Internally Perfused Research Articles

Effects of the dipolar form of phloretin on potassium conductance in squid giant axons

The effects of phloretin on membrane ionic conductances have been studied in the giant axon of the squid, Loligo pealei. Phloretin reversibly suppresses the potassium and sodium conductances and modifies their dependence on membrane potential (Em). Its effects on the potassium conductance (GK) are much greater than on the sodium conductance; no effects on sodium inactivation are observed. Internal perfusion of phloretin produces both greater shifts in GK(Em) and greater reductions maximum GK than does external perfusion; the effect of simultaneous internal and external perfusion is little greater than that of internal perfusion alone. Lowering the internal pH, which favors the presence of the neutral species of weakly acidic phloretin (pKa 7.4), potentiates the actions of internally perfused phloretin. Other organic cations with dipole moments similar to phloretin's have little effect on either potassium or sodium conductances in squid axons. These results can be explained by either of two mechanisms; on postulates a phloretin "receptor" near the voltage sensor component of the potassium channel which is accessible to drug molecules applied at either the outer or inner membrane surface and is much more sensitive to the neutral than the negatively charged form of the drug. The other mechanism proposes that neutral phloretin molecules are dispersed in an ordered array in the membrane interior, producing a diffuse dipole field which modifies potassium channel gating. Different experimental results support these two mechanisms, and neither hypothesis can be disproven.

Properties of sodium pumps in internally perfused barnacle muscle fibers.

To study the properties of the Na extrusion mechanism, giant muscle fibers from barnacle (Balanus nubilus) were internally perfused with solutions containing tracer 22Na. In fibers perfused with solutions containing adenosine 5'-triphosphate (ATP) and 30 mM Na, the Na efflux into 10 mM K seawater was approximately 25-30 pmol/cm2.s; 70% of this efflux was blocked by 50-100 microM ouabain, and approximately 30% was blocked by removal of external K. The ouabain-sensitive and K-dependent Na effluxes were abolished by depletion of internal ATP and were sigmoid-shaped functions of the internal Na concentration ([Na]i), with half-maxima at [Na]i approximately or equal to 20 mM. These sigmoid functions fit the Hill equation with Hill coefficients of approximately 3.5. Ouabain depolarized ATP-fueled fibers by 1.5-2 mV ([Na]i greater than or equal to 30 mM) but had very little effect on the membrane potential of ATP-depleted fibers; ATP depletion itself caused a 2-2.5-mV depolarization. When fueled fibers were treated with 3,4-diaminopyridine or Ba2+ (to reduce the K conductance and increase membrane resistance), application of ouabain produced a 4-5 mV depolarization. These results indicate that an electrogenic, ATP-dependent Na-K exchange pump is functional in internally perfused fibers; the internal perfusion technique provides a convenient method for performing transport studies that require good intracellular solute control.

Modification of slow sodium inactivation in nerve after internal perfusion with trypsin.

Crayfish axons, internally perfused and held at depolarized membrane potentials, exhibit an inactivation of sodium conductance with slow kinetics. Restoration of maximum peak early currents requires prepulse hyperpolarizations of up to 1 s duration. Addition of trypsin to the internal perfusate at low concentrations (0.02 mg/ml) causes a rapid and irreversible loss of slow inactivation at the resting potential and a corresponding increase in Na currents to maximum values. After trypsin action, steady-state slow Na inactivation is shifted by 20--25 mV in the depolarizing direction, with no change in fast (h) inactivation. N-ethylmaleimide (NEM), a reagent with a high specificity for sulfhydryl groups, has been shown to induce slow inactivation, modify fast inactivation, and block a fraction of the Na conductance. After trypsin action NEM no longer increases slow Na inactivation but other effects remain. Prior exposure to NEM does not protect axons against the loss of slow inactivation caused by trypsin.

Slow inactivation of the sodium conductance in squid giant axons. Pronase resistance.

1. Squid giant axons internally perfused with CsF have their Na conductance inactivated due to the low value of the resting potential. When hyperpolarized with voltage clamp to normal values of resting potential, the Na conductance recovers with an exponential time course. The time constant of recovery is of the order of 30 sec at a membrane potential of -70 mV and at 5 degrees C. The recovery from slow inactivation has a Q10 of about 3. 2. The development of inactivation during depolarization is also slow. The time constant varies between 10 and 20 sec at 5 degrees C, depending upon the value of the membrane potential. 3. Slow inactivation is also observed in NaF perfused axons and in intact axons with a low resting potential. 4. Although internal perfusion with pronase (or a purified fraction of this enzymic complex) blocks the fast (h) inactivation of the Na conductance, the slow inactivation remains. The recovery is similar before and after the proteolytic treatment. However, slow inactivation appears to develop faster after enzymic perfusion. 5. Slow inactivation develops without any apparent change in distributed or local membrane surface charge. 6. The experiments suggest that slow inactivation is a general property of the Na conductance as in many other conductance channels in excitable membranes. The experiments can be interpreted by proposing that slow inactivation is a phenomenon independent of fast inactivation, and that pronase somehow accelerates the onset of slow inactivation. 7. An alternative model, in which slow inactivation is coupled to fast inactivation, is proposed. This model is consistent with the results presented here and is very similar to one proposed to explain the frequency response of the sodium currents in Myxicola giant axons (Rudy, 1975, 1978).

