Voltage-clamp of cut-end skeletal muscle fibre: a diffusion experiment

Membrane potential and current were studied in cut end fibres of frog skeletal muscle under current and voltage clamp conditions, by the double sucrose gap technique. Similar action potentials were recorded under current clamp conditions with either the microelectrode or the double sucrose gap techniques. Under voltage clamp conditions, the control of the membrane potential was maintained adequately. The early current was sensitive to both TTX and external Na concentration suggesting that the current was carried by Na ions. Sodium current (INa) was subsequently analysed using the Hodgkin-Huxley formulae. INa half-activation and inactivation occurred at -34 mV and -60 mV, respectively. Na-rich solution applied internally by diffusion through cut ends produced a reduction of INa associated with a shift of the sodium current reversal potential (VNa) towards more negative membrane potentials. This suggested that the sodium electromotive force was reduced by the increase in internal Na content of the fibre. Iodate applied externally changed neither the activation nor the inactivation time courses of INa, but reduced the peak current. Conversely, internally applied by diffusion from the cut end of skeletal muscle fibre, iodate slowed down the time course of INa inactivation and decreased the current peak. In conclusion, the double sucrose gap technique adapted to cut end frog skeletal muscle fibre allows a satisfactory analysis of INa.     

Characterization of Na(+) currents in isolated dorsal unpaired median neurons of Locusta migratoria and effect of the alpha-like scorpion toxin BmK M1

A primary cell culture was developed for efferent dorsal unpaired median (DUM) neurons of the locust. The isolated somata were able to generate Tetrodotoxin (TTX)-sensitive action potentials in vitro. The alpha-like scorpion toxin BmK M1, from the Asian scorpion Buthus martensi Karsch, prolonged the duration of the action potential up to 50 times. To investigate the mechanism of action of BmK M1, the TTX-sensitive voltage gated Na(+) currents were studied in detail using the whole cell patch clamp technique. BmK M1 slowed down and partially inhibited the inactivation of the TTX-sensitive Na(+) current in a dose dependent manner (EC50=326.8+/-34.5 nM). Voltage and time dependence of the Na(+) current were described in terms of the Hodgkin-Huxley model and compared in control conditions and in the presence of 500 nM BmK M1. The BmK M1 shifted steady state inactivation by 10.8 mV to less negative potentials. The steady state activation was shifted by 5.5 mV to more negative potentials, making the activation window larger. Moreover, BmK M1 increased the fast time constant of inactivation, leaving the activation time constant unchanged. In summary, BmK M1 primarily affected the inactivation parameters of the voltage gated Na(+) current in isolated locust DUM neurons.     

Slow and incomplete inactivations of voltage-gated channels dominate encoding in synthetic neurons.

Electrically excitable channels were expressed in Chinese hamster ovary cells using a vaccinia virus vector system. In cells expressing rat brain IIA Na+ channels only, brief pulses (< 1 ms) of depolarizing current resulted in action potentials with a prolonged (0.5-3 s) depolarizing plateau; this plateau was caused by slow and incomplete Na+ channel inactivation. In cells expressing both Na+ and Drosophila Shaker H4 transient K+ channels, there were neuron-like action potentials. In cells with appropriate Na+/K+ current ratios, maintaining stimulation produced repetitive firing over a 10-fold range of frequencies but eventually led to "lock-up" of the potential at a positive value after several seconds of stimulation. The latter effect was due primarily to slow inactivation of the K+ currents. Numerical simulations of modified Hodgkin-Huxley equations describing these currents, using parameters from voltage-clamp kinetics studied in the same cells, accounted for most features of the voltage trajectories. The present study shows that insights into the mechanisms for generating action potentials and trains of action potentials in real excitable cells can be obtained from the analysis of synthetic excitable cells that express a controlled repertoire of ion channels.     

Molecular mechanism of protein kinase C modulation of sodium channel alpha-subunits expressed in Xenopus oocytes.

