Steady-state Inactivation Parameters Research Articles

Electronic Expression of IK1 in Human Induced Pluripotent Stem Cell Derived Cardiocytes Reveals Atrial vs Ventricular Specific Properties

Cardiomyocytes derived from human induced pluripotent stem cells (hIPSC-CMs) provide a promising platform for understanding human cardiac pathology and physiology. HIPSC-CMs differentiate into a mixed population of cells composed of ventricular, atrial, and nodal cell types; cellular phenotype is generally distinguished by morphology of the action potential (APs). However, in hIPSC-CMs a very small inward rectifying potassium channel (IK1) relative to native cardiac cells causes a depolarized membrane potential which contributes to inconsistent AP properties and misidentification of cellular phenotype.We used Electronic expression of IK1 via dynamic clamp to restore the AP and resting membrane potential back to a physiological levels. This allowed for improved discrimination of atrial and ventricular cells based on AP morphology. Using standard patch clamp techniques, we compared the inward sodium currents (INa) of cells with atrial and ventricular AP morphologies. INa current densities in cells with atrial like APs were larger than ventricular-like APs (at −30 mV, in pA/pF: −71.22 ± 6.96 (n=5) vs. −46.25 ±5.03 (n=14), p < 0.05). Analysis of steady-state inactivation parameters of INa showed that cells with an atrial like AP had a more negative steady-state inactivation (V1/2: −81.84 ± 2.75 (n=5) vs. −74.72 ±0.8 (n=14), p<0.05). Cells with atrial APs had a larger Kv1.5-like component at +50 mV than ventricular APs (in pA/pF: 3.71 ± 0.55 (n=5) vs 1.00 ± 0.10 (n=16),P<0.05) but similar peak currents: (6.89 ± 0.50 (n=5) vs. 6.58 ± 0.67 (n=14),P=N.S). IK1 current density was more than 3x smaller in cells with atrial-like APs at −120 mV (in pA/pF: −0.71 ± 0.08 (n=5) vs. −3.45 ± 0.61 (n=14), p<0.05). These data suggest that differential AP morphology in hIPSC-CM has a similar electrophysiological basis to native cells.

Reduction of the transient outward potassium current in a canine model of Chagas' disease

The effects of Chagas' disease, an important cause of cardiac arrhythmias and cardiomyopathy, on cellular electrical properties were determined in epicardial tissue from normal dogs and dogs infected with Trypanosoma cruzi for 20-25 days (25 DPI), at the time of maximum parasitemia, and for 125-140 days (140 DPI) after the parasitemia had subsided. At 25 DPI, phase 1 repolarization of the action potential was attenuated and the transient outward current (Ito) was reduced from 10.2 +/- 0.5 to 5.5 +/- 0.6 pA/pF. No differences were apparent between infected and normal cells in the time constants of current decay (25.6 +/- 4.0 and 22.8 +/- 1.3 ms, respectively) or in the steady-state inactivation parameters (V1/2 = -34.1 +/- 3.6 and -34.6 +/- 1.4 mV and k = 6.3 +/- 1.8 and 4.0 +/- 0.3, respectively). The rapid phase of recovery from inactivation was nearly eliminated in infected myocytes, whereas the slower phase was unaffected. Phase 1 repolarization and Ito density at 140 DPI were not significantly different from normal cells. Thus T. cruzi acutely inhibited Ito in epicardial myocytes, an effect that was reversed with abatement of the parasitemia.

Multiple second messenger routes enhance two high-voltage-activated calcium currents in molluscan neuroendocrine cells.

