Sodium and calcium currents in dispersed mammalian septal neurons

Voltage-gated Na+ and Ca2+ conductances of freshly dissociated septal neurons were studied in the whole-cell configuration of the patch-clamp technique. All cells exhibited a large Na+ current with characteristic fast activation and inactivation time courses. Half-time to peak current at -20 mV was 0.44 +/- 0.18 ms and maximal activation of Na+ conductance occurred at 0 mV or more positive membrane potentials. The average value was 91 +/- 32 nS (approximately 11 mS cm-2). At all membrane voltages inactivation was well fitted by a single exponential that had a time constant of 0.44 +/- 0.09 ms at 0 mV. Recovery from inactivation was complete in approximately 900 ms at -80 mV but in only 50 ms at -120 mV. The decay of Na+ tail currents had a single time constant that at -80 mV was faster than 100 microseconds. Depolarization of septal neurons also elicited a Ca2+ current that peaked in approximately 6-8 ms. Maximal peak Ca2+ current was obtained at 20 mV, and with 10 mM external Ca2+ the amplitude was 0.35 +/- 0.22 nA. During a maintained depolarization this current partially inactivated in the course of 200-300 ms. The Ca2+ current was due to the activity of two types of conductances with different deactivation kinetics. At -80 mV the closing time constants of slow (SD) and fast (FD) deactivating channels were, respectively, 1.99 +/- 0.2 and 0.11 +/- 0.03 ms (25 degrees C). The two kinds of channels also differed in their activation voltage, inactivation time course, slope of the conductance-voltage curve, and resistance to intracellular dialysis. The proportion of SD and FD channels varied from cell to cell, which may explain the differential electrophysiological responses of intracellularly recorded septal neurons.     

Voltage-dependent and odorant-regulated currents in isolated olfactory receptor neurons of the channel catfish.

The electrical properties of olfactory receptor neurons, enzymatically dissociated from the channel catfish (Ictalurus punctatus), were studied using the whole-cell patch-clamp technique. Six voltage-dependent ionic currents were isolated. Transient inward currents (0.1-1.7 nA) were observed in response to depolarizing voltage steps from a holding potential of -80 mV in all neurons examined. They activated between -70 and -50 mV and were blocked by addition of 1 microM tetrodotoxin (TTX) to the bath or by replacing Na+ in the bath with N-methyl-D-glucamine and were classified as Na+ currents. Sustained inward currents, observed in most neurons examined when Na+ inward currents were blocked with TTX and outward currents were blocked by replacing K+ in the pipette solution with Cs+ and by addition of 10 mM Ba2+ to the bath, activated between -40 and -30 mV, reached a peak at 0 mV, and were blocked by 5 microM nimodipine. These currents were classified as L-type Ca2+ currents. Large, slowly activating outward currents that were blocked by simultaneous replacement of K+ in the pipette with Cs+ and addition of Ba2+ to the bath were observed in all olfactory neurons examined. The outward K+ currents activated over approximately the same range as the Na+ currents (-60 to -50 mV), but the Na+ currents were larger at the normal resting potential of the neurons (-45 +/- 11 mV, mean +/- SD, n = 52). Four different types of K+ currents could be differentiated: a Ca(2+)-activated K+ current, a transient K+ current, a delayed rectifier K+ current, and an inward rectifier K+ current. Spontaneous action potentials of varying amplitude were sometimes observed in the cell-attached recording configuration. Action potentials were not observed in whole-cell recordings with normal internal solution (K+ = 100 mM) in the pipette, but frequently appeared when K+ was reduced to 85 mM. These observations suggest that the membrane potential and action potential amplitude of catfish olfactory neurons are significantly affected by the activity of single channels due to the high input resistance (6.6 +/- 5.2 G omega, n = 20) and low membrane capacitance (2.1 +/- 1.1 pF, n = 46) of the cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

SEA0400, a novel Na+/Ca2+ exchanger inhibitor, reduces calcium overload induced by ischemia and reperfusion in mouse ventricular myocytes