The mechanism of modulation of sodium channel alpha-subunits (Type IIA) by a protein kinase C (PKC) activator was studied on single channel level. It was found that: (i) time constants for channel activation were prolonged; (ii) inactivation remained virtually unchanged; (iii) peak sodium inward current was reduced as evidenced by calculation of average sodium currents; and (iv) time constants for current activation and decay were prolonged. (i), (iii) and (iv) were voltage dependent, being most prominent at threshold potentials. The data show that a voltage dependent action on the activation gate can account for the observed reduction of peak inward sodium current and prolongation of current decay in macroscopic experiments.     

Characterization and functional consequences of delayed rectifier current transient in ventricular repolarization

Although inactivation of the rapidly activating delayed rectifier current (I(Kr)) limits outward current on depolarization, the role of I(Kr) (and recovery from inactivation) during repolarization is uncertain. To characterize I(Kr) during ventricular repolarization (and compare with the inward rectifier current, I(K1)), voltage-clamp waveforms simulating the action potential were applied to canine ventricular, atrial, and Purkinje myocytes. In ventricular myocytes, I(Kr) was minimal at plateau potentials but transiently increased during repolarizing ramps. The I(Kr) transient was unaffected by repolarization rate and maximal after 150-ms depolarizations (+25 mV). Action potential clamps revealed the I(Kr) transient terminating the plateau. Although peak I(Kr) transient density was relatively uniform among myocytes, potentials characterizing the peak transients were widely dispersed. In contrast, peak inward rectifier current (I(K1)) density during repolarization was dispersed, whereas potentials characterizing I(K1) defined a narrower (more negative) voltage range. In summary, rapidly activating I(Kr) provides a delayed voltage-dependent (and functionally time-independent) outward transient during ventricular repolarization, consistent with rapid recovery from inactivation. The heterogeneous voltage dependence of I(Kr) provides a novel means for modulating the contribution of this current during repolarization.     

Voltage-gated transient currents in bovine adrenal fasciculata cells. II. A-type K+ current

In whole cell patch clamp recordings on enzymatically dissociated adrenal zona fasciculata (AZF) cells, a rapidly inactivating A-type K+ current was observed in each of more than 150 cells. Activation of IA was steeply voltage dependent and could be described by a Boltzmann function raised to an integer power of 4, with a midpoint of -28.3 mV. Using the "limiting logarithmic potential sensitivity," the single channel gating charge was estimated to be 7.2 e. Voltage-dependent inactivation could also be described by a Boltzmann function with a midpoint of -58.7 mV and a slope factor of 5.92 mV. Gating kinetics of IA included both voltage-dependent and -independent transitions in pathways between closed, open, and inactivated states. IA activated with voltage-dependent sigmoidal kinetics that could be fit with an n4h formalism. The activation time constant, tau a, reached a voltage- independent minimum at potentials positive to 0 mV. IA currents inactivated with two time constants that were voltage independent at potentials ranging from -30 to +45 mV. At +20 mV, tau i(fast) and tau i(slow) were 13.16 +/- 0.64 and 62.26 +/- 5.35 ms (n = 34), respectively. In some cells, IA inactivation kinetics slowed dramatically after many minutes of whole cell recording. Once activated by depolarization, IA channels returned to the closed state along pathways with two voltage-dependent time constants which were 0.208 s, tau rec-f and 10.02 s, tau rec-s at -80 mV. Approximately 90% of IA current recovered with slow kinetics at potentials between -60 and -100 mV. IA was blocked by 4-aminopyridine (IC50 = 629 microM) through a mechanism that was strongly promoted by channel activation. Divalent and trivalent cations including Ni2+ and La3+ also blocked IA with IC50's of 467 and 26.4 microM, respectively. With respect to biophysical properties and pharmacology, IA in AZF cells resembles to some extent transient K+ currents in neurons and muscle, where they function to regulate action potential frequency and duration. The function of this prominent current in steroid hormone secretion by endocrine cells that may not generate action potentials is not yet clear.     

