The influence of potassium- and sodium-free solutions on sodium efflux from squid giant axons

The sensitivity of sodium efflux to the removal of potassium ions from the external solution and the change in sodium efflux occurring when sodium ions are also removed were observed to be related. When Tris was used to replace external sodium ions, increases in sodium efflux were always observed whether the sensitivity of sodium efflux to external potassium ions was weak or strong. Greater percentage increases in sodium efflux occurred, however, the greater the sensitivity of sodium efflux to external potassium ions. When lithium ions were used to replace external sodium ions, increases in sodium efflux occurred if the sensitivity of efflux to external potassium ions was strong whereas decreases in sodium efflux took place if the sensitivity of efflux to external potassium ions was weak. Intermediate sensitivities of efflux to external potassium resulted in no change in efflux upon substitution of lithium ions for external sodium ions. In the presence of 10^-5 M ouabain, substitution of Tris for external sodium ions always resulted in a small decrease in sodium efflux. The data can be described in terms of a model which assumes the presence of efflux stimulation sites that are about 98% selective to potassium ions and about 2% selective to sodium or lithium ions.     

Magnesium efflux in dialyzed squid axons

The efflux of Mg++ from squid axons subject to internal solute control by dialysis is a function of ionized [Mg], [Na], [ATP], and [Na]o. The efflux of Mg++ from an axon with physiological concentrations of ATP, Na, and Mg inside into seawater is of the order of 2-4 pmol/cm2s but this efflux is strongly inhibited by increases in [Na]i, by decreases in [ATP]i, or by decreases in [Na]o. The efflux of Mg++ is largely independent of [Mg]i when ATP is at physiological levels, but in the absence of ATP reaches half the value of Mg efflux in be presence of ATP when [Mg]i is about 4 mM and [Na] 40 mM. Half-maximum responses to ATP occur at about 350 micronM ATP into seawater with Na either present or absent. The Mg efflux mechanism has many similarities to the Ca efflux system in squid axons especially with respect to the effects of ATP, Nao, and Na on the flux. The concentrations of free Mg and Ca in axoplasm differ, however, by a factor of 10(5) while the observed fluxes differ by a factor of 10(2).     

Sensitivity of calcium efflux from squid axons to changes in membrane potential

Squid giant axons were internally dialyzed with a medium free of metabolic substrates but containing 45Ca buffered with EGTA to concentrations of free Ca++ in the range 0.01-230 muM. At (Ca)i of 1.0 muM OR GREATER, Ca efflux was in the range of 1-3 pmol/cm2 s, was dependent on (Na)o and (Ca)o, and was sensitive to membrane potential. At lower (Ca)i, the sensitivity of Ca efflux to membrane potential was greater. Hyperpolarization of the membrane increased, and depolarization decreased Ca efflux over the range of potentials studied (-20 to -100 mV). The maximum sensitivity of Ca efflux to membrane potential was of the order of an e-fold increase in Ca efflux for a 25- mV increase in Em; this sensitivity of Ca efflux to membrane potential was lost if (Na)o was removed and was greatly reduced when (Ca)i was increased to 230 muM.     

Calcium and EDTA fluxes in dialyzed squid axons

Ca efflux in dialyzed squid axons was measured with 45Ca as a function of internal ionized Ca in the range 0.005-10 muM. Internal Ca stores were depleted by treatment with CN and dialysis with media free of high energy compounds. The [Ca]iota was stabilized with millimolar concentrations of EDTA, EGTA, or DTPA. Nonspecific leak of chelated Ca was measured with [14C]-EDTA and found to be 0.02 pmol/cm2s/mM EDTA. Correction of the measured Ca efflux for this leak of chelated calcium was made when appropriate. Ca efflux was roughly linear with internal free Ca in the range 0.005-0.1 muM. Above 0.1 muM, efflux was less than proportional to concentration but did not saturate at the highest concentration studied. Ca efflux was reduced about 50% by replacement of external Na with Li at Caiota approximately 1 muM, but was insensitive to such replacement for Ca less than 0.1 muM. Ca efflux was insensitive to internal Mg in the range 0-4 mM, indicating that the Ca pump favors Ca over Mg by a factor of about 10(6). Ca efflux was reduced about 60% by increasing internal Na from 1 to 80 mM. This effect could represent weak interference of a Ca carrier by Na or a loss of driving force because of a reduction in ENa - Em occasioned by an increase in Naiota. A few measurements were made of Ca influx in intact and in dialyzed fibers. In both cases, Ca influx increased when external Na was replaced by Li.     

