Effects of barium on the potassium conductance of squid axon

Ba++ ion blocks K+ conductance at concentrations in the nanomolar range. This blockage is time and voltage dependent. From the time dependence it is possible to determine the forward and reverse rate constants for what appears to be an essentially first-order process of Ba++ interaction. The voltage dependence of the rate constants and the dissociation constants place the site of interaction near the middle of the membrane field. Comparison of the efficacy of Ba++ block at various internal K+ concentrations suggests that Ba++ is probably a simple competitive inhibitor of K+ interaction with the K+ conductance. The character of Ba++ block in high external K+ solutions suggests that Ba++ ion may be "knocked-off" the site by inward movement of external K+. Examination of the effects of other divalent cations suggests that the channel may have a closed state with a divalent cation inside the channel. The relative blockage at different temperatures implies a strong interaction between Ba++ and the K+ conductance.     

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.     

Transglial pathway of diffusion in the Schwann sheath of the squid giant axon

Brain Cell Biology - In order to investigate the transglial pathways in the Schwann sheath of squid giant axons, an electron microscopic study of thin sections and freeze-fracture replicas was...     

Sodium, potassium, and chloride concentrations in the Schwann cell and axon of the squid nerve fiber

Sodium, potassium, and chloride concentrations were determined in the sheath cells and axoplasm of the nerve fiber of the squid Sepioteuthis sepioidea. The sheaths were obtained by slitting the nerve fiber, the extracellular electrolytes were washed out in isotonic sucrose solution, and the concentrations in the cells were determined after different soaking times in the sucrose solution. Values for the Schwann cell were calculated by extrapolation to zero time from the plots of the logarithms of the concentrations in the cells as a function of soaking time in sucrose solution. The Schwann cells made up 84 per cent of the sheath''s total cellular volume. The Schwann cell concentrations in millimols per liter, are: 312 (404-241) for sodium, 220 (308-157) for potassium, and 167 (208-138) for chloride. The concentrations in the axoplasm (mean ± SE), in millimols per liter are: 52 ± 10 for sodium, 335 ± 25 for potassium, and 135 ± 14 for chloride. The possibility that some fraction of the Schwann cell electrolytes, especially of sodium, is bound, cannot be discarded.     

The effects of homologous series of anaesthetics on a resting potassium conductance of the squid giant axon

The effects of n-alkanes (n-pentane to n-octane), n-alkanols (n-pentanol to n-undecanol) and two carboxylic esters (methyl pentanoate and methyl octanoate) on the conductance of squid giant axons in a high potassium, zero sodium bathing solution have been examined. Sodium and delayed rectifier potassium channels were as far as possible pharmacologically blocked. A substantial fraction of the measured conductance is attributed to a recently-described, voltage-independent, potassium channel. Anaesthetics block this channel but its sensitivity is markedly different from those of other squid axon ion channels.     

The effect of temperature,potassium, and sodium on the conductance change accompanying the action potential in the squid giant axon

Conductance changes associated with the response of the squid giant axon have been studied at two temperature ranges (26–27°C.; 9–10°C.) and with modified concentrations of sodium and potassium in the medium. The phase of "initial after-conductance," during which the membrane resistance increases above the resting value, is smaller at the lower temperature. At both temperature ranges it is diminished by doubling K⁺ in the medium and enhanced by removal of K⁺. Halving the Na⁺ of the medium also enhances this phase when K⁺ is absent, but not otherwise. The time course of the conductance changes alters in form with changes of the external medium. These changes indicate independent changes in the complex of ionic events associated with the response. The experiments therefore confirm the reality of the phase of increased membrane resistance. The magnitude of this change appears to be considerable and requires a transient decrease in the mobility and/or concentration of ions in the membrane. The possible cause of this decrease is discussed.     

Blockage of squid axon potassium conductance by internal tetra-N-alkylammonium ions of various sizes.

We have studied the effects of the tetra-n-alkylammonium (TAA) ions, (CnH2n+1)4N+, n = 1-6, on the potassium conductance of voltage-clamped squid giant axons. Studies using tetrahexylammonium were not quantitatively analyzed as its effect was insufficiently reversible. Each in this series of symmetric ions of graded size blocks the potassium conductance when added to the internal perfusion fluid. There is a general trend for blocking potency to increase with increasing size. We attribute this to stronger interactions of the longer alkyl side chains with hydrophobic regions of the membrane near the channels. Steady-state block by the TAA ions, n = 2-5, showed identical voltage dependence, apparently sensing about 15% of the transmembrane voltage, and kinetics block onset were qualitatively similar. We conclude that the site of action for these ions is the same. Block by TMA is about twice as steeply dependent on voltage. In its action, TMA resembles the alkali cations (French et al., 1979, Biophys, J. 25(2, pt. 2):307a) more than the larger TAA ions. Our results suggest that access to the inner mouth of the K channel is even less restricted than has been previously thought. A calculation indicates that the lumen of the channel cannot be both wide enough to admit the TAA ions and long enough to account for the voltage dependence of block. We consider possible ways to resolve this paradox.     

