Different voltage-gated sodium currents are expressed by human neuroblastoma NB69 cells when cultured in defined serum-free and in astroglial-conditioned media

Voltage-gated Na⁺ currents (I_Na) were analysed with the whole-cell patch-clamp technique in human neuroblastoma NB69 cells plated in serum-free “defined” medium (DM) or in “astroglial-conditioned” medium (CM). Cells survived in both media and expressed the microtubule associated protein 1A, indicating neuron-like differentiation. Two I_Na types with different time-, voltage-dependent properties and tetrodotoxin (TTX) sensitivities were expressed in DM and CM. The I_Na in DM-plated cells was present from day 4 and its surface density increased from 11 pA/pF (days 5–7) to 68 pA/pF (days 15–30). The underlying conductance (G_Na) half-activated (V_0A) at −24 mV. I_Na inactivation was fitted by single exponentials with 7.5 ms time constant (t_h) at the −35 mV half-inactivation voltage (V_0I). I_Na was not affected by 10 nM, was reduced (65%) by 100 nM, and not completely abolished (92%) by 300 nM tetrodotoxin (TTX). The I_Na of CM-plated cells appeared at day 3–4 and its surface density increased from 14 pA/pF (days 3–6) to 28 pA/pF (days 11–14). The G_Na V_0A was −29 mV and inactivation was fitted by single exponentials with 2.6 ms t_h at the −58 mV V_0I. This I_Na was reduced (55%) by 10 nM and totally abolished by 100 nM tetrodotoxin (TTX). In conclusion, NB69 cells displayed a slow, “TTX-resistant,” or a fast, “TTX-sensitive” I_Na in DM and CM, respectively, suggesting that the CM contained diffusible trophic factors of astroglial origin that induced the expression of a different Na⁺ channel type. About half of the CM- and DM-plated cells also displayed a persistent Na⁺ current (I_NaP). © 1997 Wiley-Liss Inc.     

Characterization of a Calcium-dependent Current Generating a Slow Afterdepolarization of CA3 Pyramidal Cells in Rat Hippocampal Slice Cultures

A depolarization-induced, slowly decaying inward current was examined in slice-cultured CA3 pyramidal cells by voltage-clamp techniques and microfluorometric measurements of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). Action potentials elicited by intracellular injection of short-lasting (50 – 100 ms) depolarizing current pulses were followed by a slowly decaying afterhyperpolarization (AHP). After switching to voltage-clamp mode, short-lasting (50 – 100 ms) depolarizing voltage jumps from –60 mV to between –30 and 0 mV induced a slowly decaying outward aftercurrent (I_AHP) which was depressed by bath application of muscarine (0.5 μM). In the presence of muscarine, the same depolarizations induced a slowly decaying afterdepolarization (ADP) or inward aftercurrent (I_ADP)in voltage-clamp mode. This current was also induced in the presence of trans(±)-1-aminc-1,3-cyclopenta-nedicarboxylic acid (t-ACPD, 5 μM), an agonist of metabotropic glutamate receptors, but not in the presence of noradrenalin (5 μM), while both of these agonists depressed I_AHP. I_ADP was depressed by reducing the external Ca²⁺ concentration from 3.8 to 0.5 mM, by external Co²⁺ (1 mM) and by external Cd²⁺ (10 – 100 μM). Combined voltage-clamp recordings and microfluorometric measurements of [Ca²⁺]_i using the Ca²⁺ indicator fura-2 revealed that the amplitude of I_ADP was correlated with the amplitude of depolarization-induced Ca²⁺ influx, I_ADP was absent at membrane potentials < –90 mV, and reached maximal amplitudes at ～–55 mV. Raising the extracellular K⁺ concentration from 2.7 to 13.5 mM increased the amplitude of I_ADP and resulted in a positively directed shift of the apparent reversal potential of I_ADP. When the external Na⁺ concentration was reduced from 157 to 33 or 18 mM the current reversed at more negative potentials and was reduced to 40 and 21%, respectively, of control amplitude. Lowering the external Cl^- concentration from 159 to 20 mM did not affect I_ADP. We conclude that I_ADP most likely represents a Ca²⁺-activated cation current, rather than a Ca²⁺ tail current, or an electrogenic Ca²⁺ extrusion current.     