Properties of internally perfused, voltage-clamped, isolated nerve cell bodies.

The membrane properties of isolated neurons from Helix aspersa were examined by using a new suction pipette method. The method combines internal perfusion with voltage clamp of nerve cell bodies separated from their axons. Pretreatment with enzymes such as trypsin that alter membrane function is not required. A platinized platinum wire which ruptures the soma membrane allows low resistance access directly to the cell's interior improving the time resolution under voltage clamp by two orders of magnitude. The shunt resistance of the suction pipette was 10-50 times the neuronal membrane resistance, and the series resistance of the system, which was largely due to the tip diameter, was about 10(5) omega. However, the peak clamp currents were only about 20 nA for a 60-mV voltage step so that measurements of membrane voltage were accurate to within at least 3%. Spatial control of voltage was achieved only after somal separation, and nerve cell bodies isolated in this way do not generate all-or-none action potentials. Measurements of membrane potential, membrane resistance, and membrane time constant are equivalent to those obtained using intracellular micropipettes, the customary method. With the axon attached, comparable all-or-none action potentials were also measured by either method. Complete exchange of Cs+ for K+ was accomplished by internal perfusion and allowed K+ currents to be blocked. Na+ currents could then be blocked by TTX or suppressed by Tris-substituted snail Ringer solution. Ca2+ currents could be blocked using Ni2+ and other divalent cations as well as organic Ca2+ blockers. The most favorable intracellular anion was aspartate-, and the sequence of favorability was inverted from that found in squid axon.

The permeability of the sodium channel in Myxicola to the alkali cations.

Relative permeabilities to the alkali cations were determined, from the reversal potential (VRev), for the Na channel of internally perfused voltage-clamped Myxicola giant axons. PLi/PNa and PK/PNa are 0.94 and 0.076, respectively. Rb and Cs are not measurably permeant. VRev vs. the internal Na activity was well described by the constant field equation over a 300-fold range of internal Na concentrations. In agreement with findings on squid axons, the PK/PNa was found to increase when the K content of the internal perfusate was reduced (equivalent per equivalent substitution with TMA). Internal Rb and Cs also decreased the PK/PNa. The order of effectiveness of internal K, Rb, and Cs in increasing the Na selectivity of the Na channel was Cs greater than Rb greater than or equal to K. External Li increases the PK/PNa but this may be due to the formation of LiF internally. It may be that substances do not have to traverse the channel in order to affect the selectivity filter. Evidence is presented which suggests that the selectivity of the Na channel may be higher for Na in intact as compared to perfused giant axons. It was concluded that the channel selectivity properities do not reflect only some fixed structural features of the channel, but the selectivity filter has a labile organization.

Simultaneous measurements of magnesium, calcium and sodium influxes in perfused squid giant axons under membrane potential control.