Photodynamic Alteration of Sodium Currents in Lobster Axons

Photodynamic alteration of lobster giant axons drastically changed the magnitude and kinetics of sodium currents seen under voltage clamp using the sucrose gap technique. Illumination of axons following treatment with acridine orange or eosin Y decreased the maximum sodium conductance to a zero asymptote as an exponential function of illumination time. Normal sodium inactivation was slowed, with τ_h more than doubled depending on experimental conditions. A second slower inactivation rate developed occasionally. τ_h was altered little, if at all. Sodium current "tails" were not prolonged. At maximum light intensity and with eosin Y as sensitizer leakage current increased after 4–10 sec in light. These changes were irreversible. Decreases in maximum sodium conductance correlated highly with increases in time to peak sodium current. The magnitude of change varied linearly with light intensity. The action spectra for eosin Y and acridine orange peaked near 545 and 505 nm, respectively. The magnitude of change varied with preillumination dye exposure time in a quasi-exponential approach to a maximum effect. Sodium dithionite protected the axon from photodynamic change.     

成年蜜蜂脑神经细胞的培养和电生理特征

为了研究杀虫剂等对蜜蜂毒性作用的神经机制,需在体外建立成年蜜蜂脑神经细胞的分离培养和电生理记录技术并研究其正常电生理特征,而对成年蜜蜂脑神经细胞的分离培养和电生理特性的研究报道甚少。我们采用酶解和机械吹打相结合的方法获得了数量较多且活力较好的成年意大利蜜蜂Apis mellifera脑神经细胞,并用全细胞膜片钳技术研究了成年意大利蜜蜂脑神经细胞对电流和电压刺激的反应,获得了成年意蜂脑神经细胞的基本电生理特征以及钠电流和钾电流的特性。全细胞电流钳的记录结果表明,在体外培养条件下,细胞无自发放电发生,注射电流后仅引起细胞单次放电,引起细胞放电的阈电流平均为60.8±63 pA; 细胞动作电位产生的阈电位平均为−27.4±2.3 mV。用全细胞电压钳记录了神经细胞的钠电流和钾电流。钠电流的分离是在电压刺激下通过阻断钾通道和钙通道实现。细胞的内向钠电流在指令电压为−40～−30 mV左右激活,−10 mV达峰值,钠通道的稳态失活电压V_1/2为−58.4 mV; 外向钾电流成份至少包括较小的快速失活钾电流和和较大的缓慢失活钾电流（占总钾电流的80%）,其半激活膜电位V_1/2为3.86 mV,无明显的稳态失活。结果提示缓慢失活钾电流的特征可能是细胞单次放电的机制之一。     

Effects of pressure overload-induced hypertrophy on TTX-sensitive inward currents in guinea pig left ventricle

We investigated the effects of pressure overload hypertrophy on inward sodium (I Na) and calcium currents (I Ca) in single left ventricular myocytes to determine whether changes in these current systems could account for the observed prolongation of the action potential. Hypertrophy was induced by pressure overload caused by banding of the abdominal aorta. Whole-cell patch clamp experiments were used to measure tetrodotoxin (TTX)-sensitive inward currents. The main findings were that I Ca density was unchanged whereas I Na density after stepping from -80 to -30 mV was decreased by 30% (-9.0 +/- 1.16 pA pF(-1) in control and -6.31 +/- 0.67 pA pF(-1) in hypertrophy, p < 0.05, n = 6). Steady-state activation/inactivation variables of I Na, determined by using double-pulse protocols, were similar in control and hypertrophied myocytes, whereas the time course of fast inactivation of I Na was slowed (p < 0.05) in hypertrophied myocytes. In addition, action potential clamp experiments were carried out in the absence and presence of TTX under conditions where only Ca2+ was likely to enter the cell via TTX-sensitive channels. We show for the first time that a TTX-sensitive inward current was present during the plateau phase of the action potential in hypertrophied but not control myocytes. The observed decrease in I Na density is likely to abbreviate rather than prolong the action potential. Delayed fast inactivation of Na+ channels was not sustained throughout the voltage pulse and may therefore merely counteract the effect of decreased I Na density so that net Na+ influx remains unaltered. Changes in the fast I Na do not therefore appear to contribute to lengthening of the action potential in this model of hypertrophy. However, the presence of a TTX-sensitive current during the plateau could potentially contribute to the prolongation of the action potential in hypertrophied cardiac muscle.     