Given the potential clinical benefit of inhibiting Na+/Ca2+ exchanger (NCX) activity during myocardial ischemia reperfusion (I/R), pharmacological approaches have been pursued to both inhibit and clarify the importance of this exchanger. SEA0400 was reported to have a potent NCX selectivity. Thus, we examined the effect of SEA0400 on NCX currents and I/R induced intracellular Ca2+ overload in mouse ventricular myocytes using patch clamp techniques and fluorescence measurements. Ischemia significantly inhibited inward and outward NCX current (from -0.04+/-0.01 nA to 0 nA at -100 mV; from 0.23+/-0.08 nA to 0.11+/-0.03 nA at +50 mV, n=7), Subsequent reperfusion not only restored the current rapidly but enhanced the current amplitude obviously, especially the outward currents (from 0.23+/-0.08 nA to 0.49+/-0.12 nA at +50 mV, n=7). [Ca2+]i, expressed as the ratio of Fura-2 fluorescence intensity, increased to 138+/-7% (P<0.01) during ischemia and to 210+/-11% (P<0.01) after reperfusion. The change of NCX current and the increase of [Ca2+]i during I/R can be blocked by SEA0400 in a dose-dependent manner with an EC50 value of 31 nM and 28 nM for the inward and outward NCX current, respectively. The results suggested that SEA0400 is a potent NCX inhibitor, which can protect mouse cardiac myocytes from Ca2+ overload during I/R injuries.     

A voltage-dependent persistent sodium current in mammalian hippocampal neurons

Currents generated by depolarizing voltage pulses were recorded in neurons from the pyramidal cell layer of the CA1 region of rat or guinea pig hippocampus with single electrode voltage-clamp or tight-seal whole-cell voltage-clamp techniques. In neurons in situ in slices, and in dissociated neurons, subtraction of currents generated by identical depolarizing voltage pulses before and after exposure to tetrodotoxin revealed a small, persistent current after the transient current. These currents could also be recorded directly in dissociated neurons in which other ionic currents were effectively suppressed. It was concluded that the persistent current was carried by sodium ions because it was blocked by TTX, decreased in amplitude when extracellular sodium concentration was reduced, and was not blocked by cadmium. The amplitude of the persistent sodium current varied with clamp potential, being detectable at potentials as negative as -70 mV and reaching a maximum at approximately -40 mV. The maximum amplitude at -40 mV in 21 cells in slices was -0.34 +/- 0.05 nA (mean +/- 1 SEM) and -0.21 +/- 0.05 nA in 10 dissociated neurons. Persistent sodium conductance increased sigmoidally with a potential between -70 and -30 mV and could be fitted with the Boltzmann equation, g = gmax/(1 + exp[(V' - V)/k)]). The average gmax was 7.8 +/- 1.1 nS in the 21 neurons in slices and 4.4 +/- 1.6 nS in the 10 dissociated cells that had lost their processes indicating that the channels responsible are probably most densely aggregated on or close to the soma. The half-maximum conductance occurred close to -50 mV, both in neurons in slices and in dissociated neurons, and the slope factor (k) was 5-9 mV. The persistent sodium current was much more resistant to inactivation by depolarization than the transient current and could be recorded at greater than 50% of its normal amplitude when the transient current was completely inactivated. Because the persistent sodium current activates at potentials close to the resting membrane potential and is very resistant to inactivation, it probably plays an important role in the repetitive firing of action potentials caused by prolonged depolarizations such as those that occur during barrages of synaptic inputs into these cells.     

Developmental changes in time course of recovery from inactivation in L-type calcium currents of rabbit ventricular myocytes

The mechanisms of recovery from inactivation of the L-type calcium current (I(Ca)) are not well established, and recovery is affected by many experimental conditions. Little is known about developmental changes of recovery from inactivation of I(Ca). We studied developmental changes of recovery from inactivation in I(Ca) using isolated adult and newborn (1-4 days) rabbit ventricular myocytes. We used broken-patch and perforated-patch techniques with physiological extracellular ionic concentrations of calcium and sodium and interpulse conditioning potentials of -80 or -50 mV. We also maximized I(Ca) with forskolin. We found that recovery from inactivation did not differ between adult and newborn cells when either EGTA or BAPTA was used to buffer intracellular calcium. Maximizing I(Ca) with forskolin slowed recovery from inactivation in newborn but not in adult cells. In contrast, when the intracellular buffering of the cell was left nearly intact (perforated patch), recovery from inactivation (half-time of recovery) in the newborn cells was significantly slower than for the adult cells when either a conditioning potential of -80 mV (140 +/- 9 vs. 58 +/- 4 ms, newborn vs. adult; P < 0.05) or -50 mV (641 +/- 106 vs. 168 +/- 15 ms, newborn vs. adult; P < 0.05) was used. Forskolin significantly increased half-time of recovery for both adult and newborn cells. Dialysis with no calcium buffer showed a slower recovery from inactivation in newborn cells. Intracellular dialysis with a calcium buffer masked differences in recovery from inactivation of I(Ca) between newborn and adult rabbit ventricular cells.     