Calcium influx in internally dialyzed squid giant axons

A method has been developed to measure Ca influx in internally dialyzed squid axons. This was achieved by controlling the dialyzed segment of the axon exposed to the external radioactive medium. The capacity of EGTA to buffer all the Ca entering the fiber was explored by changing the free EGTA at constant [Ca++]i. At a free [EGTA]i greater than 200 microM, the measured resting Ca influx and the expected increment in Ca entry during electrical stimulation were independent of the axoplasmic free [EGTA]. To avoid Ca uptake by the mitochondrial system, cyanide, oligomycin, and FCCP were included in the perfusate. Axons dialyzed with a standard medium containing: [ATP] = 2 mM, [Ca++]i = 0.06 microM, [Ca++]o = 10 mM, [Na+]i = 70 mM, and [Na+]o = 465 mM, gave a mean Ca influx of 0.14 +/- 0.012 pmol.cm-2.s-1 (n = 12. Removal of ATP drops the Ca influx to 0.085 +/- 0.007 pmol.cm-2.s-1 (n = 12). Ca influx increased to 0.35 pmol.cm-2,s-1 when Nao was removed. The increment was completely abolished by removing Nai+ and (or) ATP from the dialysis medium. At nominal zero [Ca++]i, no Nai-dependent Ca influx was observed. In the presence of ATP and Nai [Ca++]i activates the Ca influx along a sigmoid curve without saturation up to 1 microM [Ca++]i. Removal of Nai+ always reduced the Ca influx to a value similar to that observed in the absence of [Ca++]i (0.087 +/- 0.008 pmol.cm-2.s-1; n = 11). Under the above standard conditions, 50-60% of the total Ca influx was found to be insensitive to Nai+, Cai++, and ATP, sensitive to membrane potential, and partially inhibited by external Co++.     

Calcium entry in squid axons during voltage clamp pulses

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with sodium ion sensitive, current and voltage electrodes. The axons were usually bathed in a solution of varying Ca2+ concentration ([Ca2+]o) containing 150mM each of Na+, K+ and an inert cation such as Li+, Tris or N-methylglucamine and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic Ca2+ level, [Ca2+]i. The effect of membrane voltage on [Ca2+]i was found to depend on the concentration of internal Na+ ([Na+]i). Voltage clamp hyperpolarizing pulses were found to cause a reduction of [Ca2+]i. For depolarizing pulses a relationship between [Ca2+]i gain and [Na+]i indicates that Ca2+ entry is sigmoid with a half maximal response at 22 mM Na+. This Ca2+ entry is a steep function of [Na+]i suggesting that 4 Na+ ions are required to promote the influx of 1 Ca2+. There was little change in Ca2+ entry with depolarizing pulses when [Ca2+]o is varied from 1 to 10mM, while at 50mM [Ca2+]o calcium entry clearly increases suggesting an alternate pathway from that of Na+/Ca2+ exchange. This entry of Ca2+ at high [Ca2+]o, however, was not blocked by Cs+o. The results obtained lend further support to the notion that Na+/Ca2+ exchange in squid giant axon is sensitive to membrane voltage no matter whether this is applied as a constant change in membrane potential or as an intermittent one.     