Regulation of Ca-activated nonselective cationic currents in rat pituitary GH3 cells: involvement in L-type Ca current

Ionic currents were investigated by a patch clamp technique in a clonal strain of pituitary (GH₃) cells, using the whole cell configuration with Cs⁺ internal solution. Depolarizing pulses positive to 0 mV from a holding potential of −50 mV activated the voltage-dependent L-type Ca²⁺ current (I_Ca,L) and late outward current. Upon repolarization to the holding potential, a slowly decaying inward tail current was also observed. This inward tail current upon repolarization following a depolarizing pulse was found to be enhanced by Bay K 8644, but blocked by nifedipine or tetrandrine. This current was eliminated by Ba²⁺ replacement of external Ca²⁺ as the charge carrier through Ca²⁺ channels, removal of Ca²⁺ from the bath solution, or buffering intracellular Ca²⁺ with EGTA (10 mM). The reversal potential of inward tail current was approximately −25 mV. When intracellular Cl⁻ was changed, the reversal potential of the Ca²⁺-activated currents was not shifted. Thus, this current is elicited by depolarizing pulses that activate I_Ca,L and allow Ca²⁺ influx, and is referred to as Ca²⁺-activated nonselective cationic current (I_CAN). Without including EGTA in the patch pipette, the slowly decaying inward current underlying the long-lasting depolarizing potential after Ca²⁺ spike was also observed with a hybrid current–voltage protocol. Thus, the present studies clearly indicate that Ca²⁺-activated nonselective cationic channels are expressed in GH₃ cells, and can be elicited by the depolarizing stimuli that lead to the activation of I_Ca,L.     

Carbachol Potentiates Q Current and Activates a Calcium-dependent Non-specific Conductance in Rat Hippocampus In Vitro

Intracellular recordings were made from CA1 neurons in rat hippocampal slices maintained in vitro. When Na⁺ currents were blocked with tetrodotoxin and K⁺ conductances known to be sensitive to suppression by muscarinic agonists were blocked by 2 mM Ba²⁺, CA1 cells were depolarized by carbachol (3 – 10 μM) with an attendant conductance increase, whereas prior to Ba²⁺ the agonist produced a decrease or no change in conductance. Under voltage clamp at ～–60 mV and in the presence of tetrodotoxin and Ba²⁺, carbachol (3 – 10 μM) induced a variable-latency biphasic inward current of up to 380 pA associated with a conductance increase of ～50%. The first phase was associated with an increase (more than 2-fold) of the Cs⁺-sensitive, hyperpolarization-activated cationic current, I_Q. Carbachol also accelerated the kinetics of I_Q at – 100 mV with an average 24% reduction in its activation time constant. The second phase reflected an additional inward current that was Cs⁺-resistant, displayed little apparent voltage sensitivity and had a mean extrapolated reversal potential, determined in the presence of external Cs⁺ (>5 mM), of ～–20 mV. In a small proportion of cells the second phase of inward current was followed (or overlapped) by an outward current, also associated with a conductance increase, which reversed at ～–70 mV. These carbachol actions were prevented by extracellular 300 μM Cd²⁺ and 2 mM Mn²⁺, by high levels (>5 mM) of extracellular Mg²⁺ or Ca²⁺, and by omission of Ca²⁺ or reduction of extracellular Na⁺ to 25 mM by substitution of NaCl with Tris or N-methyl-d -glucamine. Carbachol action was not mimicked by oxotremorine (≤60 μM), but was irreversibly blocked by this drug. Likewise, atropine (100 nM) irreversibly and gallamine (10 μM) reversibly antagonized carbachol's action. The action of carbachol was blocked shortly after prior exposure of slices to 2 – 5 mM caffeine. Chronic or acute incubation of slices with 2 mM Li⁺ potentiated (between 1- and 2-fold) carbachol responses. The data indicate that muscarinic activation increases cationic flux by a calcium-dependent potentiation of I_Q and activation of a non-selective conductance. The probability that inositol phospholipid metabolism is involved in triggering these events is discussed.     