Two distinct modulatory effects on calcium channels in adult rat sensory neurons.

D-ala2-D-leu5-enkephalin (100 to 1000 nM) reduces HVA Ca2+ currents of approximately 60% in 92% of the adult rat sensory neurons tested. In 80% of the cells sensitive to enkephalin, the reduction in Ca2+ current amplitude was associated with a prolongation of the current activation that was relieved by means of conditioning pulses in a potential range only about 10 mV positive to the current activation range in control conditions. The time course of the current activation was fitted to a single exponential in control, (tau = 2.23 msec +/- 0.14 n = 38) and double exponential with enkephalin, (tau 1 = 2.18 msec +/- 0.25 and tau 2 = 9.6 msec +/- 1, test pulse to -10 mV, 22 degrees C). A strong conditioning depolarizing prepulse speeded up the activation time course, completely eliminating the slow, voltage-sensitive exponential component, but it was only partial effective in restoring the current amplitude to control values. The voltage-independent inhibitory component that was not relieved could be recovered only by washing out enkephalin. In the remaining 20% of the cells affected, enkephalin decreased Ca2+ current amplitude without prolongation of Ca2+ channel activation. In these cases the conditioning voltage pulse was not effective in relieving the inhibition that persisted also at strong positive test potentials, on the outward currents. The voltage-dependent inhibition occurred slowly after enkephalin superfusion (tau congruent to 12 sec), whereas the voltage-independent one developed about ten times more rapidly. Dopamine (100 microM) could also induce both voltage-dependent and independent modulations.(ABSTRACT TRUNCATED AT 250 WORDS)     

L-type but not T-type calcium current changes during postnatal development in rabbit sinoatrial node

Although the neonatal sinus node beats at a faster rate than the adult, when a sodium current (I(Na)) present in the newborn is blocked, the spontaneous rate is slower in neonatal myocytes than in adult myocytes. This suggests a possible functional substitution of I(Na) by another current during development. We used ruptured [T-type calcium current (I(Ca,T))] and perforated [L-type calcium current (I(Ca,L))] patch clamps to study developmental changes in calcium currents in sinus node cells from adult and newborn rabbits. I(Ca,T) density did not differ with age, and no significant differences were found in the voltage dependence of activation or inactivation. I(Ca,L) density was lower in the adult than newborn (12.1 +/- 1.4 vs. 17.6 +/- 2.5 pA/pF, P = 0.049). However, activation and inactivation midpoints were shifted in opposite directions, reducing the potential contribution during late diastolic depolarization in the newborn (activation midpoints -17.3 +/- 0.8 and -22.3 +/- 1.4 mV in the newborn and adult, respectively, P = 0.001; inactivation midpoints -33.4 +/- 1.4 and -28.3 +/- 1.7 mV for the newborn and adult, respectively, P = 0.038). Recovery of I(Ca,L) from inactivation was also slower in the newborn. The results suggest that a smaller but more negatively activating and rapidly recovering I(Ca,L) in the adult sinus node may contribute to the enhanced impulse initiation at this age in the absence of I(Na).     

