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++.     

ATP-dependent chloride influx into internally dialyzed squid giant axons

Summary Measurements were made of³⁶Cl influx into squid giant axons whose internal solutes were controlled by means of internal dialysis. When the intracellular chloride concentration was 50mm and the internal concentration of adenosine 5-triphosphate (ATP) was 4mm, the average chloride influx was 11.6 pmoles/cm²×sec. When the axons were dialyzed with an ATP-free solution, the average influx fell to 5.1 pmoles/cm²×sec. The effect was fully reversible upon the return of ATP to the dialysis fluid. Chloride-36 influx in the presence and absence of ATP was found to be inversely related to the internal chloride concentration.     

K+-selective microelectrode study of internally dialyzed squid giant axons.

Intracellular potassium activity, (aK)i, and axoplasmic K+ concentration, [K+]i, were measured by means of K+-selective microelectrodes and atomic absorption spectroscopy, respectively, in squid giant axons dialyzed with K+-free dialysis solution and bathed in K+-free artificial sea water. (aK)i measurements indicated that axoplasmic free K+ could be depleted by dialysis, whereas [K+]i measurements on axoplasm extruded from these axons suggest substantial retention of K+ (15.5 +/- 1.7 mmol/kg axoplasm K+; n = 9). In comparison, [K+]i in axoplasm extruded from freshly dissected axons was 330 +/- 16 mmol/kg axoplasm (n = 6). These data suggest that approximately 5% of the axoplasmic K+ ions are not easily removed by dialysis and that these ions are either bound to macromolecular sites or sequestered into membrane-enclosed organelles.     

Sodium fluxes in internally dialyzed squid axons

The effects which alterations in the concentrations of internal sodium and high energy phosphate compounds had on the sodium influx and efflux of internally dialyzed squid axons were examined. Nine naturally occurring high energy phosphate compounds were ineffective in supporting significant sodium extrusion. These compounds were: AcP, PEP, G-3-P, ADP, AMP, GTP, CTP, PA, and UTP.¹ the compound d-ATP supported 25–50% of the normal sodium extrusion, while ATP supported 80–100%. The relation between internal ATP and sodium efflux was nonlinear, rising most steeply in the range 1 to 10 µM and more gradually in the range 10 to 10,000 µM. There was no evidence of saturation of efflux even at internal ATP concentrations of 10,000 µM. The relation between internal sodium and sodium efflux was linear in the range 2 to 240 mM. The presence of external strophanthidin (10 µM) changed the sodium efflux to about 8–12 pmoles/cm² sec regardless of the initial level of efflux; this changed level was not altered by subsequent dialysis with large concentrations of ATP. Sodium influx was reduced about 50 % by removal of either ATP or Na and about 70 % by removing both ATP and Na from inside the axon.     

Sodium extrusion by internally dialyzed squid axons

A method has been developed which allows a length of electrically excitable squid axon to be internally dialyzed against a continuously flowing solution of defined composition. Tests showed that diffusional exchange of small molecules in the axoplasm surrounding the dialysis tube occurred with a half-time of 2–5 min, and that protein does not cross the wall of the dialysis tube. The composition of the dialysis medium was (mM): K isethionate 151, K aspartate 151, taurine 275, MgCI₂ 4–10, NaCl 80, KCN 2, EDTA 0.1, ATP 5–10, and phosphoarginine 0–10. The following measurements were made: resting Na influx 57 pmole/cm²sec (n = 8); resting potassium efflux 59 pmole/ cm²sec (n = 4); stimulated Na efflux 3.1 pmole/cm²imp (n = 9); stimulated K efflux 2.9 pmole/cm²imp (n = 3); resting Na efflux 48 pmole/cm²sec (n = 18); Q ₁₀ Na efflux 2.2 (n = 5). Removal of ATP and phosphoarginine from the dialysis medium (n = 4) or external application of strophanthidin (n = 1) reversibly reduced Na efflux to 10–13 pmole/cm²sec. A general conclusion from the study is that dialyzed squid axons have relatively normal passive permeability properties and that a substantial fraction of the Na efflux is under metabolic control although the Na extrusion mechanism may not be working perfectly.     

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.     

Measurements of amino acid transport in internally dialyzed giant axons

Summary It is shown that the axoplasmic composition of acidic and neutral amino acids can be controlled effectively by the method of internal dialysis. Direct assay for specific binding and measurement of diffusion coefficients in axoplasm show that there is no significant binding or compartmentalization of amino acids. The dependence of amino acid efflux on substrate concentration can be measured under well-defined, true steady-state conditions. The taurine efflux-concentration relation in theMyxicola giant axon conforms to a second-order Hill equation. This fact is consistent with either a cooperative process or a mechanism in which membrane translocation is not the rate-controlling step. The effluxes of taurine and glycine from squid axon are an order of magnitude smaller than inMyxicola. The efflux-concentration relations are essentially linear up to 200mm substrate concentration. This result may be produced by specific transporters which have very high asymmetry, or by simple diffusive leak in the absence of specific transporters.     