An analysis of conductance changes in squid axon

The membrane of the squid axon is considered on the basis of a pore model in which the distribution of the pore sizes strongly favors K⁺ transfer when there is no potential. Electrical asymmetry causes non-penetrating ions on the membrane capacitor to exert a mechanical force on both membrane surfaces and this force results in a deformation of the membrane pore system such that it assumes a distribution of sizes favoring the ions exerting mechanical force. The ions involved appear to be Ca⁺⁺ on the outside of the membrane and isethionate^-, (i^-) on the inside; as Ca⁺⁺ is equivalent in size to Na⁺, the charged membrane is potentially able to transfer Na⁺, when the ions deforming the membrane pore distribution are removed. A depolarization of the membrane leads to an opening of pores that will allow Na⁺ penetration and a release of the membrane from deformation. The pores revert to the zero-potential pore size distribution hence the Na permeability change is a transient. Calculation shows that the potassium conductance vs. displacement of membrane potential curve for the squid axon and the "inactivation" function, h, can be obtained directly from the assumed membrane distortion without the introduction of arbitrary parameters. The sodium conductance, because it is a transient, requires assumptions about the time constants with which ions unblock pores at the outside and the inside of the membrane.     

Potassium conductance of the squid giant axon. Single-channel studies

The patch-clamp technique was implemented in the cut-open squid giant axon and used to record single K channels. We present evidence for the existence of three distinct types of channel activities. In patches that contained three to eight channels, ensemble fluctuation analysis was performed to obtain an estimate of 17.4 pS for the single-channel conductance. Averaged currents obtained from these multichannel patches had a time course of activation similar to that of macroscopic K currents recorded from perfused squid giant axons. In patches where single events could be recorded, it was possible to find channels with conductances of 10, 20, and 40 pS. The channel most frequently encountered was the 20-pS channel; for a pulse to 50 mV, this channel had a probability of being open of 0.9. In other single-channel patches, a channel with a conductance of 40 pS was present. The activity of this channel varied from patch to patch. In some patches, it showed a very low probability of being open (0.16 for a pulse to 50 mV) and had a pronounced lag in its activation time course. In other patches, the 40-pS channel had a much higher probability of being open (0.75 at a holding potential of 50 mV). The 40-pS channel was found to be quite selective for K over Na. In some experiments, the cut-open axon was exposed to a solution containing no K for several minutes. A channel with a conductance of 10 pS was more frequently observed after this treatment. Our study shows that the macroscopic K conductance is a composite of several K channel types, but the relative contribution of each type is not yet clear. The time course of activation of the 20-pS channel and the ability to render it refractory to activation only by holding the membrane potential at a positive potential for several seconds makes it likely that it is the predominant channel contributing to the delayed rectifier conductance.     

Modification of K conductance of the squid axon membrane by SITS

The effects of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) on the K conductance, gK, were studied in internally perfused giant axons from squid, Doryteuthis. SITS at 3-200 microM was applied intracellularly by adding the reagent to the internal perfusion fluid. Three remarkable changes in gK were noted: there was a slowing of the opening and closing rates of the K channel in the whole voltage region; K channels modified with SITS started to open at voltages below -100 mV, and thus 30% of total K channels were open at the level of normal resting potential (approximately -60 mV) after the maximal drug effect was attained (less than 30 microM); there was a disappearance of gK inactivation that became distinct at relatively high temperature (greater than 8 degrees C). These drug effects depended solely on the drug concentration, not on factors such as repetitive alterations of the membrane potential, and the changes in gK were almost irreversible. Another disulfonic stilbene derivative, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), had similar effects on gK, but the effects were approximately 1.5 times stronger. These changes in gK were somewhat similar to alterations in gNa produced by an application of veratridine, batrachotoxin, and grayanotoxin, which are known as Na channel openers.     

The rate of action of tetrodotoxin on sodium conductance in the squid giant axon.