L-type Ca2+ currents in ventricular myocytes from neonatal and adult rats

Postnatal changes in the slow Ca2+ current (I(Ca)(L)) were investigated in freshly isolated ventricular myocytes from neonatal (1-7 days old) and adult (2-4 months old) rats, using whole-cell voltage clamp and single-channel recordings. The membrane capacitance (mean+/-SEM) averaged 23.2+/-0.5 pF in neonates (n = 163) and 140+/-4.1 pF in adults (n = 143). I(Ca)(L) was measured as the peak inward current at a test potential of +10 mV (or +20 mV) by applying a 300-ms pulse from a holding potential of -40 mV; 1.8 mM Ca2+ was used as charge carrier. The basal ICa(L) density was 6.7+/-0.2 pA/pF in neonatal and 7.8+/-0.2 pA/pF in adult cells (p < 0.05). The time course of inactivation of the fast component (at +10 ms) was significantly longer in the neonatal (10.7+/-1.4 ms) than in the adult (6.6+/-0.4 ms) cells (p < 0.05). Ryanodine (10+/-M) significantly increased this value to 18.0+/-1.9 in neonate (n = 8) and to 17.7+/-2.0 in adult (n = 9). For steady-state inactivation, the half-inactivation potential (Vh) was not changed in either group. For steady-state activation, Vh was 5.1 mV in the neonatal (n = 6) and -7.9 mV in the adult cells (n = 7). Single-channel recordings revealed that long openings (mode-2 behavior) were occasionally observed in the neonatal cells (11 events from 1080 traces/11 cells), but not in the adult cells (400 traces/4 cells). Slope conductance was 24 pS in both the neonatal and adult cells. Results in rat ventricular myocytes suggest the following: (i) the peak Ca2+ current density is already well developed in the neonatal period (being about 85% of the adult value); (ii) the fast component of inactivation is slower in neonates than in adults; and (iii) naturally occurring long openings are occasionally observed in the neonatal stage but not in the adult. Thus, the L-type Ca2+ channels of the neonate were slightly lower in density, were inactivated more slowly, and occasionally exhibited mode-2 behavior as compared with those of the adult.     

The Resting Potential of Mouse Leydig Cells: Role of an Electrogenic Na+/K+ Pump

Resting potentials (Vm) were measured in mouse Leydig cells, using the whole-cell patch-clamp technique. In contrast to conventional microelectrode measurements, where a biphasic potential was observed, we recorded a stable Vm around -32.2 +/- 1.2 mV (mean +/- SEM, n = 159), at 25 degrees C, and an input resistance larger than 2.7 x 109 W. Although Vm is sensitive to changes in the extracellular concentrations of potassium and chloride, the relationship between Vm and these ions' concentrations cannot be described by either the Goldman-Hodgkin-Katz or the Nernst equation. Perifusing cells with potassium-free solution or 10?3 M ouabain induced a marked depolarization averaging 20.1 +/- 3.2 mV (n = 9) and 23.1 +/- 2.8 mV, (n = 7), respectively. Removal of potassium or addition of ouabain with the cell voltage-clamped at its Vm, resulted in an inwardly directed current, due to inhibition of the Na+K+ATPase. The pump current increased with temperature with a Q10 coefficient of 2.3 and had an average value of -6.5 +/- 0.4 pA (n = 21) at 25 degrees C. Vm also varied strongly with temperature, reaching values as low as -9.2 +/- 1.2 mV (n = 22) at 15 degrees C. Taking the pump current at 25 degrees C and a minimum estimate for the membrane input resistance, we can see that the Na+K+ATPase could directly contribute with 17.7 mV to the Vm of Leydig cells, which is a major fraction of the ?32.2 +/- 1.2 mV (n = 159) observed.     

T-type Ca2+ channel lowers the threshold of spike generation in the newt olfactory receptor cell

Mechanisms underlying action potential generation in the newt olfactory receptor cell were investigated by using the whole-cell version of the patch-clamp technique. Isolated olfactory cells had a resting membrane potential of -70 +/- 9 mV. Injection of a depolarizing current step triggered action potentials under current clamp condition. The amplitude of the action potential was reduced by lowering external Na+ concentration. After a complete removal of Na+, however, cells still showed action potentials which was abolished either by Ca2+ removal or by an application of Ca2+ channel blocker (Co2+ or Ni2+), indicating an involvement of Ca2+ current in spike generation of newt olfactory receptor cells. Under the voltage clamp condition, depolarization of the cell to -40 mV from the holding voltage of -100 mV induced a fast transient inward current, which consisted of Na+ (INa) and T-type Ca2+ (ICa.T) currents. The amplitude of ICa,T was about one fourth of that of INa. Depolarization to more positive voltages also induced L-type Ca2+ current (ICa,L). ICa,L was as small as a few pA in normal Ringer solution. The activating voltage of ICa,T was approximately 10 mV more negative than that of INa. Under current clamp, action potentials generated by a least effective depolarization was almost completely blocked by 0.1 mM Ni2+ (a specific T-type Ca2+ channel blocker) even in the presence of Na+. These results suggest that ICa,T contributes to action potential in the newt olfactory receptor cell and lowers the threshold of spike generation.     

