K+ currents generated by NMDA receptor activation in rat hippocampal pyramidal neurons

Long lasting outward currents mediated by Ca2+-activated K+ channels can be induced by Ca2+ influx through N-methyl-D-aspartate (NMDA)-receptor channels in voltage-clamped hippocampal pyramidal neurons. Using specific inhibitors, we have attempted to identify the channels that underlie these outward currents. At a holding potential of -50 mV, applications of 1 mM NMDA to the soma of cultured hippocampal pyramidal neurons induced the expected inward currents. In 44% of cells tested, these were followed by outward currents (average amplitude 60 +/- 7 pA) that peaked 2.5 s after the initiation of the inward NMDA currents and decayed with a time constant of 1.4 s. In 43% of those cells exhibiting an outward current, SK channel inhibitors, UCL 1848 (100 nM) and apamin (100 nM) abolished the outward current. In the remainder of the cells, the outward currents were either insensitive or only partly inhibited (44 +/- 4%) by 100 nM UCL 1848. In these cells, the outward currents were reduced by the slow afterhyperpolarization (sAHP) inhibitors, muscarine (3 microM; 43 +/- 9%), UCL 1880 (3 microM; 34 +/- 10%), and UCL 2027 (3 microM; 57 +/- 6%). Neither the BK channel inhibitor, charybdotoxin (100 nM), nor the Na+/K+ ATPase inhibitor, ouabain (100 microM), reduced these outward currents. Irrespective of the pharmacology, the time course of the outward current did not differ. Interestingly, no correlation was observed between the presence of a slow apamin-insensitive afterhyperpolarization and an outward current insensitive to SK channel blockers following NMDA-receptor activation. It is concluded that an NMDA-mediated rise in [Ca2+]i can result in the activation of apamin-sensitive SK channels and of the channels that underlie the sAHP. The activation of these channels may, however, depend on their location relative to NMDA receptors as well as on the spatial Ca2+ buffering within individual neurons.     

Inspiratory-activated and inspiratory-inhibited airway vagal preganglionic neurons in the ventrolateral medulla of neonatal rat are different in intrinsic electrophysiological properties

This study investigates the firing properties of the inspiratory-activated and inspiratory-inhibited airway vagal preganglionic neurons located in the external formation of the nucleus ambiguus. The results showed that inspiratory-activated and inspiratory-inhibited neurons are distributed with different density and site preference in this area. Inspiratory-inhibited neurons exhibit significantly more positive resting membrane potential, more negative voltage threshold and lower minimal current required to evoke an action potential under current clamp. The afterhyperpolarization in inspiratory-activated neurons was blocked by apamin, a blocker of the small-conductance Ca(2+)-activated K(+) channels; and that in inspiratory-inhibited neurons by charybdotoxin, a blocker of the large-conductance Ca(2+)-activated K(+) channels. Under voltage clamp, depolarizing voltage steps evoked tetrodotoxin-sensitive rapid inward sodium currents, 4-aminopyridine-sensitive outward potassium transients and lasting outward potassium currents. 4-Aminopyridine partially blocked the lasting outward potassium currents of inspiratory-activated neurons but was ineffective on those of inspiratory-inhibited neurons. These findings suggest that inspiratory-activated and inspiratory-inhibited neurons are differentially organized and express different types of voltage-gated ion channels.     

Properties of a calcium-activated K(+) current on interneurons in the developing rat hippocampus