Two types of high-voltage-activated calcium currents were identified in whole-cell voltage-clamp recordings of the neuroendocrine caudodorsal cells, which control egg-laying in the freshwater snail Lymnaea stagnalis. The currents were: (i) a rapidly inactivating high-voltage-activated current, with an activation threshold of -40 mV and maximal amplitude at +10 mV; and (ii) a slowly inactivating high-voltage-activated current, with a threshold of -10 mV and a peak at +30 mV. Both currents were reduced by nifedipine and verapamil, but not by omega-conotoxin GVIA, suggesting that they belong to the L-type family of calcium currents. The voltage-dependence of inactivation of the rapidly inactivating high-voltage-activated current was bell-shaped. Time-constants of inactivation ranged from 10 to 25 ms. Steady-state inactivation was characterized by a potential of half maximal inactivation of -21.7 +/- 3.4 mV and a slope factor of 8.1 +/- 1.7 mV. The voltage-dependence of inactivation of the slowly inactivating high-voltage-activated current was S-shaped. Time-constants of inactivation increased with depolarization up to a maximum of 300 ms. The steady-state inactivation parameters were a potential of half maximal inactivation of +6.8 +/- 2.2 mV and a slope factor of 6.0 +/- 1.1 mV. The membrane-permeable analog of cAMP, 8-chlorophenylthio-cyclic AMP, predominantly increased the slowly inactivating high-voltage-activated current, and shifted its voltage-dependence of activation and inactivation 10 mV to the left. The rapidly inactivating high-voltage-activated current was slightly increased by 8-chlorophenylthio-cyclic AMP. 8-Bromo-cyclic GMP and the phorbol ester, 12-O-tetradecanoyl-13-phorbol acetate, had qualitatively similar effects. Both agents enhanced the rapidly inactivating current and, to a lesser degree, the slowly inactivating current, without affecting their voltage-dependence. The cyclic AMP-dependent protein kinase inhibitor, Walsh inhibitor peptide, antagonized the stimulating effect of 8-chlorophenylthio-cyclic AMP. The broad-spectrum protein kinase inhibitor 1-(5-isoquino-linylsulfonyl)-2-methyl-piperazine (H-7) strongly attenuated the effects of 8-chlorophenylthio-cyclic AMP, 8-bromo-cyclic GMP and 12-O-tetradecanoyl-13-phorbol acetate, suggesting that all treatments increase both types of high-voltage-activated calcium currents through phosphorylation of the channel-complex.

Ca2+ channel modulating effects of heparin in mammalian cardiac myocytes.

1. The effect of heparin on L-type Ca2+ channels in rabbit, rat and guinea-pig cardiac myocytes was studied using the whole-cell patch clamp method. 2. Sodium salts of heparin uniformly suppressed the Ca2+ current, ICa, independent of their molecular weight, in the rat and guinea-pig ventricular and rabbit atrial myocytes. The suppression of ICa by heparin was dose dependent and reached its maximum, about 30%, around 10 microM. Heparin did not alter the voltage-dependence or the steady-state inactivation properties of ICa. These effects were specific to heparin as another polysaccharide, dextran, failed to have any effect on ICa. 3. The suppressive effect of heparin was not diminished when [Ca2+]o was increased to 10 mM, or when Ba2+ was the charge carrier through the Ca2+ channel. 4. Spectrophotometric assays showed that heparin-induced changes in [Ca2+]o generally were too small to alter ICa significantly. 5. In myocytes buffered with 0.1 mM EGTA, the suppressive effect of heparin was more prominent on the inactivating than on the maintained component of ICa. 6. When extracellular Na+ was replaced by Cs+, the heparin suppressive effect was accompanied by a 10 mV shift of both the voltage dependence of activation and the steady-state inactivation parameters toward more negative potentials. 7. When both Mg2+ and Na+ were omitted from the bathing solutions, the suppressive effect of heparin was significantly enhanced such that almost 80% of the current was blocked. 8. In Cs(+)-based solutions 10 mM [Mg2+]o suppressed ICa by about 70% and heparin partially relieved this block. Heparin, however, did not counteract the Mg(2+)-induced suppression of ICa in Na(+)-based solution. 9. Extracellularly applied heparin did not alter the isoprenaline-induced enhancement of ICa or interfere with the blocking effect of phorbol esters on ICa. 10. Heparin thus appears to interfere with the permeation of Ca2+ through the channel by a mechanism regulating the Ca(2+)-induced inactivation of the Ca2+ channel. Na+ and Mg2+ appear to alter the kinetics and the magnitude of the suppressive effect of heparin on the Ca2+ channel, suggesting an interaction of these cations with either the Ca2+ or the heparin-binding sites of the channel.