Differential expression of voltage-gated K+ and Ca2+ currents in bipolar cells in the zebrafish retinal slice

Whole-cell voltage-gated currents were recorded from bipolar cells in the zebrafish retinal slice. Two physiological populations of bipolar cells were identified. In the first, depolarizing voltage steps elicited a rapidly activating A-current that reached peak amplitude ≤ 5 ms of step onset. I_A was antagonized by external tetraethylammonium or 4-aminopyridine, and by intracellular caesium. The second population expressed a delayed rectifying potassium current (I_K) that reached peak amplitude ≥ 10 ms after step onset and did not inactivate. I_K was antagonized by internal caesium and external tetraethylammonium. Bipolar cells expressing I_K also expressed a time-dependent h-current at membrane potentials < – 50 mV. I_h was sensitive to external caesium and barium, and was also reduced by Na⁺-free Ringer. In both groups, a calcium current (I_Ca) and a calcium-dependent potassium current (I_K(Ca)) were identified. Depolarizing voltage steps > – 50 mV activated I_Ca, which reached peak amplitude between – 20 and – 10 mV. I_Ca was eliminated in Ca⁺²-free Ringer and blocked by cadmium and cobalt, but not tetrodotoxin. In most cells, I_Ca was transient, activating rapidly at – 50 mV. This current was antagonized by nickel. The remaining bipolar cells expressed a nifedipine-sensitive sustained current that activated between – 40 and – 30 mV, with both slower kinetics and smaller amplitude than transient I_Ca. I_K(Ca) was elicited by membrane depolarizations > – 20 mV. Bipolar cells in the zebrafish retinal slice preparation express an array of voltage-gated currents which contribute to non-linear I–V characteristics. The zebrafish retinal slice preparation is well-suited to patch clamp analyses of membrane mechanisms and provides a suitable model for studying genetic defects in visual system development.     

Blockade of bepridil on IA and IK in acutely isolated hippocampal CA1 neurons

The effects of bepridil, an antianginal agent with antiarrhythmic action, on voltage-dependent K⁺ currents in the CA1 pyramidal neurons acutely isolated from rat hippocampus were studied by means of whole-cell patch clamp techniques. Current recordings were made in the presence of TTX to block Na⁺ current. Depolarizing test pulses activated two components of outward K⁺ currents: a rapidly activating and inactivating component, I_A; and a delayed component, I_K. Results showed that bepridil reduced the amplitude of I_A and I_K, and exerted its inhibitory action in time- and dose-dependent manner. Half-blocking concentrations (IC₅₀) of bepridil on I_A and I_K were 17.8 μM and 1.7 μM, respectively. 10 μM bepridil suppressed I_A and I_K by 46.7% and 77.1% at +30 mV of depolarization, respectively. When I_K was activated nearly uncontaminated with I_A by holding at −50 mV, 10 μM bepridil inhibited I_K by 71.6% at +30 mV of depolarization; 10 μM bepridil positively shifted the voltage-dependent of activation curves of I_A and I_K 12.1 mV and 28.7 mV, respectively. These results suggested that blockade on K⁺ currents by bepridil is preferential for I_K, and contributes to the protection brain against ischemic damage.     

Interaction between Low Voltage-activated Currents in Reticular Thalamic Neurons in a Rat Model of Absence Epilepsy