Calcium-activated potassium currents have an essential role in regulating excitability in a variety of neurons. Although it is well established that mature CA1 pyramidal neurons possess a Ca(2+)-activated K(+) conductance (I(K(Ca))) with early and late components, modulation by various endogenous neurotransmitters, and sensitivity to K(+) channel toxins, the properties of I(K(Ca)) on hippocampal interneurons (or immature CA1 pyramidal neurons) are relatively unknown. To address this problem, whole-cell voltage-clamp recordings were made from visually identified interneurons in stratum lacunosum-moleculare (L-M) and CA1 pyramidal cells in hippocampal slices from immature rats (P3-P25). A biphasic calcium-activated K(+) tail current was elicited following a brief depolarization from the holding potential (-50 mV). Analysis of the kinetic properties of I(K(Ca)) suggests that an early current component differs between these two cell types. An early I(K(Ca)) with a large peak current amplitude (200.8 +/- 13.2 pA, mean +/- SE), slow time constant of decay (70.9 +/- 3.3 ms), and relatively rapid time to peak (within 15 ms) was observed on L-M interneurons (n = 88), whereas an early I(K(Ca)) with a small peak current amplitude (112.5 +/- 7.3 pA), a fast time constant of decay (39.4 +/- 1.6 ms), and a slower time-to-peak (within 26 ms) was observed on CA1 pyramidal neurons (n = 85). Removal of extracellular calcium or addition of inorganic Ca(2+) channel blockers (cadmium, nickel, or cobalt) was used to demonstrate the calcium dependence of these currents. Addition of norepinephrine, carbachol, and a variety of channel toxins (apamin, iberiotoxin, verruculogen, paxilline, penitrem A, and charybdotoxin) were used to further distinguish between I(K(Ca)) on these two hippocampal cell types. Verruculogen (100 nM), carbachol (100 microM), apamin (100 nM), TEA (1 mM), and iberiotoxin (50 nM) significantly reduced early I(K(Ca)) on CA1 pyramidal neurons; early I(K(Ca)) on L-M interneurons was inhibited by apamin and TEA. Combined with previous work showing that the firing properties of hippocampal interneurons and pyramidal cells differ, our kinetic and pharmacological data provide strong support for the hypothesis that different types of Ca(2+)-activated K(+) current are present on these two cell types.     

Inhibition of apamin-sensitive K+ current by hypoxia in adult rat adrenal chromaffin cells

The effect of hypoxia on small-conductance Ca(2+)-activated K+ current was investigated in a study of adult rat adrenomedullary chromaffin cells (AMCs), which were maintained in short-term culture. The nystatin-perforated, whole-cell patchclamp technique was used to study the effect of hypoxia with minimum perturbation of the intracellular milieu. Under voltage-clamp conditions, acute hypoxia (P(O2) approximately equal to 25 mmHg) suppressed the whole-cell outward currents of more than half the AMCs (24/46). This suppression was eliminated after application of apamin (400 nM), a selective inhibitor of small-conductance Ca(2+)-activated K+ current (I(SK)(Ca)) (n=5), suggesting that an apamin-sensitive component of whole-cell currents is suppressed during hypoxia. In contrast to I(SK)(Ca), Ca2+ current (I(Ca)) (n=10) was not affected by hypoxia. Finally, under current-clamp conditions, hypoxia reversibly depolarized the resting membrane potential of adult AMCs (34/40). Apamin, however, eliminated the hypoxia-induced depolarization (400 nM) (7/8), suggesting that hypoxic depolarization is related to the suppression of I(SK(Ca). From the above results, we conclude that adult AMCs are sensitive to hypoxia, and that I(SK)(Ca) contributes to the hypoxia-induced suppression of whole-cell outward current and depolarization of the resting membrane potential in adult AMCs.     

Voltage-activated potassium outward currents in two types of spider mechanoreceptor neurons.

We studied the properties of voltage-activated outward currents in two types of spider cuticular mechanoreceptor neurons to learn if these currents contribute to the differences in their adaptation properties. Both types of neurons adapt rapidly to sustained stimuli, but type A neurons usually only fire one or two action potentials, whereas type B neurons can fire bursts lasting several hundred milliseconds. We found that both neurons had two outward current components, 1) a transient current that activated rapidly when stimulated from resting potential and inactivated with maintained stimuli and 2) a noninactivating outward current. The transient outward current could be blocked by 5 mM tetraethylammonium chloride, 5 mM 4-aminopyridine, or 100 microM quinidine, but these blockers also reduced the amplitude of the noninactivating outward current. Charybdotoxin or apamin did not have any effect on the outward currents, indicating that Ca2+-activated K+ currents were not present or not inhibited by these toxins. The only significant differences between type A and type B neurons were found in the half-maximal activation (V50) values of both currents. The transient current had a V50 value of 9. 6 mV in type A neurons and -13.1 mV in type B neurons, whereas the V50 values of noninactivating outward currents were -48.9 mV for type A neurons and -56.7 mV for type B neurons. We conclude that, although differences in the activation kinetics of the voltage-activated K+ currents could contribute to the difference in the adaptation behavior of type A and type B neurons, they are not major factors.     