Characterization of TTX-sensitive and TTX-resistant sodium currents in small cells from adult rat dorsal root ganglia.

1. The whole-cell patch-clamp technique was used to investigate the characteristics of two types of sodium current (INa) recorded at room temperature from small diameter (13-25 microns) dorsal root ganglion (DRG) cells, isolated from adult rats and maintained overnight in culture. 2. Sodium currents were isolated pharmacologically. Internal Cs+ and external tetraethylammonium (TEA) ions were used to suppress potassium currents. A combination of internal EGTA, internal F-, a low (10 microM) concentration of external Ca2+ and a relatively high (5 mM) concentration of internal and external Mg2+ was used to block calcium channels. The remaining voltage-dependent currents reversed direction at the calculated sodium equilibrium potential. Both the reversal potential and magnitude of the currents exhibited the expected dependence on the external sodium concentration. 3. INa subtypes were characterized initially in terms of their sensitivity to tetrodotoxin (TTX). TTX-sensitive (TTXs) currents were at least 97% suppressed by 0.1 microM TTX. TTX-resistant (TTXr) INa were recorded in the presence of 0.3 microM TTX and appeared to be reduced in amplitude by less than 50% in 75 microM TTX (n = 1). 4. As in earlier studies, the peak of the current-voltage relationship, the mid-point of the normalized conductance curve and the potential (Vh) at which the steady-state inactivation parameter (h infinity) was 0.5 were found to be significantly more depolarized for the TTXr INa (by ca 10, 14 and 37 mV respectively). There was little difference in the slope at the mid-point of the normalized conductance curves (the mean slope factors were 5.1 mV for the TTXs INa and 4.9 mV for the TTXr current) but the h infinity curves for TTXr currents were significantly steeper than those for TTXs currents (mean slope factors of 3.8 and 11.5 mV respectively). Both the time to peak and the decay time constant of the peak current recorded from a holding potential of -67 mV were more than a factor of three slower for the TTXr INa than for the TTXs current. 5. However, in direct contrast to the difference in activation and decay kinetics, 'slow' TTXr INa recovered from inactivation at -67mV, or reprimed, more than a factor of ten faster than 'fast' TTXs INa. 6. The differences apparent in both the repriming kinetics of TTXs and TTXr INa at -67 mV and the kinetics of the decay phase of the peak INa are shown to be explicable largely in terms of the voltage dependence of their respective inactivation systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanism of the increase in intracellular sodium during metabolic inhibition: Direct evidence against mediation by voltage-dependent sodium channels

During ischemia or metabolic inhibition, intracellular Na + concentration ([Na +] i) increases considerably. Elevation of [Na +] i figures critically in the mechanism of cellular injury by promoting Ca 2+ influx via the Na +Ca 2+ exchanger, but the exact mechanism of this intracellular Na + accumulation remains unknown. To test directly the hypothesis that voltage-dependent Na + channels are involved, we measured Na + currents ( I Na) in isolated guinea-pig ventricular myocytes using the patch-clamp technique. The cell-attached configuration was used in order to avoid disturbing the intracellular milieu. Metabolic inhibition was induced by exposing the cells to either iodoacetate (IAA, 1 m m) to inhibit glycolysis or 2,4-dinitrophenol (DNP, 0.2 m m) to uncouple oxidative phosphorylation. The amplitude of I Na was measured in multichannel patches before and during exposure to IAA or DNP, by depolarizing the cell to different membrane potentials from a holding potential of −135 mV. Analysis of current-voltage relations before and during metabolic inhibition revealed a modest but significant reduction of peak I Na at test potentials positive to −40 mV with DNP; no change was observed with IAA. The voltage dependence of steady-state parameters of inactivation was not altered by either intervention; specifically, no steady-state (“window”) current was induced. Although we cannot exclude the possibility that other factors not explored here might lead to different conclusions during genuine ischemia, metabolic inhibition alone does not up-regulate the function of Na + channels. Thus, we conclude that other mechanisms underlie the accumulation of intracellular Na + observed during metabolic inhibition.