When tetrodotoxin is applied to or washed away from the squid giant axon, the rates at which the sodium conductatnce is blocked and unblocked are an order of magnitude smaller than those reported for the isolated node of Ranvier. This slowing is to be expected if in squid the tetrodotoxin binding sites act as a saturable sink in series with the barrier to free diffusion imposed by the presence of the Schwann cell. A comparison has been made between the rates observed experimentally and those calculated for a computer model of the system, in order to estimate the apparent density in the membrane of both specific and non-specific tetrodotoxin binding sites. The figure thus obtained for the number of sodium channels in the squid giant axon, several hundred per square micrometre, agrees well with those derived from other lines of argument.     

Cation permeation through the voltage-dependent potassium channel in the squid axon. Characteristics and mechanisms

Characteristics of cation permeation through voltage-dependent delayed rectifier K channels in squid giant axons were examined. Axial wire voltage-clamp measurements and internal perfusion were used to determine conductance and permeability properties. These K channels exhibit conductance saturation and decline with increases in symmetrical K+ concentrations to 3 M. They also produce ion- and concentration-dependent current-voltage shapes. K channel permeability ratios obtained with substitutions of internal Rb+ or NH+4 for K+ are higher than for external substitution of these ions. Furthermore, conductance and permeability ratios of NH+4 or Rb+ to K+ are functions of ion concentration. Conductance measurements also reveal the presence of an anomalous mole fraction effect for NH+4, Rb+, or Tl+ to K+. Finally, internal Cs+ blocks these K channels in a voltage-dependent manner, with relief of block by elevations in external K+ but not external NH+4 or Cs+. Energy profiles for K+, NH+4, Rb+, Tl+, and Cs+ incorporating three barriers and two ion-binding sites are fitted to the data. The profiles are asymmetric with respect to the center of the electric field, have different binding energies and electrical positions for each ion, and (for K+) exhibit concentration-dependent barrier positions.     

The effect of sodium and potassium ions on the impedance change accompanying the spike in the squid giant axon

Decrease of the sodium concentration of the medium depresses both the spike and the associated impedance change in almost identical fashion. Elevation of the potassium level also depresses both phenomena, but affects the impedance change more than the spike; it slows the return to the initial impedance level. The effects on the threshold to brief square waves are also described. These results appear largely accounted for by the observations of Hodgkin and Huxley with the voltage clamp technique and by their recent hypothesis as to nature of the spike processes.     

Pressure dependence of the potassium currents of squid giant axon

Summary The effect of pressure upon the delayed, K, voltage-clamp currents of giant axons from the squidLoligo vulgaris was studied in axons treated with 300nm TTX to block the early, Na, currents. The effect of TTX remained unaltered by pressure. The major change produced by pressures up to 62 MPa is a slowing down of the rising phase of the K currents by a time scaling factor which depends on pressure according to an apparent activation volume, V, of 31 cm³/mole at 15°C; V increased to about 42 cm³/mole at 5°C.Pressure slightly increased the magnitude, but did not produce any obvious major change in the voltage dependence, of the steady-state K conductance estimated from the current jump at the end of step depolarizations of small amplitude (to membrane potentials,E, 20 mV) and relatively short duration. At higher depolarizations, pressure produced a more substantial increase of the late membrane conductance, associated with an apparent enhancement of a slow component of the K conductance which could not be described within the framework of the Hodgkin-Huxley (HH)n ⁴ kinetic scheme.The apparent V values that characterize the pressure dependence of the early component of the K conductance are very close to those that describe the effect of pressure on Na activation kinetics, and it is conceivable that they are related to activation volumes involved in the isomerization of the normal K channels. The enhancement of the slow component of membrane conductance by pressure implies either a large increase in the conductance of the ionic channels that are responsible for it or a strong relative hastening of their turn-on kinetics.     

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.     

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.     

An approach to the physical basis of negative conductance in the squid axon

In considering the problem of steady-state negative conductance in the squid axon from the standpoint of electrodiffusion, the following assumptions produce results which are in reasonable agreement with experimental observations: (1) The major ion distributions are not significantly perturbed by current flows (2) The electric field in the membrane is essentially uniform. (3) The membrane has certain properties appropriate to solids, particularly with respect to chemical potentials. (4) Na⁺ and K⁺ move according to a single-file interstitialcy migration mechanism and independently of each other. (5) The interaction energy of Na⁺ with membrane sites is about 1.4 times that for K⁺. Assumptions 1 and 2 are sufficient for the appearance of a negative conductance. Experimental test of the theory is possible and is specifically suggested.