Electrical activity in white adipose tissue of rat

Intracellular recording of white adipocytes was performed in an in vitro preparation. Resting potential, input resistance and membrane time constant averaged: -34 +/- 9 mV, 295 +/- 161 M omega, and 58 +/- 19 ms respectively (mean +/- SD, n = 32). Intracellular injection of positive and negative square current pulses elicited membrane voltage responses, characterized by a rectification of the voltage change evoked by positive pulses, and a slow return to baseline at the offset of hyperpolarizing pulses. The amplitude and duration of the slow return to resting potential was dependent on membrane potential, pulse duration, and extracellular K+ concentration. This response was depressed when external Ca2+ was replaced by Co2+, and by external application of 4-aminopyridine. These results indicate that white adipocytes can generate membrane voltage responses which may mostly be a consequence of the activity of ionic channels. The properties of the slow return to baseline suggest that it may be due to a transient K+ current.     

Electrical properties of neurons recorded from the rat supraoptic nucleus in vitro

The electrical properties of neurons in the supraoptic nucleus (so.n.) have been studied in the hypothalamic slice preparation by intracellular and extracellular recording techniques, with Lucifer Yellow CH dye injection to mark the recording site as being the so.n. Intracellular recordings from so.n. neurons revealed them to have an average membrane potential of -67 +/- 0.8 mV (mean +/- s.e.m.), membrane resistance of 145 +/- 9 M omega with linear current-voltage relations from 40 mV in the hyperpolarizing direction to the level of spike threshold in the depolarizing direction. Average cell time constant was 14 +/- 2.2 ms. So.n. action potentials ranged in amplitude from 55 to 95 mV, with a mean of 76 +/- 2 mV, and a spike width of 2.6 +/- 0.5 ms at 30% of maximal spike height. Both single spikes and trains of spikes were followed by a strong, long-lasting hyperpolarization with a decay fitted by a single exponential having a time constant of 8.6 +/- 1.8 ms. Action potentials could be blocked by 10(-6) M tetrodotoxin. Spontaneously active so.n. neurons were characterized by synaptic input in the form of excitatory and inhibitory postsynaptic potentials, the latter being apparently blocked when 4 M KCl electrodes were used. Both forms of synaptic activity were blocked by application of divalent cations such as Mg2+, Mn2+ or Co2+. 74% of so.n. neurons fired spontaneously at rates exceeding 0.1 spikes per second, with a mean for all cells of 2.9 +/- 0.2 s-1. Of these cells, 21% fired slowly and continuously at 0.1 - 1.0 s-1, 45% fired continuously at greater than 1 Hz, and the remaining 34% fired phasically in bursts of activity followed by silence or low frequency firing. Spontaneously firing phasic cells showed a mean burst length of 16.7 +/- 4.5 s and a silent period of 28.2 +/- 4.2 s. Intracellular recordings revealed the presence of slow variations in membrane potential which modified the neuron's proximity to spike threshold, and controlled phasic firing. Variations in synaptic input were not observed to influence firing in phasic cells.     

Voltage-activation of high-conductance K+ channel in the insulin-secreting cell line RINm5F is dependent on local extracellular Ca2+ concentration

Patch-clamp single-channel current recording experiments have been carried out on intact insulin-secreting RINm5F cells. Voltage-activation of high-conductance K+ channels were studied by selectively depolarizing the electrically isolated patch membrane under conditions with normal Ca2+ concentration in the bath solution but with or without Ca2+ in the patch pipette solution. When Ca2+ was present in the pipette, 40 mV to 120 mV depolarizing pulses (100 ms) from the normal resting potential (-70 mV) regularly evoked tetraethylammonium-sensitive large outward single-channel currents and the average open state probability during the pulses varied from about 0.015 (40 mV pulses) to 0.1 (120 mV pulses). In the absence of Ca2+ in the pipette solution the same protocol resulted in fewer and shorter K+ channel openings and the open-state probability varied from about 0.0015 (40 mV pulses) to about 0.03 (120 mV pulses). It is concluded that Ca2+ entering voltage-gated channels raises [Ca2+]i locally and thereby markedly enhances the open-state probability of tetraethylammonium-sensitive voltage-gated high-conductance K+ channels.     

A low voltage-activated calcium conductance in embryonic chick sensory neurons.