1. Giant axons from the squids Dosidicus gigas, Loligo forbesi and Loligo vulgaris were internally perfused with 550 or 275 mM KF plus sucrose and bathed in artificial sea water containing 45Ca, 28Mg or mixtures of 45Ca-28Mg or 45Ca-22Na. Resting influxes and extra influxes during voltage-clamp pulses were measured by collecting and counting the internal perfusate. 2. For Dosidicus axons in 10 mM-CaCl2 the resting influx of calcium was 0-016 +/- 0-007 p-mole/cm2 sec and a linear function of external concentration. For two experiments in 10 and 84-7 mM-CaCl2, 100 nM tetrodotoxin had no effect. Resting calcium influx in 10 mM-CaCl2 was 0-017 +/- 0-013 p-mole/cm2 sec for Loligo axons. 3. With 55 mM-MgCl2 outside the average resting magnesium influx was 0-124 +/- 0-080 p-mole/cm2 sec for Loligo axons. Discarding one aberrant point the value is 0-105 +/- 0-046 which is not significantly different from the resting calcium influx for Dosidicus fibres in 55 mM-CaCl2, given as 0-094 p-mole/cm2 sec by the regression line shown in Fig. 1. In two experiments 150 nM tetrodotoxin had no effect. 4. With 430 mM-NaCl outside 100 nM tetrodotoxin reduced the average resting influx of sodium in Dosidicus axon from 27-7 +/- 4-5 to 25-1 +/- 6-2 p-mole/cm2 sec and for Loligo fibres in 460 mM-NaCl from 50-5 +/- 4 to 20 +/- 8 p-mole/cm2 sec. 5. Using depolarizing pulses of various durations, the extra calcium influx occurred in two phases. The early phase was eliminated by external application of tetrodotoxin. The results of analysis are consistent with, but do not rigorously demonstrate, the conclusion that the tetrodotoxin sensitive calcium entry is flowing through the normal sodium channels (cf. Baker, Hodgkin & Ridgway, 1971). 6. Measurements of extra influxes using 22Na and 45Ca simultaneously indicate that the time courses of tetrodotoxin sensitive calcium and sodium entry are similar but not necessarily identical. It is very doubtful that any significant calcium entry occurs before the sodium or is involved in the activation of the sodium system. 7. These measurements confirm for Loligo, as previously shown for Dosidicus axons, that the magnitude and time course of the sodium entry during a depolarizing pulse deduced from electrical measurements is the same as that measured with 22Na. 8. Using 28Mg, or mixtures of 45Ca and 28Mg, we observed a single phase of magnesium entry which was insensitive to external tetrodotoxin or internal tetraethyl ammonium. The magnitude of the magnesium influx was considerably greater than the calcium extra entry and large enough to have been detected in the experiments of Meves & Vogel (1973) if it represented current. 9. We suggest the possibility that the calcium and magnesium extra influxes, after external treatment with tetrodotoxin, during a depolarizing pulse, do not contribute to the measured current.

Potentiometric estimation of charges in barnacle muscle fibers under internal perfusion.

Single barnacle muscle fibers fromMegabalanus Psittacus (Darwin) were internally perfused with a number of solutions of K-acetate of concentrationC 2 which was increased from 100 to 600mm maintaining a pH of 7.5. The external solutions at the same pH contained 40mm MgAc2 and K-acetate of concentrationC 1 which was varied from 10 through 60mm. The tonicity of both internal and external fluids was maintained at 1,000 mosmole with sucrose. The membrane potentialE m (the potential was referred to the external solution as ground) arising across the fiber membrane was measured using those internal and external solutions which maintained the ratio (C 2/C1) at 10. Similar measurements but now maintaining the ratio (C 2/C1) at 2, whereC 1 was the concentration of Tris-Cl in the internal perfusion fluid and was increased from 100 to 250mm andC 2 was the concentration of total Cl in the external fluid which contained 40mm MgCl2 and different amounts of Tris-Cl (120 to 420mm) were also made using various isotonic solutions at pH 4.0. The measured values ofE m corrected for the liquid junction potentials, on interpolation with the theoretical curves ofE m plotted against log (1/C 1) derived from the theory of membrane potential developed by Teorell and by Meyer and Sievers, gave values of 0.019 and 0.7m for the density of fixed charges present on the membrane at pH 7.5 (cation selective) and 4.0 (anion selective), respectively. Also, values of 2.3 at pH 7.5 and 1.0 at pH 4.0 were derived for the mobility ratio of cation to anion.

Excitability, instability and phase transitions in squid axon membrane under internal perfusion with dilute salt solutions

Using giant axons of squid, Doryteuthis, available in Hokkaido, Japan, it was shown that axons internally perfused with a dilute sodium salt solution undergo an abrupt transition from a resting to a depolarized state on addition of KCl to an external medium containing CaCl 2. Under internal perfusion with a dilute solution of sodium or cesium salt, it was possible to induce abrupt transitions between the two ( i.e., resting and depolarized) states of the membrane by changing the temperature. “Giant fluctuations” in the state of the axon membrane were demonstrated at and near the critical points of the axon membrane. These findings are interpreted as supporting the view that an abrupt change in the membrane potential and conductance is an electrochemical manifestation of a phase transition of the membrane macromolecules.

Internal perfusion of crayfish, giant axons: action of tannic acid, DDT, and TEA.

AbstractCrayfish giant axons remain viable following internal perfusion with a mixture of fluoride and citrate salts. The relative favorability of various internal anions, and the dependence of resting and action potentials on internal cations are both similar to results on internally perfused squid axons. TEA widens the falling phase of the spike only from inside the axon, while DDT is active from either side of the membrane. Records of impedance changes show that effects of TEA and DDT on components of ionic conductances are similar to those found in other axons by voltage clamp measurements.Tannic acid perfused internally at a concentration of the order of 10 μM produces spontaneous activity, and a progressive increase in spike width. After 30 minutes, action potentials are “cardiac” type and are up to several minutes in duration. Records of impedance changes, and data from rapid changes in external ionic concentrations, suggest that the plateau phase of the spike is due to a maintained increase in sodium conductance. Since tannic acid is capable of crosslinking proteins and “rigidifying” protein monolayers, it is suggested that its effects on the axon may be the result of an interference with a conformational change in a membrane protein or protein‐phospholipid complex during excitation.

