The binding and release of 45Ca by axoplasm isolated from Myxicola giant axons were examined. Two distinct components of binding were observed, one requiring ATP and one not requiring ATP. The ATP-dependent binding was largely prevented by the addition of mitochondrial inhibitors, whereas the ATP-independent component was unaffected by these inhibitors. The ATP-independent binding accounted for roughly two-thirds of the total 45Ca uptake in solutions containing an ionized [Ca2+] = 0.54 microM and was the major focus of this investigation. This fraction of bound 45Ca was released from the axoplasm at a rate that increased with increasing concentrations of Ca2+ in the incubation fluid. The ions Cd2+ and Mn2+ were also able to increase 45Ca efflux from the sample, but Co2+, Ni2+, Mg2+, and Ba2+ had no effect. The concentration-response curves relating the 45Ca efflux rate coefficients to the concentration of Ca2+, Cd2+, and Mn2+ in the bathing solution were S-shaped. The maximum rate of efflux elicited by one of these divalent ions could not be exceeded by adding a saturating concentration of a second ion. Increasing EGTA concentration in the bath medium from 100 to 200 microM did not increase 45Ca efflux; yet increasing the concentration of the EGTA buffer in the uptake medium from 100 to 200 microM and keeping ionized Ca2+ constant caused more 45Ca to be bound by the axoplasm. These results suggest the existence of high-affinity, ATP-independent binding sites for 45Ca in Myxicola axoplasm that compete favorably with 100 microM EGTA. The 45Ca efflux results are interpreted in terms of endogenous sites that interact with Ca2+, Cd2+, or Mn2+.

Relations are derived that describe the combined effects of electrodiffusion, the Na/K pump, and Na/Ca transport by carrier on the resting membrane potential. Equations are derived that apply to both steady-state and non-steady-state conditions. Some example calculations from the equations are plotted at different permeability coefficient ratios, PK:PCa:PNa. The equations predict a depolarizing action of Na/Ca transport when more than two Na ions per Ca ion are transported by the carrier. For all permeability ratios examined, a steady state for Ca ions is achieved with at most a few millivolts of depolarization.

In microinjected Myxicola giant axons with elevated [Na]i, Na efflux was sensitive to Cao under some conditions. In Li seawater, sensitivity to Cao was high whereas in Na seawater, sensitivity to Cao was observed only upon elevation of [Ca]o above the normal value. In choline seawater, the sensitivity of Na efflux to Cao was less than that observed in Li seawater whereas Mg seawater failed to support any detectable Cao-sensitive Na efflux. Addition of Na to Li seawater was inhibitory to Cao-sensitive Na efflux, the extent of inhibition increasing with rising values of [Na]o. The presence of 20 mM K in Li seawater resulted in about a threefold increase in the Cao-activated Na efflux. Experiments in which the membrane potential, Vm, was varied or held constant when [K]o was changed showed that the augmentation of Ca-activated Na efflux by Ko was not due to changes in Vm but resulted from a direct action of K on activation by Ca. The same experimental conditions that favored a large component of Cao-activated Na efflux also caused a large increase in Ca influx. Measurements of Ca influx in the presence of 20 mM K and comparison with values of Ca-activated Na efflux suggest that the Na:Ca coupling ratio may be altered by increasing external [K]o. Overall, the results suggest that the Cao-activated Na efflux in Myxicola giant axons requires the presence of an external monovalent cation and that the order of effectiveness at a total monovalent cation concentration of 430 mM is K + Li greater than Li greater than Choline greater than Na.

Several properties of the Na pump in giant axons from the marine annelid Myxicola infundibulum have been determined in an attempt to characterize this preparation for membrane transport studies. Both NaO and KO activated the Na pump of normal microinjected Myxicola axons. In this preparation, the KO activation was less and the NaO activation much greater than that found in the squid giant axon. However, when the intracellular ATP:ADP ratio of the Myxicola axon was elevated by injection of an extraneous phosphagen system, the K sensitivity of Na efflux increased to the magnitude characteristic of squid axons and the activating effect of NaO disappeared. Several axons were injected with Na2SO4 in order to determine the effect of elevated Nai on the Na efflux. Increasing Nai enhanced a component of Na efflux which was insensitive to ouabain and dependent on [Ca] in Na-free (Li) seawater. After subtracting the CaO-dependent fraction, Na efflux was related linearly to [Na]i in all solutions except in K-free (Li) seawater, where it appeared to reach saturation at high [Na]i.