Blockade of SK-type Ca2+-activated K+ channels uncovers a Ca2+-dependent slow afterdepolarization in nigral dopamine neurons

Sharp electrode current-clamp recording techniques were used to characterize the response of nigral dopamine (DA)-containing neurons in rat brain slices to injected current pulses applied in the presence of TTX (2 microM) and under conditions in which apamin-sensitive Ca2+-activated K+ channels were blocked. Addition of apamin (100-300 nM) to perfusion solutions containing TTX blocked the pacemaker oscillation in membrane voltage evoked by depolarizing current pulses and revealed an afterdepolarization (ADP) that appeared as a shoulder on the falling phase of the voltage response. ADP were preceded by a ramp-shaped slow depolarization and followed by an apamin-insensitive hyperpolarizing afterpotential (HAP). Although ADPs were observed in all apamin-treated cells, the duration of the response varied considerably between individual neurons and was strongly potentiated by the addition of TEA (2-3 mM). In the presence of TTX, TEA, and apamin, optimal stimulus parameters (0.1 nA, 200-ms duration at -55 to -68 mV) evoked ADP ranging from 80 to 1,020 ms in duration (355.3 +/- 56.5 ms, n = 16). Both the ramp-shaped slow depolarization and the ensuing ADP were markedly voltage dependent but appeared to be mediated by separate conductance mechanisms. Thus, although bath application of nifedipine (10-30 microM) or low Ca2+, high Mg2+ Ringer blocked the ADP without affecting the ramp potential, equimolar substitution of Co2+ for Ca2+ blocked both components of the voltage response. Nominal Ca2+ Ringer containing Co2+ also blocked the HAP evoked between -55 and -68 mV. We conclude that the ADP elicited in DA neurons after blockade of apamin-sensitive Ca2+-activated K+ channels is mediated by a voltage-dependent, L-type Ca2+ channel and represents a transient form of the regenerative plateau oscillation in membrane potential previously shown to underlie apamin-induced bursting activity. These data provide further support for the notion that modulation of apamin-sensitive Ca2+-activated K+ channels in DA neurons exerts a permissive effect on the conductances that are involved in the expression of phasic activity.     

A long lasting Ca2+ -activated outward current in guinea-pig atrial myocytes

Among other characteristics, the steady-state current-voltage relationship of patch-clamped single atrial myocytes from guinea-pig hearts is defined by an outward current hump in the potential region -15 to +40 mV. This hump was reversibly suppressed by Co2+ (3 mM) or nitrendipine (5 microM) and enhanced by Bay K 8644 (5 microM). The maintained outward current component suppressed by Co2+ extended between -15.2 +/- 1.9 mV and +39.5 +/- 1.7 mV (mean +/- SEM of 14 cells) and has an amplitude of 95.7 +/- 9.4 pA at +10 mV. In isochronal I-V curves, the hump was already visible at 400 ms with essentially the same amplitude as at 1500 ms. The Co2+-sensitive outward current underlying the hump was poorly time-dependent during 1.5 s voltage pulses but slowly relaxed upon repolarization. Tail currents reversed near the K+ equilibrium potential under our experimental conditions. The current hump of the steady-state I-V curve was also abolished by caffeine (10 mM) or ryanodine (3 microM), both drugs that interfere with sarcoplasmic reticulum function. Apamin (1 microM) or quinine (100 microM) but not TEA (5-50 mM) markedly reduced its amplitude. However, at similar concentrations as required to inhibit the hump, both apamin and quinine appeared to be poorly specific for Ca2+-activated K+ currents in heart cells since they also inhibited the L-Type Ca2+ current. It is concluded that a long lasting Ca2+-activated outward current, probably mainly carried by K+ ions but not sensitive to TEA, exists in atrial myocytes which is responsible for the current hump of the background I-V curve.     

Ionic currents in crustacean neurosecretory cells.