Changes in cytosolic calcium monitored by inward currents during action potentials in guinea-pig ventricular cells

Action potentials were recorded from single cells isolated from guinea-pig ventricular muscle. Contraction was measured with an optical technique. Tail currents thought to be activated by cytosolic calcium were recorded when action potentials were interrupted by application of a voltage-clamp. A family of tail currents was recorded by interrupting the action potential at various times after the upstroke. The envelope of tail current amplitudes was taken as an index of changes in cytosolic calcium. Consistent with this interpretation, tail currents were negligible following intracellular loading with the calcium chelator BAPTA to suppress calcium transients. The cytosolic calcium transient estimated from the envelope of tails reached a peak approximately 50 ms after the upstroke of the action potential, and fell close to diastolic levels before repolarization was complete; 10 mM caffeine delayed the time to peak contraction, and caused a prolongation of the cytosolic calcium transient estimated from the envelope of tail currents. Caffeine also induced the appearance of a distinct late plateau phase of the action potential. Intracellular BAPTA suppressed the late plateau, contraction and tail currents in cells exposed to caffeine. Exposure to caffeine increased the time constant for decay of tail currents (from approximately 25 to 70 ms). When action potentials were greatly abbreviated by interruption with a voltage-clamp, a progressive decline occurred in the subsequent three contractions and tail currents. There was a progressive reversal of these effects over four responses when the full action potential duration was restored. None of these effects was observed in cells exposed to caffeine. Calcium-activated tail currents appear to be a useful qualitative index of changes in cytosolic calcium. The observations are consistent with the suggestion that cytosolic calcium is reduced during the plateau by a combination of calcium extrusion through Na-Ca exchange and calcium uptake into caffeine-sensitive stores. It also appears that reduction of stores loading during abbreviated action potentials reduces subsequent contraction in cells not exposed to caffeine.     

Serotonin decreases a background current and increases calcium and calcium-activated current in pedal neurons ofHermissenda

The effects of serotonin (5-HT) on membrane potential, membrane resistance, and select ionic currents were examined in large pedal neurons (LP1, LP3) of the mollusk Hermissenda. Calcium (Ca) action potentials were evoked in sodium-free artificial seawater containing tetramethylammonium, tetraethylammonium, and 4-aminopyridine (0-Na, 4-AP, TEA ASW). They failed at stimulation rates greater than 0.5/sec and were blocked by cadmium (Cd). Under voltage clamp the calcium current (ICa) responsible for them also failed with repeated stimulation. Thus, ICa inactivation accounts for refractoriness of the Ca action potential. The addition of 10 microM 5-HT to 0-Na, 4-AP, TEA ASW produced a slight depolarization and increased excitability and input resistance. Under voltage clamp the background current decreased. The voltage-dependent inward, late outward, and outward tail currents, sensitive to Cd, increased. ICa inactivation persisted. Under voltage clamp with Ca influx blocked by Cd, the addition of 10 microM 5-HT decreased the remaining current uniformly over membrane potentials of -10 to -100 mV. Thus, 5-HT reduces a background current that is active within the physiological range of the membrane potential, voltage insensitive, independent of Ca influx, noninactivating, and not blocked by 4-AP or TEA.     