The influence of charge on the effects of n-octyl derivatives on sodium current inactivation in rat sensory neurones.

1. The whole-cell patch-clamp technique was used to determine the actions of n-octyl sulphate (OS-) anions and n-octyl trimethylammonium (OTMA+) cations on sodium current steady-state inactivation and peak amplitude in cells isolated from dorsal root ganglia of neonatal rats and maintained in short-term tissue culture. This paper concentrates on the effects of external addition but the actions of internal OS- and OTMA+ are briefly considered. 2. The main action of external OS- was to cause a hyperpolarizing shift in the voltage dependence of the steady-state inactivation parameter, h infinity. At 1-6 mM OS- caused a shift in the mid-point of the h infinity curve of around -30 mV. The shape of the h infinity curve was altered in a concentration-dependent manner. Internal OS- had no discernible effect on the shape or position of the h infinity curve. 3. External OS- produced a relatively small (less than 25%) reduction in the maximum current achieved following pre-pulses sufficiently negative to remove resting steady-state inactivation. 4. By contrast, external OTMA+ had little effect on the voltage dependence of h infinity and produced a small, but significant, increase in the maximum sodium current. 2 mM-external OTMA+ moved the mid-point of the h infinity curve (Vh) 5 mV in the depolarizing direction (relative to the mean of control and reversal curves) and increased the maximum current by 13%. One millimolar internal OTMA+ induced a frequency-dependent current block. 5. Raising the external calcium concentration from 2 to 20 mM (in the presence of 2 mM-magnesium and 5 mM-cobalt) caused an 18 mV depolarizing shift in Vh, consistent with a reduction in the negativity of an external surface charge. The maximum current was reduced by 22%. 6. One millimolar OS- reduced the surface potential of egg phosphatidylcholine (EPC) monolayers (at an air-0.5 M-NaCl interface) by 35 mV but 1 or 2 mM-OTMA+ produced only a 2-3 mV increase. The quantitative agreement between the effects of OS-, on Vh in the rat and on monolayer surface potential, decreased with increasing concentration.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of sodium current in developing rat diencephalic neurons in serum-free culture.

1. Dissociated, synchronized (G1 phase of cell cycle), and birth-dated fetal rat diencephalic neurons were grown in a serum-free defined medium. The gigaseal whole-cell voltage-clamp technique was used to measure the inward Na+ currents (INa) from morphologically identified bipolar neurons. The earliest expressed somatic INa has been characterized and compared with that present at a later date. 2. The identity of the INa was established on the basis of its reversal potential and reversible blockade by tetrodotoxin (TTX). Close agreement between the measured reversal potentials (68.5 +/- 1.3 and 38.3 +/- 2.4 mV, mean +/- SE) and calculated Nernst equilibrium potentials (64.6 and 34.7 mV) at two different bath Na+ concentrations (120 and 35 mM, respectively) suggests that the channels are highly selective for Na+. 3. The peak INa density increased from 47.7 +/- 2.9 pA/pF in younger neurons (5-6 days in culture) to 93.9 +/- 6.4 pA/pF in older neurons (12-13 days in culture). The activation voltage and the voltage for peak current were also shifted by 10 mV in the hyperpolarizing direction, from -30 and +10 mV in younger neurons to -40 and 0 mV in older neurons, respectively. However, the reversal potential did not change (69.2 +/- 2.3 and 68.5 +/- 1.3 mV in younger and older neurons, respectively). 4. In older neurons the steady-state inactivation parameters (V1/2, the voltage at which inactivation was 50% of maximum, and kh, the voltage at which there is an e-fold change in inactivation) were significantly altered. V1/2 was shifted from -41.5 +/- 2.3 to -48.8 +/- 1.8 mV, and kh was increased from 6.2 +/- 0.5 to 8.9 +/- 0.4 mV. However, the time course of activation and the rates of inactivation and recovery from inactivation were unchanged. 5. In both groups, the INa decays were best described by a sum of two exponentials. The corresponding time constants were voltage dependent. Also, the amplitudes of the two components were differentially affected by membrane potential and niflumic acid. 6. The extrapolated amplitudes of both the fast and the slow components of INa were larger in older neurons, but the ratio of the amplitudes of the two components did not change with age. The voltage dependencies of the time constants of both components were altered. 7. We conclude that INa in fetal rat diencephalic neurons grown in a defined medium with only essential nutrients undergoes in vitro changes in current density and in some, but not all, kinetic parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of inactivation in the effects of n‐alkanols on the sodium current of cultured rat sensory neurones.