1. The patterns of electrical activity and membrane characteristics of a population of neurosecretory-cell somata in the X-organ of the crayfish were investigated with microelectrodes and whole-cell, voltage-clamp techniques. Some neurons (56%) were silent but could be excited by intracellular current injection: other cells showed spontaneous tonic activity (35%), and some had spontaneous bursting activity (9%). The spiking activity was abolished by tetrodotoxin (TTX) exposure and by severing the axon near the cell body. After axotomy, only a small, slow, regenerative depolarization remained that could be blocked by Cd2+. 2. Under voltage clamp the steady-state I-V curve in low [Ca2+]i (9 X 10(-9) M) showed a slope conductance of 16.7 +/- 3.9 (SD) nS (n = 10) at -50 mV and zero current potential of -50.1 +/- 7.7 mV. In current-clamp mode these neurons were either silent or fired tonically. With high [Ca2+]i (1.7 X 10(-6) M) both the slope conductance and inward and outward currents were reduced. In some neurons high [Ca2+]i reveals a negative slope resistance in the range of -46 to -41 mV. It could be supressed by removing [Na+]o, but it was TTX insensitive. These are the neurons that under current clamp showed bursting activity. 3. The main inward current in cell somata was a Ca2+ current of 2 +/- 0.6 nA (n = 18), activated at -40 mV and peaking at 20 mV. It showed relaxation with prolonged pulses. No Na(+)-dependent, TTX-sensitive inward currents were recorded with short (100-ms) pulses in axotomized neurons. 4. Two outward currents could be distinguished.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of an apamin-sensitive potassium current in suprachiasmatic nucleus neurons

In neurons of the suprachiasmatic nucleus, spike frequency adaptation and membrane afterhyperpolarization occur during a train of action potentials. Extracellular Ca2+ may regulate neuronal excitability by several mechanisms, including activation of small conductance and large conductance Ca(2+)-activated K+ channels. The overall goal of this study was to examine the role of Ca(2+)-activated K+ currents in individual suprachiasmatic nucleus neurons. To this end, we used the nystatin-perforated patch technique to record currents from suprachiasmatic nucleus neurons. Iberiotoxin and tetraethylammonium, antagonists of large conductance Ca(2+)-activated K+ channels, had no effect on the membrane afterhyperpolarization. However, antagonists of small conductance Ca(2+)-activated K+ channels, apamin and d-tubocurarine, reduced the amplitude of the membrane afterhyperpolarization and inhibited the spike frequency adaptation that occurred during a train of action potentials. Although there was no significant difference in membrane AHP between different portions of the circadian day, apamin and d-tubocurarine increased the spontaneous firing frequency of suprachiasmatic nucleus neurons during the daytime. In voltage-clamp mode, membrane depolarization-activated currents were followed by an outward tail current reversing near the K+ equilibrium potential. The tail current decayed with a time constant of 220 ms at +20 mV and 149 ms at -40 mV. Apamin irreversibly and d-tubocurarine reversibly inhibited the tail current. The tail current amplitude was also reduced by the GABAA receptor antagonist, bicuculline methiodide, while picrotoxin (another GABAA receptor antagonist) was without effect. Removal of extracellular Ca2+ or the addition of Cd2+ reversibly inhibited the tail current. These results indicate that apamin- and d-tubocurarine-sensitive small conductance Ca(2+)-activated K+ channels have a modulatory function on the action potential firing frequency as well as the membrane afterhyperpolarization that follows a train of action potentials in suprachiasmatic nucleus neurons. Importantly, our data also indicate that a portion of the effects of bicuculline methiodide on suprachiasmatic nucleus neurons may be mediated by inhibition of small conductance Ca(2+)-activated K+ channels.     

Cloning and expression of a small-conductance Ca(2+)-activated K+ channel from the mouse cochlea: coexpression with alpha9/alpha10 acetylcholine receptors

Functional interactions between ligand-gated, voltage-, and Ca(2+)-activated ion channels are essential to the properties of excitable cells and thus to the working of the nervous system. The outer hair cells in the mammalian cochlea receive efferent inputs from the brain stem through cholinergic nerve fibers that form synapses at their base. The acetylcholine released from these efferent fibers activates fast inhibitory postsynaptic currents mediated, to some extent, by small-conductance Ca(2+)-activated K+ channels (SK) that had not been cloned. Here we report the cloning, characterization, and expression of a complete SK2 cDNA from the mouse cochlea. The cDNAs of the mouse cochlea alpha9 and alpha10 acetylcholine receptors were also obtained, sequenced, and coexpressed with the SK2 channels. Human cultured cell lines transfected with SK2 yielded Ca(2+)-sensitive K+ current that was blocked by dequalinium chloride and apamin, known blockers of SK channels. Xenopus oocytes injected with SK2 in vitro transcribed RNA, under conditions where only outward K+ currents could be recorded, expressed an outward current that was sensitive to EGTA, dequalinium chloride, and apamin. In HEK-293 cells cotransfected with cochlear SK2 plus alpha9/alpha10 receptors, acetylcholine induced an inward current followed by a robust outward current. The results indicate that SK2 and the alpha9/alpha10 acetylcholine receptors are sufficient to partly recapitulate the native hair cell efferent synaptic response.     