A transient potassium (K⁺) outward current (I_A) contributes to the distinctive patterns of low-threshold spike firing observed in various classes of thalamic neurons through a functional interaction with a calcium (Ca²⁺)-mediated inward current (I_T). The present study was undertaken to investigate the properties of transient K⁺ currents and their interaction with I_T in neurons of the reticular thalamic nucleus, and to compare these properties in reticular thalamic nucleus neurons from a rat model of absence epilepsy, designated the Genetic Absence Epilepsy Rat from Strasbourg (GAERS), with those from a Non-epileptic Control strain (NEC). This comparative approach appeared to be particularly important in view of the recent finding of a selective increase in I_T in reticular thalamic nucleus neurons from GAERS. Neurons were acutely isolated from the reticular thalamic nucleus through enzymatic procedures, and identified by morphological and immunocytochemical criteria. Ionic currents were analysed using whole-cell patch-clamp techniques. Transient K⁺ currents in reticular thalamic nucleus neurons with properties indicative of I_A activated at ～?55 mV (with half-activation at ?27 and ?33 mV in NEC and GAERS respectively), declined rapidly with a voltage-dependent time constant (τ= 4 ms at +45 mV), were 50% steady-state-inactivated at ?81 and ?86 mV in the two strains of rats respectively, and rapidly recovered from inactivation with a monoexponential time course (τ= 31 and 37 ms respectively). No significant differences in I_A properties or densities were found between reticular thalamic nucleus neurons from GAERS and NEC rats. Analysis of the interaction between I_A and I_T indicated a shift in the balance between the two opposing membrane conductances towards the generation of a low-voltage-activated inward current in reticular thalamic nucleus neurons from GAERS compared with NEC, and a lack of I_A to functionally compensate for this shift, which in turn may contribute to pathological forms of low-threshold spike firing characterizing spike-and-wave discharges.     

Inositol 1,4,5-trisphosphate activates non-selective cation conductance via intracellular Ca2+ increase in isolated frog taste cells

The effect of intracellular Ca²⁺ increase was analysed in isolated frog taste cells under the whole-cell patch clamp. External application of a Ca²⁺-ionophore, ionomycin (3 μm ) induced the sustained inward current of ?200 ± 17 pA (mean ± SE, n = 23) at – 50 mV in taste cells. The ionomycin-induced response was observed in most of the cells exposed in the drug, but not when 10 mm BAPTA (1,2-bis (o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) was included in the pipette (eight cells). Steady-state I–V relationships of ionomycin-induced currents were almost linear and reversed at – 8 ± 1 mV (n = 23). The simultaneous removal of Na⁺ and Ca²⁺ from the external solution eliminated the response completely (three cells). Intracellular dialysis with 1 mm Ca²⁺ or 50 μm inositol 1,4,5-trisphosphate (IP₃) in K⁺-internal solution also induced an inward current in the taste cells. The Ca²⁺-induced and IP₃-induced responses were observed in 82% and 36% of the cells dialysed with the drugs, respectively. The Ca²⁺-induced and IP₃-induced currents were inhibited by external Cd²⁺ (1–2 mm ). The reversal potentials of the inward currents were – 15 ± 3 mV (n = 9) in Ca²⁺ dialysis and – 11 ± 3 mV (n = 13) in IP₃ dialysis. The half-maximal Ca²⁺ concentration in the pipette to induce the inward current was ≈ 170 μm . The results suggest that IP₃ can depolarize the taste cell with mediation by intracellular Ca²⁺.     

Selective block of Ca2+-dependent K+ current in crayfish neuromuscular system and chromaffin cells by sea anemone Bunodosoma cangicum venom

The effects of the nematocyst venom of the sea anemone Bunodosoma cangicum on depolarization-activated currents were studied in opener crayfish muscle fibers and in cultured bovine chromaffin cells. The venom selectively and reversibly blocked the Ca²⁺ -dependent K⁺ current (I_K(Ca)) present in crayfish muscle in a dose-dependent manner without affecting voltage-gated Ca²⁺ or K⁺ currents. Furthermore, the venom also reduced I_K(Ca) in chromaffin cells, without modifying voltage-gated Na⁺, Ca²⁺, or K⁺ currents. Synaptic transmission in crayfish muscle was also affected by the venom. Repetitive excitatory and inhibitory postsynaptic currents (each associated with a presynaptic action potential) were evoked by each nerve stimulus, suggesting that presynaptic I_K(Ca) may control the electrical activity of excitatory and inhibitory presynaptic fibers. We conclude that B. cangicum venom includes a toxin that selectively and reversibly blocks Ca²⁺ -dependent K⁺ currents in crayfish muscle and in bovine chromaffin cells, and modifies excitatory and inhibitory synaptic transmission, probably abolishing a similar conductance at the presynaptic fibers. © 1995 Wiley-Liss, Inc.     