1. The whole-cell patch-clamp technique has been used to investigate the actions of n-butanol, n-pentanol, n-hexanol and n-octanol on the sodium current of cells isolated from the dorsal root ganglia (DRGs) of neonatal rats and maintained in short-term tissue culture. 2. The influence of n-alkanols on the level of steady-state inactivation of the sodium current was investigated by a standard two-pulse protocol. All alkanols increased the level of resting inactivation and this was manifested as a hyperpolarizing shift of the relationship between the steady-state inactivation parameter (h infinity) and membrane potential. The mid-point of the h infinity curve was moved by up to -30 mV. 3. The relationship between the shift in the mid-point of the inactivation curve (delta Vh) and aqueous n-alkanol concentration has been derived for each n-alkanol. These are complex in shape and do not appear consistent with a hypothesis that the increase in inactivation results from 1:1 binding of an alkanol molecule to a single site on the channel protein. 4. The aqueous concentrations used ranged from 70 mM-n-butanol to 0.05 mM-n-octanol. However, equal fractional saturations of n-alkanols produced approximately equal shifts in the h infinity curve, particularly in the range 0.01-0.07 saturated. This implies a hydrophobic site of action, with a standard free energy per methylene group for adsorption to the site from the aqueous phase of ca -3.2 kJ/mol. 5. The increase in resting inactivation was not the sole means by which n-alkanols reduced the sodium current. The current was still reduced in cells pre-pulsed to sufficiently negative potentials to remove steady-state inactivation even in the presence of alkanols. The concentration required to reduce the current by 50% (ED50) has been interpolated for each n-alkanol. From these data it was estimated that the standard free energy per methylene group for adsorption to the site of action was ca -3.1 kJ/mol, similar to that calculated for the effect on inactivation. The concentration dependence of this residual block indicated the involvement of more than one n-alkanol molecule. 6. The n-alkanols increase the level of inactivation of rat DRG cell sodium channels at potentials around the resting membrane potential and this effect contributes to their local anaesthetic action.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of Ba currents in Xenopus oocyte injected with pregnant rat myometrium mRNA

Using the single electrode voltage-clamp technique, stage V and VI Xenopus oocytes showed a Ba inward current (endogenous IBa) with a peak amplitude of -15 +/- 2 nA in a Cl and Na-free Ba methane sulphonate medium. When oocytes were injected with pregnant rat (18 days gestation) myometrium mRNA, an additional component of Ba current could be detected (exogenous IBa). This inward current could be distinguished from the native IBa by several means: i) peak amplitude (-75 +/- 3 nA); ii) activation voltage threshold; iii) steady-state inactivation parameter; iv) sensitivity to dihydropyridines. The features of the exogenous IBa were compared to those of the inward Ca channel current recorded with the tight-seal patch-clamp technique in single myometrial cells maintained in primary culture.