Voltage-dependent ion channel currents in putative neuroendocrine cells dissociated from the ventral prostate of rat

Prostate neuroendocrine (NE) cells play important roles in the growth and differentiation of the prostate. Following enzymatic digestion of rat ventral prostate, the whole-cell patch-clamp technique was applied to dark, round cells that exhibited chromogranin-A immunoreactivity, a representative marker of NE cells. Under zero current-clamp conditions, putative NE cells showed hyperpolarized resting membrane potentials of some -70 mV, and spontaneous action potentials were induced by an increase in external [K+] or by the injection of current. Using a CsCl pipette solution, step-like depolarization activated high-voltage-activated Ca2+ current (HVA I(Ca)) and tetrodotoxin-resistant voltage-activated Na+ current. The HVA I(Ca) was blocked by nifedipine and omega-conotoxin GVIA, L-type and N-type Ca2+ channel blockers, respectively. Using a KCl pipette solution, the transient outward K+ current (I(to)), Ca2+ -activated K+ currents (I(K,Ca)), the non-inactivating outward current and an inwardly rectifying K+ current (I(Kir)) were identified. I(K,Ca) was suppressed by charybdotoxin (50 nM), iberiotoxin (10 nM) or clotrimazol (1 microM), but not by apamine (100 nM). I(to) was inhibited by 4-aminopyridine (5 mM). I(Kir) was identified as a Ba2+ -sensitive inwardly rectifying current in the presence of a high-K+ bath solution. The voltage- and Ca2+ -activated ion channels could play significant roles in the regulation of neurohormonal secretion in the prostate.     

Potassium currents contributing to action potential repolarization and the afterhyperpolarization in rat vagal motoneurons.

1. Intracellular recordings were made from neurons in the dorsal motor nucleus of the vagus (DMV) in transverse slices of rat medulla maintained in vitro at 30 degrees C. Neurons had a resting potential of -59.8 +/- 1.4 (SE) mV (n = 39) and input resistance of 293 +/- 23 M omega (n = 44). 2. Depolarization elicited overshooting action potentials that were blocked by tetrodotoxin (TTX; 1 microM). In the presence of TTX, two types of action potentials having low and high thresholds could be elicited. The action potentials were blocked by cobalt (2 mM) indicating they were mediated by calcium currents. 3. Under voltage clamp, depolarization of the cell from membrane potentials negative of the resting potential activated a transient potassium current. This current was selectively blocked by 4-aminopyridine (4-AP) (5 mM) and catechol (5 mM) indicating that it is an A-type current. This current inactivated with a time constant of 420 ms and recovered from inactivation with a time constant of 26 ms. 4. When calcium currents were blocked by cadmium or cobalt, the rate of action potential repolarization was slower. In the presence of tetraethylammonium (TEA; 200-400 microM) or charybdotoxin (CTX; 30 nM) a small "hump" appeared on the repolarizing phase of the action potential that was abolished by addition of cadmium. These results indicate that a calcium-activated potassium current (IC) contributes to action potential repolarization. 5. Actions potentials elicited from hyperpolarized membrane potentials repolarized faster than those elicited from resting membrane potential. This effect could be blocked by catechol, indicating that voltage-dependent potassium currents (IA) can also contribute to action-potential repolarization. In the presence of catechol and calcium channel blockers, action potentials still had a significant early afterhyperpolarization suggesting that another calcium independent outward current is also active during repolarization. This fast afterhyperpolarizations (AHP) was not blocked by TEA. 6. Action potentials were followed by prolonged AHPs, which had two phases. The initial part of the AHP was blocked by apamin (100 nM) indicating that it results from activation of SK type calcium-activated potassium channels. The slow phase was selectively blocked by catechol suggesting that it is due to activation of IA. 7. It is concluded that a TTX-sensitive sodium current and two calcium currents contribute to the action potential in rat DMV neurons. At least three different currents contribute to action-potential repolarization: IC, IA, and a third unidentified calcium-insensitive outward current.(ABSTRACT TRUNCATED AT 400 WORDS)     