When frog sartorius muscles recover from Na enrichment in the presence of external K, net K entry into the fibers occurs by both passive movement and active inward transport via a K pump. Under normal conditions, it has not been possible to experimentally distinguish these processes. Fractionation into the flux components must be accomplished from inferences concerning the K conductance or permeability during a period of rapid Na extrusion. The best estimates indicate that 60-80% of the K entry occurs via the K pump. In the presence of Ba ions, the membrane permeability to K is very much reduced. Under these conditions, Na-enriched muscles underwent a normal recovery in the presence of external K, and the amount of inward K movement due to the K pump rose to over 90% of the total K entry. The characteristics of the K pump studied by this means were: (a) essentially complete inhibition by 10(-4) M ouabain, (b) inhibition by [Na]omicron, (c) activation by [K]omicron according to a rectangular hyperbola in the absence of [Na]omicron, (d) linear activation by [Na]iota over a wide range in concentration, (e) zero or undetectably low pumping rate as [Na]iota leads to 0, (f) the number of Na ions actively transported per K ion actively transported is 1.4-1.7 normally and 1.1 in the presence of Ba.

Net sodium influx under K-free conditions was independent of the intracellular sodium ion concentration, [Na] i , and was increased by ouabain. Unidirectional sodium influx was the sum of a component independent of [Na] i and a component that increased linearly with increasing [Na] i . Net influx of sodium ions in K-free solutions varied with the external sodium ion concentration, [Na] o , and a steady-state balance of the sodium ion fluxes occurred at [Na] o = 40 mM. When solutions were K-free and contained 10 -4 M ouabain, net sodium influx varied linearly with [Na] o and a steady state for the intracellular sodium was observed at [Na] o = 13 mM. The steady state observed in the presence of ouabain was the result of a pump-leak balance as the external sodium ion concentration with which the muscle sodium would be in equilibrium, under these conditions, was 0.11 mM. The rate constant for total potassium loss to K-free Ringer solution was independent of [Na] i but dependent on [Na] o . Replacing external NaCl with MgCl 2 brought about reductions in net potassium efflux. Ouabain was without effect on net potassium efflux in K-free Ringer solution with [Na] o = 120 mM, but increased potassium efflux in a medium with NaCl replaced by MgCl 2 . When muscles were enriched with sodium ions, potassium efflux into K-free, Mg ++ -substituted Ringer solution fell to around 0.1 pmol/cm 2 ·s and was increased 14-fold by addition of ouabain.

After a 20 min initial washout, the rate of loss of radioactively labeled sodium ions from sodium-enriched muscle cells is sensitive to the external sodium and potassium ion concentrations. In the absence of external potassium ions, the presence of external sodium ions increases the sodium efflux. In the presence of external potassium ions, the presence of external sodium ions decreases the sodium efflux. In the absence of external potassium ions about one-third of the Na + efflux that depends upon the external sodium ion concentration can be abolished by 10 -5 M glycoside. The glycoside-insensitive but external sodium-dependent Na + efflux is uninfluenced by external potassium ions. In the absence of both external sodium and potassium ions the sodium efflux is relatively insensitive to the presence of 10 -5 M glycoside. The maximal external sodium-dependent sodium efflux in the absence of external potassium ions is about 20% of the magnitude of the maximal potassium-dependent sodium efflux. The magnitude of the glycoside-sensitive sodium efflux in K-free Ringer solution is less than 10% of that observed when sodium efflux is maximally activated by potassium ions. The inhibition of the potassium-activated sodium efflux by external sodium ions is of the competitive type. Reducing the external sodium ion concentration displaces the plots of sodium extrusion rate vs. [K] o to the left and upwards.

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.

"Low sodium" muscles were prepared which contained around 5 mmoles/kg fiber of intracellular sodium. "High sodium" muscles containing between 15 and 30 mmoles/kg fiber of intracellular sodium were also prepared. In low sodium muscles application of 10 -5 M strophanthidin reduced potassium influx by about 5%. Potassium efflux was unaffected by strophanthidin under these conditions. In high sodium muscles, 10 -5 M strophanthidin reduced potassium influx by 45% and increased potassium efflux by 70%, on the average. In low sodium muscles sodium efflux was reduced by 25% during application of 10 -5 M strophanthidin while in high sodium muscles similarly treated, sodium efflux was reduced by about 60%. Low sodium muscles showed a large reduction in sodium efflux when sodium ions in the Ringer solution were replaced by lithium ions. The average reduction in sodium efflux was 4.5-fold. Of the amount of sodium efflux remaining in lithium. Ringer's solution, 40% could be inhibited by application of 10 -5 M strophanthidin. The total sodium efflux from low sodium muscles exposed to Ringer's solution in which lithium had been substituted for sodium ions for a period of 1 hr can be fractionated as 78% Na-for-Na interchange, 10% strophanthidin-sensitive sodium pump, and 12% residual sodium efflux. It is concluded that large strophanthidin-sensitive components of sodium and potassium flux can be expected only at elevated sodium concentrations within the muscle cells.