Effects of some chemical reagents on sodium current inactivation in myelinated nerve fibers of the frog

The effect of several chemical reagents on the sodium current was studied in voltage-clamped single nerve fibers of the frog. The oxidants halazone and hypochlorous acid drastically inhibited inactivation. Their effect was similar to that of chloramine T (Wang, 1984a). The curve relating the steady-state inactivation parameter h infinity to the conditioning potential E became nonmonotonic after treatment with the oxidants, i.e., dh infinity/dE greater than 0 for E greater than -20 mV. By contrast, the oxidants periodate, iodate, and hydrogen peroxide (applied for the same time, but at higher concentrations) merely produced a parallel shift of the h infinity(E) curve to more negative values of membrane potential. Diethylpyrocarbonate, a reagent that preferentially modifies histidine groups, had one marked effect: a strong shift of the h infinity(E) curve to more negative values of membrane potential. Almost no effect was observed after application of the tyrosine-reactive reagent N-acetylimidazole. Similarly, the arginine-reactive reagent glyoxal had only minor effects on the Na permeability. The results suggest that methionine is not critically involved in the kinetics of Na current inactivation. Similarly, an essential tyrosine or arginine residue seems to be unavailable to chemical reagents from outside on the frog node of Ranvier. Deduced from the reactivities of (some of) the reagents used, modification of membrane lipids is a tentative explanation for the effects observed on inactivation kinetics.

Temperature Acclimation: Influence on Transient Outward and Inward Currents in an Identified Neurone of Helix Pomatia

ABSTRACT To gain insight into the mechanisms underlying compensatory changes in excitability induced by thermal acclimation we examined the effects of short-term and long-term temperature transition on a transient outward potassium current (A-current) and the transient inward current in an identified neurone (Br) of the land snail Helix pomatia. The A-current in neurones from snails acclimated to 20°C was suppressed by cooling to 3°C, with a Q10 of 4.5 between 10°C and 20°C. This is consistent with suppression of the ability of these neurones to fire repetitively at low temperatures. Neurones acclimated to low temperature (5°C) partially recovered their ability to fire repetitively, but exhibited no A-current, at this temperature. Neither the equilibrium potential for the A-current (EA), the steadystate inactivation parameters of the A-current (Bo), nor the ratio of the time constants of A-current activation and inactivation (τA/τB) were affected by cooling, leading to the conclusion that the reduction of the A-current by cooling was due to a decrease in the A-current activation parameters (GAA∞). Cooling reduced the maximum peak inward current, with a Q10 of 2, in neurones from warm-acclimated animals. After acclimation to 5°C, maximum peak inward current partially recovered. Cooling neurones from warm-acclimated animals slowed the time course of the recovery of inward current from inactivation. Acclimation to the cold caused a partial, compensatory shortening of the inactivation removal process.

Modulation of potassium current kinetics in bag cell neurons of Aplysia by an activator of adenylate cyclase

The bag cell neurons of Aplysia are neurosecretory cells which control egg-laying behavior. In their resting state, the cells have a high resting potential and show no spontaneous activity. In response to brief stimulation of a neural input, the cells depolarize and fire repetitively for up to 60 min. This afterdischarge is thought to be controlled by elevations of intracellular adenosine 3':5'-monophosphate (cAMP). A voltage clamp study of bag cells in primary culture was undertaken in order to characterize the effects of cAMP on the cells' electrical properties. The transient outward potassium current (A-current) was studied before and after the application of forskolin (an activator of adenylate cyclase) and RO20-1724 (a phosphodiesterase inhibitor). These drugs reduced the amplitude of the A-current, primarily by speeding the inactivation process. The time constants for inactivation were speeded at all potentials, but the largest effects were seen at the more positive potentials (-40 to -15 mV), where the time constants were reduced 5-fold. Neither the activation process nor the steady-state parameters of inactivation were altered by the drugs. It is suggested that these changes in the A-current could explain the ability of the bag cells to fire repetitively during the afterdischarge.

Anaesthetic action of esters and ketones: evidence for an interaction with the sodium channel protein in squid axons.