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.     

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.     

Potassium fluxes in dialyzed squid axons

Measurements have been made of K influx in squid giant axons under internal solute control by dialysis. With [ATP]_i = 1 µM, [Na]_i = 0, K influx was 6 ± 0.6 pmole/cm² sec; an increase to [ATP]_i = 4 mM gave an influx of 8 ± 0.5 pmole/cm² sec, while [ATP]_i 4, [Na]_i 80 gave a K influx of 19 ± 0.7 pmole/cm² sec (all measurements at ∼16°C). Strophanthidin (10 µM) in seawater quantitatively abolished the ATP-dependent increase in K influx. The concentration dependence of ATP-dependent K influx on [ATP]_i, [Na]_i, and [K]_o was measured; an [ATP]_i of 30 µM gave a K influx about half that at physiological concentrations (2–3 mM). About 7 mM [Na]_i yielded half the K influx found at 80 mM [Na]_i. The ATP-dependent K influx responded linearly to [K]_o from 1–20 mM and was independent of whether Na, Li, or choline was the principal cation of seawater. Substances tested as possible energy sources for the K pump were acetyl phosphate, phosphoarginine, PEP, and d-ATP. None was effective except d-ATP and this substance gave 70% of the maximal flux only when phosphoarginine or PEP was also present.     

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).     

Magnesium influx in dialyzed squid axons

Summary The influx of magnesium from seawater into squid giant axons has been measured under conditions where internal solute control in the axon was maintained by dialysis. Mg influx is smallest (1 pmol/cm² sec) when both Na and ATP have been removed from the axoplasm by dialysis. The addition of 3mm ATP to the dialysis fluid gives a Mg influx of 2.5 pmol/cm² sec while the addition of [Na] _i and [ATP] _i gives 3 pmol/cm² sec as a value for Mg influx; this corresponds well with fluxes measured in intact squid giant axons.The Mg content of squid axons is 6 mmol/kg axoplasm; this is unaffected by soaking axons in Li or Na seawater for periods of up to 100 min.     

Effects of internal and external cations and of ATP on sodium-calcium and calcium-calcium exchange in squid axons.

Calcium-45 efflux was measured in squid axons whose internal solute concentration was controlled by internal dialysis. Most of the Ca efflux requires either external Na (Na-Ca exchange) or external Ca plus in alkali metal ion (Ca-Ca exchange; cf. Blaustein & Russell, 1975). Both Na-Ca and Ca-Ca exchange are apparently mediated by a single mechanism because both are inhibited by Sr and Mn, and because addition of Na to an external medium optimal for Ca-Ca exchange inhibits Ca efflux. The transport involves simultaneous (as opposed to sequential) ion counterflow because the fractional saturation by internal Ca (Cai) does not affect the external Na (Nao) activation kinetics; also, Nao promotes Ca efflux whether or not an alkali metal ion is present inside, whereas Ca-Ca exchange requires alkali metal ions both internally and externally (i.e., internal and external sites must be appropriately loaded simultaneously). ATP increases the affinity of the transport mechanism for both Cai and Nao, but it does not affect the maximal transport rate at saturating [Ca2+]i and [Na+]o; this suggest that ATP may be acting as a catalyst of modulator, and not as an energy source. Hill plots of the Nao activation data yield slopes congruent to 3 for both ATP-depleted and ATP-fueled axons, compatible with a 3 Na+-for-1 Ca2+ exchange. With this stoichiometry, the Na electrochemical gradient alone could provide sufficient energy to maintain ionized [Ca2+]i in the physiological range (about 10(-7) M).     

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.     

Asymmetrical properties of the Na-Ca exchanger in voltage-clamped, internally dialyzed squid axons under symmetrical ionic conditions

In this work we have investigated whether the asymmetrical properties of the Na/Ca exchange process found in intact preparations are intrinsic to the exchange protein(s) or the result of the asymmetric ionic environment normally prevailing in living cells. The activation of the Na/Ca exchanger by Ca2+ ions, monovalent cations, ATP gamma S and the effect of membrane potential on the different operational modes of the exchanger (Nao/Cai, Cao/Nai, Cao/Cai, and Nao/Nai) was studied in voltage-clamped squid giant axons externally perfused and internally dialyzed with symmetrical ionic solutions. Under these conditions: (a) Ca ions activate with higher affinity from the inside (K1/2 = 22 microM) than from the outside (K1/2 = 300 microM); (b) experiments measuring the Cao-dependent Ca efflux in the conditions Lio-Trisi, Lio-Lii, Triso-Trisi, and Triso-Lii, show that the activating monovalent cation site on the exchanger faces the external surface; (c) ATP gamma S activates the Cao-dependent Ca efflux (Cao/Cai exchange) only at nonsaturating [Ca2+]i. Its effect appears to be on the Ca transport site since no alteration in the apparent affinity of the activating monovalent cation site was observed. The above results show that the Na/Ca exchange process is indeed a highly asymmetric transport mechanism. Finally, the voltage dependence of the components of the different exchange modes was measured over the range of +20 to -40 mV. The voltage dependence (approximately 26% change/25 mV) was found to be similar for all modes of operation of the exchanger except Nao/Nai exchange, which was found to be voltage insensitive. The sensitivity of the Cao/Cai exchange to voltage was found to be the same in the presence and in the complete absence of monovalent cations. This finding does not support the proposition that the voltage sensitivity of the Cao/Cao exchange is induced by the binding and transport of an external monovalent cation.     