Isolated Ca currents in cultured dorsal root ganglion (DRG) cells were studied using the patch clamp technique. The currents persisted in the presence of 30 microM tetrodotoxin (TTX) or when external Na was replaced by choline. They were fully blocked by millimolar additions of Cd2+ and Ni2+ to the bath. Two components of an inward-going Ca current were observed. In 5 mM external Ca, a current of small amplitude, turned on already during steps changes to -60 mV membrane potential, leveled off at -30 mV to a value of approximately 0.2 nA. A second, larger current component, which resembled the previously described Ca current in other cells, appeared at more positive voltages (-20 to -10 mV) and had a maximum approximately 0 mV. The current component activated at the more negative membrane potentials showed the stronger dependence on external Ca. The presence of a time- and a voltage-dependent activation was indicated by the current's sigmoidal rise, which became faster with increased depolarization. Its tail currents were generally slower than those associated with the Ca currents of larger amplitude. From -60 mV holding potential, the maximum obtainable amplitude of the low depolarization-activated current was only one-tenth of that achieved from a holding potential of -90 mV. Voltage-dependent inactivation of this current component was fast compared with that of the other component. The properties of this low voltage-activated and fully inactivating Ca current suggest it is the same as the inward current that has been postulated in several central neurons (Llinas, R., and Y. Yarom, 1981, J. Physiol. (Lond.), 315:569-584), which produce depolarizing potential waves and burst-firing only when membrane hyperpolarization precedes.     

大鼠背根节神经元后超极化中Ca^2＋激活K^＋电流的蜂毒明肽敏感成分 …

为了明确大鼠背根节(DRG)神经元中存在慢的Ca2+激活K+电流成分,本实验在新鲜分散的DRG神经元胞体上,采用全细胞电压箝技术,给予DRG神经元一定强度的去极化刺激,记录刺激结束后30 ms时的尾电流幅度.结果发现:(1)随着去极化时间从1 ms延长至180 ms时,尾电流幅度由9.3±2.8 pA逐渐增大至64.1±3.4 pA(P＜0.001);(2)当去极化结束后的复极化电位降低时,尾电流幅度先逐渐下降到零,然后改变方向,逆转电位约为-63 mV;(3)细胞外施加500μmol/L Cd2+或细胞内液中施加11 mmol/L EGYA时尾电流明显减小甚至完全消失;(4)尾电流中慢成分的幅度在细胞外给与200 nmol/L蜂毒明肽后,减小了约26.32±3.9%(P＜0.01);(5)细胞外施加10 mmol/L TEA,可明显降低尾电流中的快成分.结果提示,在DRG神经元后超极化中存在Ca2+激活K+电流的蜂毒明肽敏感成分--ⅠAiHP.     

Transcellular ionic currents studied by intracellular potential recordings in Neurospora crassa hyphae. Transfer of energy from proximal to apical cells

Membrane potentials, input resistances, and electric coupling in the apical parts of N. crassa growing hyphae were recorded with the aid of intracellular microelectrodes. It was revealed that the apical cells were always depolarized by 10 to 30 mV as compared to the adjacent proximal cells. The septal pore maintained an electrical resistance of 4 to 6 M omega. The calculated values of the endogenous electrical current passing through the septal pore varied between 0.5 and 1 nA. Electrical isolation of the apical cells resulted in their depolarization from 120-150 mV to 40-60 mV, characteristics of the membrane potential value of N. crassa adult hyphae with completely blocked electrogenic pumps. A simultaneous increase in the input resistance value from 15-20 M omega to 40-80 M omega was observed. The above data can be explained assuming that H+-ATPase activity was greatly lowered in the apical cells. Thus in the intact hyphae with electrically coupled cells energy is transferred from the proximal hyphal compartments to the apical ones.     

Single K+ channels in endocrine cells dispersed from the cricket (Gryllus bimaculatus) corpora allata

