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++.     

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.     

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.     

The binding of tritiated tetrodotoxin to squid giant axons.

The binding of tetrodotoxin to squid gian axons was determined as a function of toxin concentration, using a tritiated toxin preparation of known radiochemical purity and specific activity. From the amount of saturable binding observed, the number of toxin binding sites thought to be sodium channels was found to be 553 plus or minus 119/mum-2 of axon surface.     

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.     

Sodium-calcium exchange and calcium-calcium exchange in internally dialyzed squid giant axons.

The influx and efflux of calcium (as 45Ca) and influx of sodium (as 24Na) were studied in internally dialyzed squid giant axons. The axons were poisoned with cyanide and ATP was omitted from the dialysis fluid. The internal ionized Ca2+ concentration ([Ca2+]i) was controlled with Ca-EGTA buffers. With [Ca2+]i greater than 0.5 muM, 45Ca efflux was largely dependent upon external Na and Ca. The Nao-dependent Ca efflux into Ca-free media appeared to saturate as [Ca2+]i was increased to 160 muM; the half-saturation concentration was about 8 muM Ca2+. In two experiments 24Na influx was measured; when [Ca2+]i was decreased from 160 muM to less than 0.5 muM, Na influx declined by about 5 pmoles/cm2 sec. The Nao-dependent Ca efflux averaged 1.6 pmoles/cm2 sec in axons with a [Ca2+]i of 160 muM, and was negligible in axons with a [Ca2+]i of less than 0.5 muM. Taken together, the Na influx and Ca efflux data may indicate that the fluxes are coupled with a stoichiometry of about 3 Na+-to-1 Ca2+. Ca efflux into Na-free media required the presence of both Ca and an alkali metal ion (but not Cs) in the external medium. Ca influx from Li-containing media was greatly reduced when [Ca2+]i was decreased from 160 to 0.23 muM, or when external Li was replaced by choline. These data provide evidence for a Ca-Ca exchange mechanism which is activated by certain alkali metal ions. The observations are consistent with a mobile carrier mechanism which can exchange Ca2+ ions from the axoplasm for either 3 Na+ ions, or one Ca2+ and an alkali metal ion (but not Cs) from the external medium. This mechanism may utilize energy from the Na electrochemical gradient to help extrude Ca against an electrochemical gradient.     

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.     

Geographical distribution and inactivation kinetics in internally perfused Myxicola giant axons.

In some preparations the time constant of Na current inactivation determined with two pulses (tau c) is larger over some range of potentials than that determined from the current decay during a single pulse (tau h), while in others tau c(V) and tau h(V) are the same. Myxicola giant axons obtained from specimens collected from coastal waters of northeastern North America display a tau c - tau h difference under all conditions we have tested. In these axons tau c(V) and tau h(V) are unchanged by reduction of Na current density, addition of K-channel blockers, or internal perfusion. Specimens of the same species, Myxicola infundibulum, collected from a different geographical location, the south coast of England, have been studied under internal perfusion with K as the major cation internally, with reduced external Na concentration and in the presence of K-channel blockers. In these axons tau c(V) and tau h(V) approximately superpose, raising the possibility that dramatic differences in Na current kinetics may not necessarily reflect basic differences in the organization of the Na channel gating machinery.     

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.     

Separation of the action potential into a Na-channel spike and a K- channel spike by tetrodotoxin and by tetraethylammonium ion in squid giant axons internally perfused with dilute Na-salt solutions

Squid giant axons internally perfused with a 30 mM NaF solution and bathed in a 100 mM CaCl2 solution, which are known to produce long lasting action potentials in response to pulses of outward current, were investigated. The effects of tetrodotoxin (TTX) and of tetraethylammonium ion (TEA+) on such action potentials were studied. The results are summarized as follows: (a) An addition of 1--3 microM TTX to the external solution altered but did not block the action potentials; it increased the height of the action potential by approximately 15 mV, and it decreased the membrane conductance as the peak of excitation by about two-thirds. (b) Voltage-clamp experiments performed with both NaCl and TTX in the external CaCl2 solution revealed that the TTX-insensitive action potential does not involve a rise in gNa, whereas the experiments performed without TTX showed that the action potential is accompanied by a large rise in gNa. (c) Internally applied TEA+ was shown to selectively block the TTX- insensitive action potential, but it did not block the other component of the action potential, which is accompanied by a rise in gNa, and which is selectively suppressed by TTX. (d) The addition of a small amount of KCl to the external CaCl2 solution containing TTX greatly increased both the maximum peak inward current under voltage clamp and the maximum slope conductance. Furthermore, it was shown that K+ applied on both sides of the axon plays a dominant role in producing the membrane potential in the active state in the presence of TTX, even though a large amount of Ca2+ is presented in the bathing medium. These observations have led me to conclude that the sodium channel is responsible for the production of the TTX-sensitive component of the action potential under the ionic conditions of these experiments, and the potassium channel for the TTX-insensitive component of the action potential.     

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.     

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.     

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.     

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.