Calcium content and net fluxes in squid giant axons

Axons freshly dissected from living specimens of the tropical squid Dorytheutis plei have a calcium content of 68 mumol/kg of axoplasm. Fibers stimulated at 100 impulses/s in 100 mM Ca seawater increase their Ca content by 150 mumol/kg.min; axons placed in 3 Ca (choline) seawater increase their Ca content by 12 mumol/kg.min. Axons loaded with 0.2--1.5 mmol Ca/kg of axoplasm extruded Ca with a half time of 15--30 min when allowed to recover in 3 Ca (Na) seawater. The half time for recovery of loaded axons poisoned with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and iodoacetic acid (IAA) is about the same as control axons. Axons placed in 40 mM Na choline seawater (to reduce chemical gradient for Na) or in 40 mM Na, 410 mM K seawater to reduce the electrochemical gradient for Na to near zero either fail to lose previously loaded Ca or gain further Ca.     

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.     

Some factors influencing sodium extrusion by internally dialyzed squid axons

Squid giant axons were internally dialyzed by a technique previously described. In an axon exposed to cyanide seawater for 1 hr and dialyzed with an ATP-free medium, the Na efflux had a mean value of 1.3 pmole/cm²sec when [Na]_i was 88 mM, in quantitative agreement with flux ratio calculations for a purely passive Na movement. When ATP at a concentration of 5–10 mM was supplied to the axoplasm by dialysis, Na efflux rose almost 30-fold, while if phosphoarginine, 10 mM, was supplied instead of ATP, the Na efflux rose only about 15-fold. The substitution of Li for Na in the seawater outside did not affect the Na efflux from an axon supplied with ATP, while a change to K-free Na seawater reduced the Na efflux to about one-half. When special means were used to free an axon of virtually all ADP, the response of the Na efflux to dialysis with phosphoarginine (PA) at 10 mM was very small (an increment of ca. 3 pmole/cm²sec) and it can be concluded that more than 96% of the Na efflux from an axon is fueled by ATP rather than PA. Measurements of [ATP] in the fluid flowing out of the dialysis tube when the [ATP] supplied was 5 mM made it possible to have a continuous measurement of ATP consumption by the axon. This averaged 43 pmole/cm²sec. The ATP content of axons was also measured and averaged 4.4 mM. Estimates were made of the activities of the following enzymes in axoplasm: ATPase, adenylate kinase, and arginine phosphokinase. Values are scaled to 13°C.     

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 D-600 on sodium current in squid axons

Summary The effects of the calcium antagonist D-600 (methoxyverapamil) on the excitatory inward sodium current,I _Na, of internally perfused squid giant axons were studied under voltageclamp conditions. We observed little or no effect of the drug when it was added to the external solution at concentrations of 10–200 M. Furthermore, it did not produce a frequency, or use-dependent block ofI _Na when repetitive voltage-clamp pulses were used at rates of 2–5Hz. However, it did produce use-dependent blockade ofI _Na when it was placed internally at a concentration of 200 M. These results in conjunction with other studies suggest that D-600 is a selective blocker of calcium channels in squid axons when the drug is placed in the external solution. Its effects, when placed in the internal solution, are similar to those of permanently charged local anesthetic derivatives, which also produce use-dependent block ofI _Na.     

Hydrogen ion block of the sodium pore in squid giant axons

The block of squid axon sodium channels by H ions was studied using voltage-clamp and internal perfusion techniques. An increase in the concentration of internal permeant ions decreased the block produced by external H ions. The voltage dependence of the block was found to be nonmonotonic: it was reduced by both large positive and large negative potentials. The ability of internal ions to modify the block by external H+ is explained by a competition among these ions for a binding site within the pore. The nonmonotonic voltage dependence is consistent with this picture if the hydrogen ions are allowed to be permeant.     

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.     

Anticalmodulin drugs block the sodium gating current of squid giant axons

Summary The effects of calmodulin (CaM) antagonists (W-7, W-5, trifluoperazine, chlorpromazine, quinacrine, diazepam, propericyazine and carmidazolium) on the sodium and potassium channels were studied on the intracellularly perfused and voltage-clamped giant axon of the squid. It was found that the drugs are more potent blockers of the sodium current than of the potassium current. The drugs also reduce the sodium gating current. The blockage of the sodium and gating current can be explained by assuming that the drugs interact with the sodium gating subunit in one of its closed states. The site of action is probably the intracellular surface of the axolemma where presumably a Ca²⁺-calmodulin complex can be formed.     

