Asymmetry currents in intracellularly perfused squid giant axons.

Asymmetry currents were recorded from intracellularly perfused squid axons subjected to exactly equal positive and negative voltage clamp pulses at a temperature close to 0 degrees C. The voltage and time dependence of the asymmetry currents was studied at a holding potential of minus 80 to minus 100 mV. The effect of varying the holding potential was investigated. The latter experiments showed that the voltage dependence of the asymmetrical charge movement is different from the voltage dependence of the m system.     

Effects of external cesium and rubidium on outward potassium currents in squid axons

We have studied the effects of external cesium and rubidium on potassium conductance of voltage clamped squid axons over a broad range of concentrations of these ions relative to the external potassium concentration. Our primary novel finding concerning cesium is that relatively large concentrations of this ion are able to block a small, but statistically significant fraction of outward potassium current for potentials less than approximately 50 mV positive to reversal potential. This effect is relieved at more positive potentials. We have also found that external rubidium blocks outward current with a qualitatively similar voltage dependence. This effect is more readily apparent than the cesium blockade, occurring even for concentrations less than that of external potassium. Rubidium also has a blocking effect on inward current, which is relieved for potentials more than 20-40 mV negative to reversal, thereby allowing both potassium and rubidium ions to cross the membrane. We have described these results with a single-file diffusion model of ion permeation through potassium channels. The model analysis suggests that both rubidium and cesium ions exert their blocking effects at the innermost site of a two-site channel, and that rubidium competes with potassium ions for entry into the channel more effectively than does cesium under comparable conditions.     

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.     

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.     

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.     

Regulation of intracellular calcium in squid axons

Internal dialysis and metallochromic indicators were used to determine the free calcium concentration and calcium buffering properties of squid axoplasm. The free calcium concentration in fresh unloaded squid axons is about 30 to 50 nM. About 6% of the calcium content (ca. 50 mumol/kg axoplasm) of a fresh squid axon is held in a metabolically labile, presumably mitochondrial, component. A morphological consequence of this finding is that there should be no accumulation of calcium in mitochondria of fresh squid axons unless there is a large component of nonlabile calcium. The physiological implication is that the mitochondria are probably not buffers for physiological perturbations in free calcium concentration. When an exogenous load of several hundred mumol/kg axoplasm with an ambient ionized calcium concentration above a few hundred nanomolar is applied to axoplasm, all of it goes into organelles. About one-third of that load is found in the mitochondria and about two thirds in some other organelles. When axoplasm is poisoned with carbonyl cyanide-p-trifluoromethonyphenylhydrazone (FCCP), around 70% of the load remains in the nonmitochondrial fraction.     

Survival of K+ permeability and gating currents in squid axons perfused with K+-free media

K+ currents were recorded in squid axons internally perfused with impermeant electrolyte. Total absence of permeant ions inside and out leads to an irreversible loss of potassium conductance with a time constant of approximately 11 min at 8 degrees C. Potassium channels can be protected against this effect by external K+, Cs+, NH4+, and Rb+ at concentrations of 100-440 mM. These experiments suggest that a K+ channel is normally occupied by one or more small cations, and becomes nonfunctional when these cations are removed. A large charge movement said to be related to K+ channel gating in frog skeletal muscle is absent in squid giant axons. However, deliberate destruction of K+ conductance by removal of permeant cations is accompanied by measurable loss in asymmetric charge movement. This missing charge component is large enough to contain a contribution from K+ gating charge movements of more than five elementary charges per channel.     

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.     

Effect of specific antibodies on the excitability of internally perfused squid axons

Giant axons from the squid Dosidicus gigas were internally perfused with rabbit antiaxoplasm antibodies and their effect upon the action potential and the membrane potential was studied. Necessary requirements for the antibodies to affect these parameters in a consistent manner were: (a) removal of the bulk of axoplasm from the perfused zone, accomplished by initially perfusing with a cysteine-rich (400 mM) solution, and (b) addition of small amounts of cysteine (30 mM) to the antibody-containing solution. When these experimental conditions were met, conduction block ensued generally within 3 hr of the first contact of the axon inner surface with the antibody Antineurofilament antibodies and nonspecific antibodies had no effect. External application of antiaxoplasm antibodies had no effect.     

The control of ionized calcium in squid axons

Measurements of the Ca content, [Ca](T), of freshly isolated squid axons show a value of 60 μmol/kg axoplasm. Axons in 3 mM Ca(Na) seawater show little change in Ca content over 4 h, while axons in 3 mM Ca(Na) seawater show little change in Ca content over 4 h, while axons in 10 mM Ca(Na) seawater show gains of 18 μmol/Ca/kgxh. In 10 Ca (Choline) seawater the gain is 2,400 μmol/kgxh. Using aequorin confined to a dialysis capillary in the center of an axon, one finds that [Ca](i) is in a steady state with 3 Ca (Na) seawater, and that both 10 Ca (Na) and 3 Ca (choline) seawater cause increases in [Ca](i). In 3 Ca (Na) seawater-3 Ca (choline) seawater mixtures, 180 mM [Na](0) (40 perecent Na) is as effective as 450 mM [Na](0) (100 percent Na) in maintaining a normal [Ca](1); lower [Na] causes an increase in [Ca](i). If axons are injected with the ATP-splitting enzyme apyrase, the resulting [Ca](1) is not loading with high [Ca](0) or low [Na](0) solutions. Depolarization of an axon with 100 mM K (Na) seawater leads to an increase in the steady-state level of [Ca](1) that is reversed upon returning the axon to normal seawater. Freshly isolated axons treated with either CN or FCCP to inhibit mitochondrial Ca buffering can still maintain a normal [Ca](i) in 1 Ca (Na) seawater.     