Zinc modulation of ionic currents in the horizontal limb of the diagonal band of Broca

We examined modulation of ionic currents by Zn2+ in acutely dissociated neurons from the rat's horizontal limb of the diagonal band of Broca using the whole-cell patch-clamp technique. Application of 50 microM Zn2+ increased the peak amplitude of the transiently activated potassium current, I(A) (at + 30 mV), from 2.20+/-0.08 to 2.57+/-0.11 nA (n = 27). This response was reversible and could be repeated in 0 Ca2+/1 microM tetrodotoxin (n = 15). Zn2+ shifted the inactivation curve to the right, resulting in a shift in the half-inactivation voltage from 76.4+/-2.2 to -53.4+/-2.0 mV (n = 11), with no effect on the voltage dependence of activation gating (n = 15). There was no significant difference in the time to peak under control conditions (7.43+/-0.35 ms, n = 14) and in the presence of Zn2+ (8.20+/-0.57 ms, n = 14). Similarly, the time constant of decay of I(A) (tau(d)) at + 30 mV showed no difference (control: 38.68+/-3.68 ms, n = 15; Zn2+: 38.48+/-2.85 ms, n = 15). I(A) was blocked by 0.5-1 mM 4-aminopyridine. In contrast to its effects on I(A), Zn2+ reduced the amplitude of the delayed rectifier potassium current (I(K)). The reduction of outward K+ currents was reproducible when cells were perfused with 1 microM tetrodotoxin in a 0 Ca2+ external solution. The amplitude of the steady-state outward currents at +30 mV under these conditions was reduced from 6.40+/-0.23 (control) to 5.76+/-0.18 nA in the presence of Zn2+ (n = 16). The amplitudes of peak sodium currents (INa) were not significantly influenced (n = 10), whereas barium currents (I(Ba)) passing through calcium channels were potently modulated. Zn2+ reversibly reduced I(Ba) at -10 mV by approximately 85% from -2.06+/-0.14 nA under control conditions to -0.30+/-0.10 nA in the presence of Zn2+ (n = 14). Further analyses of Zn2+ effects on specific calcium channels reveals that it suppresses all types of high-voltage-activated Ca2+ currents. Under current-clamp conditions, application of Zn2+ resulted in an increase in excitability and loss of accommodation (n = 13), which appears to be mediated through its effects on Ca2+-dependent conductances.     

Block of potassium outward currents in the crayfish stretch receptor neurons by 4-aminopyridine, tetraethylammonium chloride and some other chemical substances.

The effects of 4-aminopyridine (4-AP) and tetraethylammonium (TEA) on the outward potassium currents in the rapidly and slowly adapting stretch receptor neurons (SRNs) of the crayfish (Pacifastacus leniusculus) were studied using a two micro-electrode voltage-clamp technique. The leakage current was not affected by either 4-AP or TEA. External 4-AP blocked the peak outward current in a dose-dependent manner (1:1 stoichiometry) with an apparent dissociation constant (Kd) of 2.3 +/- 0.2 mM (mean +/- SEM) in the slowly and 1.4 +/- 0.2 mM in the rapidly adapting SRN, the block being voltage dependent. External application of TEA resulted in a block of the steady state current enhancing the transient characteristics of the current response. The block appeared to deviate from a 1:1 stoichiometry and the apparent Kd for TEA was 9.6 +/- 3.4 mM with a cooperativity factor n = 0.43 +/- 0.03 in the slowly adapting SRN and 34.5 +/- 9.2 mM and 0.37 +/- 0.03 respectively in the rapidly adapting SRN. Low Ca2+, apamin and charybdotoxin, which are known to block Ca(2+)-dependent K-currents, had no effects on the outward current as was also the case with catechol. It is concluded that the different effects of TEA and 4-AP on the outward current in the two types of SRNs can be explained by the presence of at least two, probably heteromultimeric, channel populations having similar sensitivity to 4-AP but different sensitivity to TEA. One channel has a high affinity (Kd = 0.8-1.6 mM) for TEA and the other a low affinity (Kd = 173-213 mM) for TEA. The low-affinity channel seems to dominate in the slowly adapting SRN while both channels are equally common in the rapidly adapting SRN. Further, the present results do not support the existence of a macroscopic Ca(2+)-dependent K+ current in the SRNs.     