Stoichiometry and voltage dependence of the sodium pump in voltage- clamped, internally dialyzed squid giant axon

The stoichiometry and voltage dependence of the Na/K pump were studied in internally dialyzed, voltage-clamped squid giant axons by simultaneously measuring, at various membrane potentials, the changes in Na efflux (delta phi Na) and holding current (delta I) induced by dihydrodigitoxigenin (H2DTG). H2DTG stops the Na/K pump without directly affecting other current pathways: (a) it causes no delta I when the pump lacks Na, K, Mg, or ATP, and (b) ouabain causes no delta I or delta phi Na in the presence of saturating H2DTG. External K (Ko) activates Na efflux with Michaelis-Menten kinetics (Km = 0.45 +/- 0.06 mM [SEM]) in Na-free seawater (SW), but with sigmoid kinetics in approximately 400 mM Na SW (Hill coefficient = 1.53 +/- 0.08, K1/2 = 3.92 +/- 0.29 mM). H2DTG inhibits less strongly (Ki = 6.1 +/- 0.3 microM) in 1 or 10 mM K Na-free SW than in 10 mM K, 390 mM Na SW (1.8 +/- 0.2 microM). Dialysis with 5 mM each ATP, phosphoenolpyruvate, and phosphoarginine reduced Na/Na exchange to at most 2% of the H2DTG-sensitive Na efflux. H2DTG sensitive but nonpump current caused by periaxonal K accumulation upon stopping the pump, was minimized by the K channel blockers 3,4-diaminopyridine (1 mM), tetraethylammonium (approximately 200 mM), and phenylpropyltriethylammonium (20-25 mM) whose adequacy was tested by varying [K]o (0-10 mM) with H2DTG present. Two ancillary clamp circuits suppressed stray current from the axon ends. Current and flux measured from the center pool derive from the same membrane area since, over the voltage range -60 to +20 mV, tetrodotoxin-sensitive current and Na efflux into Na-free SW, under K-free conditions, were equal. The stoichiometry and voltage dependence of pump Na/K exchange were examined at near-saturating [ATP], [K]o and [Na]i in both Na-free and 390 mM Na SW. The H2DTG-sensitive F delta phi Na/delta I ratio (F is Faraday's constant) of paired measurements corrected for membrane area match, was 2.86 +/- 0.09 (n = 8) at 0 mV and 3.05 +/- 0.13 (n = 6) at -60 to -90 mV in Na-free SW, and 2.72 +/- 0.09 (n = 7) at 0 mV and 2.91 +/- 0.21 (n = 4) at -60 mV in 390 mM Na SW. Its overall mean value was 2.87 +/- 0.07 (n = 25), which was not significantly different from the 3.0 expected of a 3 Na/2 K pump.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of K channels in dialyzed squid axons. ATP-mediated phosphorylation

In squid axons, internally applied ATP potentiates the magnitude of the potassium conductance and slows down its activation kinetics. This effect was characterized using internally dialyzed axons under voltage-clamp conditions. Both amplitude potentiation and kinetic slow-down effects are very selective towards ATP, other nucleotides like GTP and ITP are ineffective in millimolar concentrations. The current potentiation Km for ATP is near 10 microM with no further effects for concentrations greater than 100 microM. ATP effect is most likely produced via a phosphorylative reaction because Mg ion is an obligatory requirement and nonhydrolyzable ATP analogues are without effect. In the presence of ATP, the K current presents more delay, resembling a Cole-Moore effect due to local hyperpolarization of the channel. ATP effect induces a 10-20 mV shift in both activation and inactivation parameters towards more depolarized potentials. As a consequence of this shift, conductance-voltage curves with and without ATP cross at approximately -40 mV. This result is consistent with the hyperpolarization observed with ATP depletion, which is reversed by ATP addition. At potentials around the resting value, addition of ATP removes almost completely K current slow inactivation. It is suggested that a change in the amount of the slow inactivation is responsible for the differences in current amplitude with and without ATP, possibly as a consequence of the additional negative charge carried by the phosphate group. However, a modification of the local potential is not enough to explain completely the differences under the two conditions.