Sartorius muscle cells from the frog were stored in a K-free Ringer solution at 3°C until their average sodium contents rose to around 23 m M /kg fiber (about 40 m M /liter fiber water). Such muscles, when placed in Ringer's solution containing 60 m M LiCl and 50 m M NaCl at 20°C, extruded 9.8 m M /kg of sodium and gained an equivalent quantity of lithium in a 2 hr period. The presence of 10 -5 M strophanthidin in the 60 m M LiCl/50 m M NaCl Ringer solution prevented the net extrusion of sodium from the muscles. Lithium ions were found to enter muscles with a lowered internal sodium concentration at a rate about half that for entry into sodium-enriched muscles. When sodium-enriched muscles labeled with radioactive sodium ions were transferred from Ringer's solution to a sodium-free lithium-substituted Ringer solution, an increase in the rate of tracer sodium output was observed. When the lithium-substituted Ringer solution contained 10 -5 M strophanthidin, a large decrease in the rate of tracer sodium output was observed upon transferring labeled sodium-enriched muscles from Ringer's solution to the sodium-free medium. It is concluded that lithium ions have a direct stimulating action on the sodium pump in skeletal muscle cells and that a significantly large external sodium-dependent component of sodium efflux is present in muscles with an elevated sodium content. In the sodium-rich muscles, about 23% of the total sodium efflux was due to strophanthidin-insensitive Na-for-Na interchange, about 67% being due to strophanthidin-sensitive sodium pumping.

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 42 K 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 42 K 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 m M ) 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 m M K sea water at different membrane potentials did not support the theory that the potassium fluxes obey the independence principle.

Giant axons from the squid were injected with 1.5 M cesium sulfate solutions containing the radioactive isotopes 42 K and 134 Cs. These axons, when stimulated, gave characteristic long duration action potentials lasting between 5 and 45 msec. The effluxes of 42 K and 134 Cs were measured both under resting conditions and during periods of repetitive stimulation. During the lengthened responses there were considerable increases in potassium efflux but only small increases in cesium efflux. The selectivity of the delayed rectification process was about 9 times greater for potassium ions than for cesium ions. The data suggest that internal cesium ions inhibit the outward potassium movement occurring during an action potential. The extra potassium effluxes taking place during excitation appear to be reduced in the presence of cesium ions to values between 7 and 22% of those expected in the absence of cesium inhibition.

The flux ratio of potassium ions was measured on frog sartorius muscle under conditions in which a substantial net potassium loss occurs. Muscle fiber membrane potentials were measured under identical conditions. The observed flux ratios were compared with values calculated from a theoretical relation derived on the assumptions that the unidirectional fluxes are both passive and occur independently. The results favor the conclusion that the potassium fluxes across skeletal muscle membrane occur along passive electrochemical gradients and obey the independence principle.

Experiments were performed to test the applicability of permeability kinetics to whole frog sartorius muscle using K 42 ions as tracers of potassium flux. The whole muscle was found to obey closely the kinetic laws expected to hold for single cellular units in which the potassium fluxes are membrane-limited and intracellular mixing is rapid enough not to introduce serious error. In a 5 m M K Ringer's solution, potassium efflux was very nearly equal to influx when the rate constant for K 42 loss was applied to the whole of the muscle potassium. Over a fairly wide range of external potassium concentration, the assumed unidirectional fluxes measured with tracer K 42 showed good agreement with net potassium changes determined analytically. The specific activity of potassium lost from labeled muscles to an initially K-free Ringer's solution was measured as a test of the adequacy of intracellular mixing. The results were those expected for a population of cells with uniformly distributed intracellular K 42 . A small deviation was encountered which can be attributed either to a dispersion of fiber sizes in the sartorius or to a possible small additional cellular compartment in each individual fiber. The additional cellular compartment, should it exist, contains from 0.5 to 1 per cent of the muscle potassium. This is evidently not large enough to interfere seriously with the applicability of permeability kinetics to the whole muscle.

It has been possible to treat potassium, rubidium, and cesium ion entry into frog sartorius muscle by the use of a model which assumes a limited number of sites at the cell surface. The ion concentration in an outer surface layer is regarded as the main factor determining the rate of inward movement. It is supposed that the concentration of ions in the external solution is effective in promoting inward movement only to an extent determined by the fraction of sites occupied. Equations are derived from the model which fit the inward flux versus applied concentration curves experimentally determined for the three ions. The ions were found to compete for the postulated sites in various bi-ionic mixtures, the competition being satisfactorily described by equations derived from the model. The constants assigned to each ion remain invariant and independent of gradients in electrochemical potential. The order of decreasing exchange rate found is K > Rb > Cs. The order of decreasing site affinity found is Rb > K > Cs which is the same order as that observed for the ion selectivity deduced from analytical measurements of cation preference after equilibration in various equimolal mixtures (Lubin and Schneider (21)). The manner in which such a model might affect the application of a theory which assumes electrical driving forces as well is discussed.

Squid axons impaled with a microelectrode have been treated with concentrations of xylene and benzene such that there is no change in threshold or resting potential at 20°C., while the spike height declines about 10 mv. A decrease in ambient temperature results in large, reversible, increases in threshold. While neither low temperature nor the added blocking agent induces repetitive firing from a single stimulus, the two treatments when combined do yield repetitive responses which commence at a sharply defined temperature. The alteration in the membrane responsible for the effects observed can be described by saying that there has been a large increase in the inductance of the equivalent electric circuit, and the temperature coefficient of the apparent membrane inductance has a Q 10 = 5.