The Mu class glutathione transferase is abundant in striated muscle and is an isoform-specific regulator of ryanodine receptor calcium channels

Members of the glutathione transferase (GST) structural family are novel regulators of cardiac ryanodine receptor (RyR) calcium channels. We present the first detailed report of the effect of endogenous muscle GST on skeletal and cardiac RyRs. An Mu class glutathione transferase is specifically expressed in human muscle. An hGSTM2-2-like protein was isolated from rabbit skeletal muscle and sheep heart, at concentrations of approximately 17-93 microM. When added to the cytoplasmic side of RyRs, hGSTM2-2 and GST isolated from skeletal or cardiac muscle, modified channel activity in an RyR isoform-specific manner. High activity skeletal RyR1 channels were inactivated at positive potentials or activated at negative potentials by hGSTM2-2 (8-30 microM). Inactivation became faster as the positive voltage was increased. Channels recovered from inactivation when the voltage was reversed, but recovery times were significantly slowed in the presence of hGSTM2-2 and muscle GSTs. Low activity RyR1 channels were activated at both potentials. In contrast, hGSTM2-2 and GSTs isolated from muscle (1-30 microM) in the cytoplasmic solution, caused a voltage-independent inhibition of cardiac RyR2 channels. The results suggest that the major GST isoform expressed in muscle regulates Ca2+ signalling in skeletal and cardiac muscle and conserves Ca2+ stores in the sarcoplasmic reticulum.     

K+ currents activated by depolarization in cardiac fibroblasts

K(+) currents expressed in freshly dispersed rat ventricular fibroblasts have been studied using whole-cell patch-clamp recordings. Depolarizing voltage steps from a holding potential of -90 mV activated time- and voltage-dependent outward currents at membrane potentials positive to approximately -30 mV. The relatively slow activation kinetics exhibited strong dependence on the membrane potential. Selected changes in extracellular K(+) concentration ([K(+)](o)) revealed that the reversal potentials of the tail currents changed as expected for a K(+) equilibrium potential. The activation and inactivation kinetics of this K(+) current, as well as its recovery from inactivation, were well-fitted by single exponential functions. The steady-state inactivation was well described by a Boltzmann function with a half-maximal inactivation potential (V(0.5)) of -24 mV. Increasing [K(+)](o) (from 5 to 100 mM) shifted this V(0.5) in the hyperpolarizing direction by -11 mV. Inactivation was slowed by increasing [K(+)](o) to 100 mM, and the rate of recovery from inactivation was decreased after increasing [K(+)](o). Block of this K(+) current by extracellular tetraethylammonium also slowed inactivation. These [K(+)](o)-induced changes and tetraethylammonium effects suggest an important role for a C-type inactivation mechanism. This K(+) current was sensitive to dendrotoxin-I (100 nM) and rTityustoxin Kalpha (50 nM).     

Properties of a nonjunctional current expressed from a rat connexin46 cDNA in Xenopus oocytes

Connexin46 (cxn46) is a gap junctional protein that was cloned from a rat lens cDNA library. Expression of cxn46 in solitary Xenopus oocytes resulted in the development of a large time- and voltage-dependent current that was not observed in noninjected control oocytes or in oocytes injected with mRNA for cxn43 or cxn32. The cxn46-induced current activated at potentials positive to -20 mV. On repolarization to -40 mV, the current deactivated over a period of several seconds. Removal of external calcium caused a marked increase in the amplitude of the cxn46-induced current, shifted the steady-state activation curve to more negative potentials, and altered the kinetics of activation and deactivation. Increasing external calcium had the opposite effect. The ability of cxn46 to induce the formation of cell-to-cell channels was tested in the oocyte pair system. Oocyte pairs injected with cxn46 mRNA + antisense oligonucleotides for Xenopus cxn38 were strongly coupled. In contrast, oocyte pairs injected with antisense alone showed no coupling. The inactivation kinetics of the gap junctional channels resembled the deactivation kinetics of the cxn46-induced current in solitary oocytes.     

Activation and inactivation kinetics of an E-4031-sensitive current from single ferret atrial myocytes.