Anoxia on slow inward currents of immature hippocampal neurons

1. The effects of brief anoxia (2-4 min) on membrane currents--especially the tetrodotoxin (TTX)-insensitive, Cd2+-sensitive slow inward currents, presumed to be Ca2+ currents--were studied by single-electrode voltage clamp in CA1 and CA3 neurons in submerged hippocampal slices from adult and newborn Wistar rats (PN1-13). 2. In mature neurons, anoxia had no effect on Q-type inward relaxations, but slowly activating C-type outward currents were depressed. The most striking change was the suppression of Ca inward currents (especially the slowly inactivating L-type, by greater than 95%). This effect of anoxia was not sensitive to the N-methyl-D-aspartate (NMDA) receptor blocker, D-aminophosphonovalerate. Anoxia also reversibly abolished the NMDA-evoked inward current. 3. In neurons from newborn animals (PN1-6), Q-type inward relaxations and postanoxic outward currents were very small or undetectable. The slow inward (Ca) currents were smaller than in mature cells, but they showed a clearer separation between low-threshold, fast-inactivating and high-threshold, slowly inactivating currents. Both types of current were more resistant to anoxia (mean depression of L-type was by only 53.3 +/- 5.6%, mean +/- SE). 4. In such immature neurons, the NMDA-evoked inward currents were also more resistant to anoxia. 5. By PN7-13, increasing maturation was reflected in 1) larger voltage-dependent inward currents, 2) increasingly evident Q-type relaxations and postanoxic outward currents, and 3) near-complete blockade of inward currents by anoxia (at PN11-13, mean depression of L-type currents was by 98.5 +/- 1.5%).     

Ca2+-activated Cl– channels in Ehrlich ascites tumor cells are distinct from mCLCA1, 2 and3

Using the whole-cell patch-clamp technique, we have studied the electrophysiological and pharmacological properties of the Ca(2+)-activated Cl- current present in Ehrlich cells. The currents activated slowly upon depolarization, deactivated upon hyperpolarization, and showed strong outward rectification. An increase in [Ca2+]i activated the current with an EC50 of 165.2 nM. Extracellular application of niflumic acid (100 microM) rapidly blocked the current in a voltage-dependent manner whereas sulfhydryl-modifying agents such as dithiothreitol (DTT, 1-2 mM) and N-ethylmaleimide (NEM, 100 microM) had no effect on Ca(2+)-activated currents in Ehrlich cells. Members of the recently discovered CLCA gene family are the only molecular candidates for Ca(2+)-activated Cl- channels cloned so far. Using RT-PCR we demonstrated that the appearance of a Ca(2+)-activated Cl- current in Ehrlich cells is not associated with the expression of the murine members of the CLCA family (mCLCA1-mCLCA3). Correspondingly, the kinetic and pharmacological properties of the Ca(2+)-activated current in Ehrlich cells differ from those of CLCA-associated currents, which are time independent and DTT sensitive. Thus, phenotypic differences in combination with RT-PCR data point to the existence of different molecular species for Ca(2+)-activated Cl- channels.     

Simultaneous recordings of cytosolic Ca2+ level and membrane potential and current during the response to thyroliberin in clonal rat anterior pituitary cells

The response to thyroliberin in prolactin-producing rat GH4C1 clonal cells was studied using fura-2 to monitor the cytosolic Ca2+ level ([Ca2+]i) in single cells, combined with recordings of membrane potential and current. The average value of [Ca2+]i was 109 nM (mean +/- SD, n = 112), and evoked action potentials caused transient elevations of about 100 nM. At higher firing frequencies these transients merged to a sustained elevation. In 100% of the cells thyroliberin caused an instant rise in [Ca2+]i, peaking at 795 +/- 300 nM (n = 112). This first phase of the thyroliberin response was associated with hyperpolarization in current clamp and outward current in voltage clamp, caused by the opening of Ca2(+)-activated K+ channels. In 75% of the cells the initial peak in [Ca2+]i was followed by a prolonged plateau phase at 247 +/- 76 nM (n = 84). In current clamp the second-phase elevation of [Ca2+]i was linked to either a modest depolarization in combination with enhanced firing frequency or a more pronounced depolarization in silent cells. This elevation of [Ca2+]i was reversed by hyperpolarizing current injection. No second-phase elevation of [Ca2+]i was observed during voltage clamp at a holding potential of -50 mV. Short exposure to Ca2(+)-free conditions eliminated the second-phase elevation in [Ca2+]i, whereas the first phase remained intact. Our experiments show a direct relationship between electrical activity and [Ca2+]i in the GH4C1 cells. The second-phase elevation of [Ca2+]i caused by thyroliberin is the result of influx through voltage-sensitive Ca2+ channels, without involving agonist-gated channels.     