Single channel currents were recorded from cell-attached patches of endocrine cells of the adult male cricket corpora allata. Three distinct types of K+ channels were identified; a weak inward rectifier (Type 1), a strong inward rectifier (Type 2) and a weak outward rectifier (Type 3). The type 1 channel had a slope conductance of 191 +/- 9 pS (n = 4) at negative membrane potentials (Vm) and 101 +/- 6 pS (n = 6) at positive Vm. In addition, the channel showed fast open-closed kinetics at negative Vm and slow open-closed kinetics at positive Vm. The open probability (Po) of this channel was strongly voltage-dependent at positive Vm, but less voltage-dependent at negative Vm. The reversal potential was not modified significantly by the substitution of gluconate for external Cl- but was modified after N-methyl-D-glucamine (NMDG+) was substituted for external K+, according to the Nernst equation for a K+-selective channel. The type 2 channel had a slope conductance of 44 +/- 2 pS (n = 5) at negative Vm, but no detectable outward current was observed at positive Vm. This channel showed very slow open-closed kinetics at negative Vm and its Po was not voltage-dependent. The type 3 channel had a limit conductance of 55 +/- 12 pS (n = 3) at negative Vm and 88 +/- 10 pS (n = 3) at positive Vm. This channel showed slow open-closed kinetics at negative Vm and fast open-closed kinetics at positive Vm. The Po for the channel was voltage-dependent at positive Vm but was voltage-independent at negative Vm. These three types of K+ channels may be important for the control of the resting membrane potential, and may thus participate in the regulation of Ca2+ influx and juvenile hormone secretion in corpora allata cells.     

Voltage-activated currents in guinea pig pancreatic alpha 2 cells. Evidence for Ca2+-dependent action potentials

Glucagon-secreting alpha 2 cells were isolated from guinea pig pancreatic islets and used for electrophysiological studies of voltage- activated ionic conductances using the patch-clamp technique. The alpha 2 cells differed from beta cells in producing action potentials in the absence of glucose. The frequency of these potentials increased after addition of 10 mM arginine but remained unaffected in the presence of 5- 20 mM glucose. When studying the conductances underlying the action potentials, we identified a delayed rectifying K+ current, an Na+ current, and a Ca2+ current. The K+ current activated above -20 mV and then increased with the applied voltage. The Na+ current developed at potentials above -50 mV and reached a maximal peak amplitude of 550 pA during depolarizing pulses to -15 mV. The Na+ current inactivated rapidly (tau h approximately 0.7 ms at 0 mV). Half-maximal steady state inactivation was attained at -58 mV, and currents could no longer be elicited after conditioning pulses to potentials above -40 mV. The Ca2+ current first became detectable at -50 mV and reached a maximal amplitude of 90 pA (in extracellular [Ca2+] = 2.6 mM) at about -10 mV. Unlike the Na+ current, it inactivated little or not at all. Membrane potential measurements demonstrated that both the Ca2+ and Na+ currents contribute to the generation of the action potential. Whereas there was an absolute requirement of extracellular Ca2+ for action potentials to be elicited at all, suppression of the much larger Na+ current only reduced the upstroke velocity of the spikes. It is suggested that this behavior reflects the participation of a low-threshold Ca2+ conductance in the pacemaking of alpha 2 cells.     

Action of tetanus toxin on cholinergic neuroblastoma X glioma hybrid cells: selective blockade of Ca spikes

We examined the effect of tetanus toxin on clonal neuroblastoma X glioma hybrid cells, NG108-15, by intracellular microelectrode studies of passive membrane electrical properties and action potentials generated under various conditions. Binding of tetanus toxin to the surface of the cells was demonstrated by indirect immunofluorescent staining but no morphological alteration was observed in tetanus toxin-treated cells under a phase contrast microscope. These is no significant difference between the tetanus toxin-treated and untreated cells in their passive electrical membrane properties, i.e. resting membrane potentials, input resistances, time constants and input capacities. Cells in 120 mM Na+, 2 mM Ca2+ salt solution showed Na spikes, and cells in high Ca2+ (30 mM), Na+-free salt solution showed Ca spikes in response to depolarizing current pulses. While the Na spike was not affected by tetanus toxin, the Ca spike was blocked by the toxin. The minimum dose of tetanus toxin for maximum suppression of the peak potential level of the Ca spike was 250 ng/ml. Addition of tetraethyl ammonium (TEA) to extracellular fluid enhanced the Ca spike in untreated cells. In toxin-treated cells, TEA did not alter the effect of tetanus toxin on the Ca spike. Blockade of the Ca spike by tetanus toxin could be detected even at low extracellular Ca2+ concentration (10 mM) by adding TEA to the extracellular fluid and adjusting the membrane potential to a steady hyperpolarized level (-80 mV) to ensure optimal and uniform electrical responses. The usefulness of NG108-15 hybrid cells for in vitro investigations on the mechanism of action of tetanus toxin was discussed.