociation of channel forming molecules depend on the membrane fluidity.     

A voltage-clamp study of the effects of colchicine on the squid giant axon

The effects of colchicine applied inside a squid giant axon were studied using voltage-clamp and internal perfusion techniques. It was found that colchicine selectively and reversibly suppresses the sodium conductance during excitation. The possible involvement of the microtubular structure in the functioning of the excitable channel is discussed.     

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.     

Effects of phlorizin and phloretin on passive and dynamic electrical properties in muscle membrane

The effects of phlorizin and phloretin on the cable properties were investigated in frog sartorius muscle by conventional cable analysis. Actions of phloretin on voltage-dependent ionic conductances were also studied by analysis of the phase plane trajectories. Both drugs evoked a significant decrease in specific membrane resistance (Rm) in chloride-containing Ringer's solution. The linear membrane capacitance increased by about 30%. On the contrary, in the presence of the non-penetrating anion, glutamate, a slight increase in Rm was induced by phlorizin. It is suggested that these drugs may increase the chloride conductance in the muscle membrane. Under the effect of phloretin the resting membrane potential remained unchanged but the amplitude of the action potential was lowered and the rate of repolarization was significantly reduced. The rate of depolarization during the "foot" of the action potential and the conduction velocity calculated from the rate constant of depolarization decreased. The maximum Na conductance was not altered by phloretin but K conductance was reduced. The time constant (tau K) reflecting the kinetic properties of K conductance was increased about seven-fold. It is suggested that great importance may be attributed to the dipole properties of these drugs in the actions presented above.