Dopamine modulation of honey bee (Apis mellifera) antennal-lobe neurons

Primary olfactory centers [antennal lobes (ALs)] of the honey bee brain are invaded by dopamine (DA)-immunoreactive neurons early in development (pupal stage 3), immediately before a period of rapid growth and compartmentalization of the AL neuropil. Here we examine the modulatory actions of DA on honey bee AL neurons during this period. Voltage-clamp recordings in whole cell configuration were used to determine the effects of DA on ionic currents in AL neurons in vitro from pupal bees at stages 4-6 of the nine stages of metamorphic adult development. In approximately 45% of the neurons tested, DA (5-50 x 10(-5) M) reduced the amplitude of outward currents in the cells. In addition to a slowly activating, sustained outward current, DA reduced the amplitude of a rapidly activating, transient outward conductance in some cells. Both of the currents modulated by DA could be abolished by the removal of Ca2+ from the external medium or by treatment of cells with charybdotoxin (2 x 10(-8) M), a blocker of Ca2+-dependent K+ currents in the cells. Ca2+ currents were not affected by DA, nor were A-type K+ currents (I(A)). Results suggest that the delayed rectifier-like current (I(KV)) also remains intact in the presence of DA. Taken together, our data indicate that Ca2+-dependent K+ currents are targets of DA modulation in honey bee AL neurons. This study lends support to the hypothesis that DA plays a role in the developing brain of the bee.     

Calcium-dependent potassium currents in neurons from cat sensorimotor cortex.

1. Ca(2+)-dependent K+ currents were studied in large pyramidal neurons (Betz cells) from layer V of cat sensorimotor cortex by use of an in vitro brain slice and single microelectrode voltage clamp. The Ca(2+)-dependent outward current was taken as the difference current obtained before and after blockade of Ca2+ influx. During step depolarizations in the presence of tetrodotoxin (TTX), this current exhibited a fast onset of variable amplitude and a prominent slowly developing component. 2. The Ca(2+)-dependent outward current first appeared when membrane potential was stepped positive to -40 mV. Downsteps from a holding potential of -40 mV revealed little or no time-, voltage-, or Ca(2+)-dependent current. When membrane potential was stepped positive to -40 mV, a prolonged Ca(2+)-dependent outward tail current followed repolarization. The decay of this tail current at -40 mV was best described by a single exponential function having a time constant of 275 +/- 75 (SD) ms. The tail current reversed at 96 +/- 5 mV in 3 mM extracellular K+ concentration ([K+]o) and at more positive potentials when [K+]o was raised, suggesting that it was carried predominantly by K+. 3. The Ca(2+)-dependent K+ current consisted of two pharmacologically separable components. The slowly developing current was insensitive to 1 mM tetraethylammonium (TEA), but a substantial portion was reduced by 100 nM apamin. Most of the remaining current was blocked by the addition of isoproterenol (20-50 microM) or muscarine (10-20 microM). 4. The time courses of the apamin- and transmitter-sensitive components were similar when activated by step depolarizations in voltage clamp, but they were quite different when activated by a train of action potentials. Applying the voltage clamp at the end of a train of 90 spikes (evoked at 100-200 Hz) resulted in an Ca(2+)-dependent K+ current with a prominent rapidly decaying portion (time constant approximately 50 ms at -64 mV) and a smaller slowly decaying portion (time constant approximately 500 ms at -64 mV). The rapidly decaying portion was blocked by apamin (50-200 nM), and the slowly decaying portion was blocked by isoproterenol (20-50 microM). 5. When recorded with microelectrodes containing 2 mM dimethyl-bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (dimethyl-BAPTA), which causes prolonged afterhyperpolarizations, the Ca(2+)-dependent K+ current evoked by step depolarizations had an extremely slow onset and decay. The current recorded after a train of evoked spikes had a similar slow decay.(ABSTRACT TRUNCATED AT 400 WORDS)