Ferret atrial myocytes can display an E-4031-sensitive current (IKr) that is similar to that previously described for guinea pig cardiac myocytes. We examined the ferret atrial IKr as the E-4031-sensitive component of current using the amphotericin B perforated patch-clamp technique. Steady-state IKr during depolarizing pulses showed characteristic inward rectification. Activation time constants during a single pulse were voltage dependent, consistent with previous studies. However, for potentials positive to +30 mV, IKr time course became complex and included a brief transient component. We examined the envelope of tails of the drug-sensitive current for activation in the range -10 to +50 mV and found that the tail currents for IKr do not activate with the same time course as the current during the depolarizing pulse. The activation time course determined from tail currents was relatively voltage insensitive over the range +30 to +50 mV (n = 5), but was voltage sensitive for potentials between -10 and +30 mV and appeared to show some sigmoidicity in this range. These data indicate that activation of IKr occurs in at least two steps, one voltage sensitive and one voltage insensitive, the latter of which becomes rate limiting at positive potentials. We also examined the rapid time-dependent inactivation process that mediates rectification at positive potentials. The time constants for this process were only weakly voltage dependent over the range of potentials from -50 to +60 mV. From these data we constructed a simple linear four-state model that reproduces the general features of ferret IKr, including the initial transient at positive potentials and the apparent discrepancy between the currents during the initial depolarizing pulse and the tail current.     

Omega-conotoxin blockade of calcium currents in cultured neonatal rat cardiomyocytes: different action on EGTA-modified calcium channels

Calcium currents from neonatal rat ventricular heart muscle cells grown in primary culture were examined using the "whole-cell" voltage clamp technique. An inward current characterized by large amplitude and slow inactivation decay was induced when the extracellular Ca2+ concentration was reduced by EGTA. This current was suppressed by extracellular Na+ removal, or by calcium antagonists, and increased by epinephrine and BAY K 8644. These findings suggest that this current is carried by sodium ions through Ca channels. Both Ca and Na currents through calcium channels were irreversibly blocked by omega-conotoxin. Complete blockade developed 10-15 minutes after the toxin introduction in the extracellular solution. Blockade of Na currents through calcium channels was characterized by a transient increase of current amplitude without any changes in its kinetics and voltage-dependent properties. Structural differences between calcium channels in rat and guinea-pig and frog cardiomyocytes were suggested.     

Dynamic Coupling of Voltage Sensor and Gate Involved in Closed-State Inactivation of Kv4.2 Channels

Voltage-gated potassium channels related to the Shal gene of Drosophila (Kv4 channels) mediate a subthreshold-activating current (I_SA) that controls dendritic excitation and the backpropagation of action potentials in neurons. Kv4 channels also exhibit a prominent low voltage–induced closed-state inactivation, but the underlying molecular mechanism is poorly understood. Here, we examined a structural model in which dynamic coupling between the voltage sensors and the cytoplasmic gate underlies inactivation in Kv4.2 channels. We performed an alanine-scanning mutagenesis in the S4-S5 linker, the initial part of S5, and the distal part of S6 and functionally characterized the mutants under two-electrode voltage clamp in Xenopus oocytes. In a large fraction of the mutants (>80%) normal channel function was preserved, but the mutations influenced the likelihood of the channel to enter the closed-inactivated state. Depending on the site of mutation, low-voltage inactivation kinetics were slowed or accelerated, and the voltage dependence of steady-state inactivation was shifted positive or negative. Still, in some mutants these inactivation parameters remained unaffected. Double mutant cycle analysis based on kinetic and steady-state parameters of low-voltage inactivation revealed that residues known to be critical for voltage-dependent gate opening, including Glu 323 and Val 404, are also critical for Kv4.2 closed-state inactivation. Selective redox modulation of corresponding double-cysteine mutants supported the idea that these residues are involved in a dynamic coupling, which mediates both transient activation and closed-state inactivation in Kv4.2 channels.     