Salt transport in Valonia: inhibition of potassium uptake by small hydrostatic pressures.

Internally perfused cells of the marine alga Valonia actively transport potassium from external seawater into the cell vacuole. This active uptake of potassium is reduced by restoring the normal turgor pressure of 1 atmosphere by means of a mercury manometer attached to the internal perfusion system. This inhibition of salt uptake by a small hydrostatic pressure suggests that Valonia and other walled cells may regulate their turgor pressures by adjusting their rates of salt uptake.

Bi-ionic action potentials in squid giant axons internally perfused with sodium saltssalts.

presence of univalent cations (such as sodium) externally is not essential for production of all-or-none action potentials under these conditions. The term action potentials has been introduced2 to designate electric responses produced under these conditions in which only two critical cations (a univalent ion internally and a divalent ion externally) are involved in the process of excitation. The present report deals mainly with the electrophysiological properties of squid giant axons internally perfused with dilute solutions of a sodium salt. With sodium as the sole intracellular cation, squid giant axons were found to maintain excitability in external media containing a calcium salt as the sole electrolyte species.2 It is to be noted that the gradient of the sodium ion concentration in these axons is opposite to that of unperfused axons immersed in sea water. These experimental findings may be explained in terms of the hypothesis postulating a conformational change (phase transition) of membrane macromolecules associated with a cooperative ion exchange process between uni- and divalent cations at negatively charged sites of the macromolecules.3, 4 Methods.-Giant axons from Loligo pealii, available at the Marine Biological Laboratory in Woods Hole, Massachusetts, were used. The major portion of small nerve fibers and other adherent tissue surrounding the giant axon were removed under a dissecting microscope during the process of dissection. The technique of internal perfusion used in the present study was substantially the same as that used in previous studies from this laboratory.1 The giant axon was mounted horizontally in a Lucite chamber (approximately 33 mm in length) containing natural sea water, and a glass cannula was inserted into each end of the preparation. Initially, the tip of the smaller, inlet cannula (150 , in diameter) was placed concentrically within the lumen of the larger, outlet cannula (250-300 A in diameter). Internal perfusion was initiated by separating the two cannulae. The inlet cannula was connected to a reservoir of internal perfusion fluid by means of a small polyethylene tubing. In most of the present experiments, the length of the internal perfusion zone was between 14 and 21 mm. Internal perfusion solutions were usually prepared by mixing a 12 (vol) % glycerol solution with a 600 mM sodium phosphate solution (pH 7.3 ? 0.1).4a In order to remove the axoplasm from the perfusion zone, perfusion was instituted with a sodium phosphate solution containing 0.05 mg/ml Pronasel, 5 (Calbiochem) for a period of 1 to 1.5 min and then switched to enzymefree solution. The flow rate of the internal perfusate, controlled by adjusting the height of the fluid reservoir, was maintained at 15-20 ,l/min. External fluid medium was, as a rule, a mixture of 400 mM CaC12 and 12 (vol) % glycerol; the pH was adjusted to 8.0 4= 0.1 with tris(hydroxymethyl)aminomethane buffer (less than 1 mM). (Note that the presence of a pH buffer in the external medium is not essential for the maintenance of bi-ionic action potentials.') Continuous flow of the external medium was maintained at 10-30 ml/min throughout the course of the experiments. A trap served to electrically isolate the suction apparatus used to drain the chamber from the external fluid medium.

Ion fluxes and short-circuit current in internally perfused cells of Valonia ventricosa.

Ion transport in the giant celled marine alga, Valonia ventricosa, was studied during internal perfusion and short-circuiting of the vacuole potential. The perfusing and bathing solutions were similar to natural Valonia sap and contained the following concentrations of major ions: Na 51, K 618, and Cl 652 mM. The average short-circuit current (I(sc)) was 97 pEq/cm(2) sec (inward positive current), and the average open-circuit potential difference (PD) was 74 mv (vacuole positive to external solution). Perfused and short-circuited cells showed a small net influx of Na (2.0 pEq/cm(2) sec) and large net influxes of K (80 pEq/cm(2) sec) and Cl (50 pEq/cm(2) sec). Unidirectional K influx was proportional to I(sc), but more than one-half of the I(sc) remained unaccounted for. Both the I(sc) and PD were partly light-dependent, declining rapidly during the first 1-2 min of darkness. Ouabain (5 x 10(-4)M) had little effect on the influx of Na or K and had no effect on I(inf) or PD. Fluid was absorbed at a rate of about 93 pliter/cm(2) sec. Reversing the direction of fluid movement by adding mannitol to the outside solution had little effect on ion movements. The ionic and electrical properties of normal and perfused cells of Valonia are compared.