The effects of methyl butyl ketone, methyl heptyl ketone and methyl pentanoate on the sodium current of the squid giant axon have been examined. The peak inward current in intact axons was reduced reversibly by each substance. Sodium currents were recorded in intracellularly perfused axons before and during exposure to the test substances and the records were fitted with equations similar to those proposed by Hodgkin & Huxley (1952). Shifts in the voltage dependence of the steady-state activation and inactivation parameters (m infinity and h infinity), reductions in the peak heights of the activation and inactivation time constants (tau m and tau h) and changes in the maximum sodium conductance (gNa) caused by these substances have been tabulated and compared with the effects of methyl octanoate (Haydon & Urban, 1983b). Each compound shifted the voltage dependence of the steady-state inactivation parameter in the hyperpolarizing direction and that of the steady-state activation parameter in the depolarizing direction. The shifts produced by the ketones are compared with those produced by methyl pentanoate and by methyl octanoate. The possible role of an interaction between the carbonyl oxygen of the test substance and the sodium channel protein in producing the h infinity shift is discussed. The peak time constants are reduced and the voltage dependences of tau m and tau h are shifted in a direction commensurate with the shifts in steady-state properties. The maximum sodium conductance is not much affected either by the ketones or by methyl pentanoate. Large reductions in peak inward current coupled with little effect on gNa have been reported for the n-alkanols and other surface-active compounds (Haydon & Urban, 1983b). This lack of a large effect on gNa indicates that whatever direct interaction does take place between the test substance and the channel protein, it does not result in a blockage of the channel.

The effect of temperature on the nerve-blocking action of benzyl alcohol on the squid giant axon.

The action of benzyl alcohol was studied on the voltage-clamped giant axons of Loligo forbesi. The depressant effect of 7.5 mM-benzyl alcohol on the action potential amplitude and peak rate of rise was more marked at a low (8 degrees C) than at a high temperature (16.5 degrees C). A small depolarization (approximately 5 mV) was also produced. These effects were usually reversible. Benzyl alcohol (7.5 mM) selectively depressed the amplitude of the peak early current. The maximum inward current was depressed to 0.65 and 0.67 of the control value at 20 degrees C and 7 degrees C respectively. This effect was usually reversible. Benzyl alcohol also depressed the peak inward conductance (gNa but had no effect on the time to peak early current. A small reversible decrease in the time constant of inactivation (tau h) was caused by benzyl alcohol (7.5 mM). This was usually reversible on washout of the anaesthetic. Benzyl alcohol (7.5 mM) had no effect on the time course of activation or the amplitude of the delayed outward current. The curve of the steady-state inactivation parameter (h infinity) for the Na current against the conditioning membrane potential was shifted by benzyl alcohol in a hyperpolarizing direction (at h infinity = 0.5 the shift was an average of 3.3 mV and was reversible). Increasing the temperature from 7 to 20 degrees C shifted the curve in a depolarizing direction by approximately 10 mV. The reason for the increased nerve-blocking action of benzyl alcohol at the lower temperature is discussed.

Some effects of n-pentane on the sodium and potassium currents of the squid giant axon.

1. Sodium and potassium currents have been recorded in intracellularly perfused squid giant axons before, during and after exposure to solutions of n-pentane in artificial sea water. 2. The currents were fitted with equations similar to those proposed by Hodgkin & Huxley (1952) and the changes in the parameters of these equations in the presence of pentane were calculated. 3. In the range of membrane potential -40 to 40 mV, the time constants for activation (tau m) and inactivation (tau h) of the Na current, and for activation (tau n) of the K current were all reduced by the pentane. 4. The curve of the steady-state inactivation parameter (h infinity) for the Na current against membrane potential was shifted by the pentane in a hyperpolarizing direction (at h infinity = 0.5 this shift was approx. -15 mV in 275 microM-pentane) and the slope at all potentials was reduced. 5. The curve of the steady-state activation parameter (m infinity) for the Na current against membrane potential was also shifted by the pentane in a hyperpolarizing direction (in 153 microM-pentane, 10 mV at m infinity = 0.5). 6. The maximum Na and K conductances gNa and gK were lowered by the pentane, though not usually completely reversibly. 7. The changes in position and slope of the steady-state inactivation curve have been tentatively accounted for in terms of an increase in membrane thickness.