Characterization of two distinct depolarization-activated K+ currents in isolated adult rat ventricular myocytes

Depolarization-activated outward K+ currents in isolated adult rat ventricular myocytes were characterized using the whole-cell variation of the patch-clamp recording technique. During brief depolarizations to potentials positive to -40 mV, Ca(2+)-independent outward K+ currents in these cells rise to a transient peak, followed by a slower decay to an apparent plateau. The analyses completed here reveal that the observed outward current waveforms result from the activation of two kinetically distinct voltage-dependent K+ currents: one that activates and inactivates rapidly, and one that activates and inactivates slowly, on membrane depolarization. These currents are referred to here as Ito (transient outward) and IK (delayed rectifier), respectively, because their properties are similar (although not identical) to these K+ current types in other cells. Although the voltage dependences of Ito and IK activation are similar, Ito activates approximately 10-fold and inactivates approximately 30-fold more rapidly than IK at all test potentials. In the composite current waveforms measured during brief depolarizations, therefore, the peak current predominantly reflects Ito, whereas IK is the primary determinant of the plateau. There are also marked differences in the voltage dependences of steady-state inactivation of these two K+ currents: IK undergoes steady-state inactivation at all potentials positive to -120 mV, and is 50% inactivated at -69 mV; Ito, in contrast, is insensitive to steady-state inactivation at membrane potentials negative to -50 mV. In addition, Ito recovers from steady-state inactivation faster than IK: at -90 mV, for example, approximately 70% recovery from the inactivation produced at -20 mV is observed within 20 ms for Ito; IK recovers approximately 25-fold more slowly. The pharmacological properties of Ito and IK are also distinct: 4-aminopyridine preferentially attenuates Ito, and tetraethylammonium suppresses predominantly IK. The voltage- and time-dependent properties of these currents are interpreted here in terms of a model in which Ito underlies the initial, rapid repolarization phase of the action potential (AP), and IK is responsible for the slower phase of AP repolarization back to the resting membrane potential, in adult rat ventricular myocytes.     

Effect of sea anemone toxins on the sodium inactivation process in crayfish axons

The effect of sea anemone toxins from Parasicyonis actinostoloides and Anemonia sulcata on the Na conductance in crayfish giant axons was studied under voltage-clamp conditions. The toxin slowed the Na inactivation process without changing the kinetics of Na activation or K activation in an early stage of the toxin effect. An analysis of the Na current profile during the toxin treatment suggested an all-or-none modification of individual Na channels. Toxin-modified Na channels were partially inactivated with a slower time course than that of the normal inactivation. This slow inactivation in steady state decreased in its extent as the membrane was depolarized to above -45 mV, so that practically no inactivation occurred at the membrane potentials as high as +50 mV. In addition to inhibition of the normal Na inactivation, prolonged toxin treatment induced an anomalous closing in a certain population of Na channels, indicated by very slow components of the Na tail current. The observed kinetic natures of toxin-modified Na channels were interpreted based on a simple scheme which comprised interconversions between functional states of Na channels. The voltage dependence of Parasicyonis toxin action, in which depolarization caused a suppression in development of the toxin effect, was also investigated.     

Asymmetric modulation and blockade of the delayed rectifier in squid giant axons by divalent cations.

The effects of intracellular magnesium ions and extracellular calcium and magnesium ions on the delayed rectifier potassium ion channel, IK, were investigated from intracellularly perfused squid giant axons. Cao+2 and Mgo+2 both blocked IK in a voltage-independent manner with a KD of approximately 100 and 500 mM, respectively. This effect was obscured at potentials in the vicinity of the resting potential (approximately -60 mV) by a rightward shift of the steady-state IK inactivation curve along the voltage axis. The addition of either calcium or magnesium ions to the extracellular solution also produced the well known shift of the IK activation curve along the voltage axis. Cao+2 was approximately twice as effective in this regard as Mgo+2. The IK activation kinetics were slowed by Cao+2, but deactivation kinetics were not altered, as shown previously. Similar results were obtained with Mgo+2. The addition of magnesium ions to the intracellular perfusate shifted the activation curve along the voltage axis in the negative direction (without producing block) by approximately the same among as the Mgo+2 shift of this curve in the positive direction. Moreover, Mgi+2 substantially slowed the deactivation kinetics, whereas the effects of Mgi+2 on activation kinetics at strongly depolarized potentials were relatively minor. At modest depolarizations, Mgi+2 significantly reduced the delay before IK activation. These results are essentially the mirror image of the effects on gating of extracellular divalent cations.