The effects of vanadate on calcium transport in dialyzed squid axons Sidedness of vanadate-cation interactions

(1) Vanadate (VO₃^?) fully inhibits the ATP-dependent uncoupled Ca efflux (Ca pump) in dialyzed squid axons. (2) Vanadate inhibits with high affinity. The mean apparent affinity ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) obtained was 7 μM. (3) Inhibition by vanadate is dependent on Ca_o. External Ca lead to a release of the inhibitory effect. ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 3}\text{mM}$). This antagonic effect can be reverted by increasing the vanadate concentration. Internal K⁺ increases the affinity of the intracellular vanadate binding site. External K⁺ has no effect on the inhibition. (4) Vanadate has no effect on the Na_o-dependent Ca efflux component (forward Na-Ca exchange) in the absence of ATP. In axons containing ATP vanadate modified this component.     

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.     

Comparison of tetrodotoxin and procaine in internally perfused squid giant axons

Squid giant axons were internally perfused with tetrodotoxin and procaine, and excitability and electrical properties were studied by means of current-clamp and sucrose-gap voltage-clamp methods. Internally perfused tetrodotoxin was virtually without effect on the resting potential, the action potential, the early transient membrane ionic current, and the late steady-state membrane ionic current even at very high concentrations (1,000–10,000 nM) for a long period of time (up to 36 min). Externally applied tetrodotoxin at a concentration of 100 nM blocked the action potential and the early transient current in 2–3 min. Internally perfused procaine at concentrations of 1–10 mM reversibly depressed or blocked the action potential with an accompanying hyperpolarization of 2–4 mv, and inhibited both the early transient and late steady-state currents to the same extent. The time to peak early transient current was increased. The present results and the insolubility of tetrodotoxin in lipids have led to the conclusion that the gate controlling the flow of sodium ions through channels is located on the outer surface of the nerve membrane.     

Dependence of calcium influx in squid axons on rhythmic excitation frequency

Calcium-45 influx was measured in squid axons during excitation. Different stimulation frequencies changed this influx and general concentration of calcium ions in squid axons. The maximum influx was recorded at the frequency 10 imp/s and 30 imp/s. Calcium influx and general content of calcium ions in axoplasma during excitation was independent of the number of excitation impulses. The role of sodium and calcium channels during excitation is discussed.     

Characterization of the ATP-dependent calcium efflux in dialyzed squid giant axons

The magnitude of the activating effect of ATP on the Ca efflux was explored at different [Ca++]i in squid axons previously exposed to cyanide seawater and internally dialyzed with a medium free of ATP and containing p-trifluoro methoxy carbonyl cyanide phenyl hydrazine. At the lowest [Ca++]i used (0.06 micron more than 95% of the Ca efflux depends on ATP. At high [Ca++]i (100 micron), 50-60% of the Ca efflux still depends on ATP. The apparant affinity constant for ATP was not significantly affected in the range of [Ca++]i from 0.06 to 1 micron. Axons dialyzed to reduce their internal magnesium failed to show the usual activation of the Ca efflux when the Tris or the sodium salt of ATP was used. Only in the presence of internal magnesium is ATP able to stimulate the Ca efflux. Nine naturally occurring high-energy phosphate compounds were ineffective in supporting calcium efflux. These compounds were: UTP, GTP, CTP, UDP, CDP, ADP, AMP, CAMP, and acetyl phosphate. The compounds 2' deoxy-ATP and the hydrolyzable analog alpha,beta-methylene ATP were able to activate the Ca efflux. The nonhydrolyzable analog beta,gamma-methylene ATP competes with ATP for the activating site, but is unable to activate the Ca efflux. The results are discussed in terms of the specificity of the nucleotide site responsible for the ATP-dependent Ca efflux.     

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.     

Tracer and nontracer potassium fluxes in squid giant axons and the effects of changes in external potassium concentration and membrane potential

The efflux of labeled and unlabeled potassium ions from the squid giant axon has been measured under a variety of experimental conditions. Axons soaked in sea water containing 42K ions lost radioactivity when placed in inactive sea water according to kinetics which indicate the presence of at least two cellular compartments. A rapidly equilibrating superficial compartment, probably the Schwann cell, was observed to elevate the specific activity of 42K lost from such axons to K-free sea water for a period of hours. The extra radioactive potassium loss from such axons during stimulation, however, was shown to have a specific activity identical within error to that measured in the axoplasm at the end of the experiment. The same was shown for the extra potassium loss occurring during passage of a steady depolarizing current. Axons placed in sea water with an elevated potassium ion concentration (50 mM) showed an increased potassium efflux that was in general agreement with the accompanying increase in membrane conductance. The efflux of potassium ions observed in 50 mM K sea water at different membrane potentials did not support the theory that the potassium fluxes obey the independence principle.