Dynamics of 9-aminoacridine block of sodium channels in squid axons

The interactions of 9-aminoacridine with ionic channels were studied in internally perfused squid axons. The kinetics of block of Na channels with 9-aminoacridine varies depending on the voltage-clamp pulses and the state of gating machinery of Na channels. In an axon with intact h gate, the block exhibits frequency- and voltage-dependent characteristics. However, in the pronase-perfused axon, the frequency- dependent block disappears, whereas the voltage-dependent block remains unchanged. A time-dependent decrease in Na currents indicative of direct block of Na channel by drug molecule follows a single exponential function with a time constant of 2.0 +/- 0.18 and 1.0 +/- 0.19 ms (at 10 degrees C and 80 m V) for 30 and 100 microM 9- aminoacridine, respectively. A steady-state block can be achieved during a single 8-ms depolarizing pulse when the h gate has been removed. The block in the h-gate intact axon can be achieved only with multiple conditioning pulses. The voltage-dependent block suggests that 9-aminoacridine binds to a site located halfway across the membrane with a dissociation constant of 62 microM at 0 m V. 9-Aminoacridine also blocks K channels, and the block is time- and voltage-dependent.     

Removal of sodium channel inactivation in squid giant axons by n-bromoacetamide

The group-specific protein reagents, N-bromacetamide (NBA) and N- bromosuccinimide (NBS), modify sodium channel gating when perfused inside squid axons. The normal fast inactivation of sodium channels is irreversibly destroyed by 1 mM NBA or NBS near neutral pH. NBA apparently exhibits an all-or-none destruction of the inactivation process at the single channel level in a manner similar to internal perfusion of Pronase. Despite the complete removal of inactivation by NBA, the voltage-dependent activation of sodium channels remains unaltered as determined by (a) sodium current turn-on kinetics, (b) sodium tail current kinetics, (c) voltage dependence of steady-state activation, and (d) sensitivity of sodium channels to external calcium concentration. NBA and NBS, which can cleave peptide bonds only at tryptophan, tyrosine, or histidine residues and can oxidize sulfur- containing amino acids, were directly compared with regard to effects on sodium inactivation to several other reagents exhibiting overlapping protein reactivity spectra. N-acetylimidazole, a tyrosine-specific reagent, was the only other compound examined capable of partially mimicking NBA. Our results are consistent with recent models of sodium inactivation and support the involvement of a tyrosine residue in the inactivation gating structure of the sodium channel.     

Vanadate selectively inhibits the Ko+-activated Na+ efflux in squid axons.

The effects of internally applied 1 mM vanadate on the Na+ efflux in dialysed squid axons were found to depend on the presence of external K+. In K+-free artificial sea water, vanadate did not produce any change in the rate of Na+ efflux, whereas in the presence of 10 mM K+ the Na+ efflux was reduced to values even lower than those observed in the absence of K+ (inversion of the K+-free effect). In vanadate-poisoned axons, K+ and NH+4 at low concentrations activated Na+ efflux, but at high concentrations both cations were inhibitory. However, NH+4 was always a better activator and a poorer inhibitor than K+.     

Vanadate selectively inhibits the K0+-activated Na+ efflux in squid axons

The effects of internally applied 1 mM vanadate on the Na⁺ efflux in dialysed squid axons were found to depend on the presence of external K⁺. In K⁺-free artificial sea water, vanadate did not produce any change in the rate of Na⁺ efflux, whereas in the presence of 10 mM K⁺ the Na⁺ efflux was reduced to values even lower than those observed in the absence of K⁺ (inversion of the K⁺-free effect). In vanadate-poisoned axons, K⁺ and NH₄⁺ at low concentrations activated Na⁺ efflux, but at high concentrations both cations were inhibitory. However, NH₄⁺ was always a better activator and a poorer inhibitor than K⁺.