Different Types of Potassium Outward Current in Relay Neurons Acutely Isolated from the Rat Lateral Geniculate Nucleus

Different classes of potassium (K+) outward current activated by depolarization were characterized in relay neurons acutely isolated from the rat lateral geniculate nucleus (LGN), using the whole-cell version of the patch-clamp technique. A fast-transient current (IA), activated at around - 70 mV, declined rapidly with a voltage-dependent time constant (tau=6 ms at + 45 mV), was 50% steady-state inactivated at - 70 mV, and rapidly recovered from inactivation with a monoexponential time course (tau=21 ms). IA was blocked by 4-aminopyridine (4-AP, 2 - 8 mM) and was relatively insensitive to tetraethylammonium (TEA, 2 - 10 mM). After elimination of IA by a conditioning prepulse (30 ms to - 50 mV), a slow-transient K+ current could be studied in isolation, and was separated into three components, IKm, IKs and a calcium (Ca2+)-dependent current, IK[Ca]. The slow-transient current was not consistently affected by 4-AP (up to 8 mM), while TEA (2 - 10 mM) predominantly blocked IKs and IK[Ca]. The component IKm persisted in a solution containing TEA and 4-AP, activated at around - 55 mV, declined monoexponentially during maintained depolarization (tau=98 ms at + 45 mV), was 50% inactivated at - 39 mV, and recovered with tau=128 ms from inactivation. IKs activated at a similar threshold, but declined much slower with tau=2662 ms at + 45 mV. Steady-state inactivation of IKs was half-maximal at - 49 mV, and recovery from inactivation occurred relatively fast with tau=116 ms. From these data and additional current-clamp recordings it is concluded that the K+ currents, due to their wide range of kinetics and dependence on membrane voltage or internal Ca2+ concentration, are capable of cooperatively controlling the firing threshold and of shaping the different states of electrophysiological behaviour in LGN relay cells.     

4-aminopyridine causes apoptosis and blocks an outward rectifier K+ channel in malignant astrocytoma cell lines

Among the ion channels and pumps activated by growth factor stimulation, K⁺ channels have been implicated in the growth and proliferation of several cancer cell lines. The role of these channels in central nervous system tumors, however, has not been described. This study used the malignant astrocytoma cell lines U87 and A172. 4-Aminopyridine (4-AP) inhibition of proliferation was dose dependent, and assessment using a TUNEL in situ assay revealed that apoptosis occurred in U87 cells with wild-type p53 but not in A172 cells with mutant p53 (24-hr incubation with 4 mM 4-AP). In patch clamp experiments, we identified two types of K⁺ currents in both cell lines, a charybdotoxin-sensitive Ca²⁺-activated K⁺ channel and a 4-AP-sensitive outward rectifier K⁺ current. The outward rectifier current was blocked by 4-AP in a dose-dependent manner, with half-maximal block occurring at 3.9 mM. The blocking effect of 4 mM 4-AP was noticeable at potentials as low as −65 mV and was statistically significant at −60 mV and above, suggesting that 4-AP-sensitive current is active at physiological potentials. By contrast, charybdotoxin (1 μM) and tetraethylammonium · Cl (2 mM) blocked the Ca²⁺-activated K⁺ channel in both cell lines but had no appreciable effect on cell growth. Our findings reveal that 4-AP inhibits proliferation and the outward rectifier K⁺ channel in both U87 and A172 cells. More studies are needed, however, to describe the mechanism by which K⁺ channels influence proliferation and induce apoptosis. J. Neurosci. Res. 48:122–127, 1997. © 1997 Wiley-Liss, Inc.     

Modulation of presynaptic Ca2+ currents in frog motor nerve terminals by high pressure

Presynaptic Ca²⁺‐dependent mechanisms have already been implicated in depression of evoked synaptic transmission by high pressure (HP). Therefore, pressure effects on terminal Ca²⁺ currents were studied in Rana pipiens peripheral motor nerves. The terminal currents, evoked by nerve or direct stimulation, were recorded under the nerve perineurial sheath with a loose macropatch clamp technique. The combined use of Na⁺ and K⁺ channel blockers, [Ca²⁺]_o changes, voltage‐dependent Ca²⁺ channel (VDCC) blocker treatments and HP perturbations revealed two components of presynaptic Ca²⁺ currents: an early fast Ca²⁺ current (I_CaF), possibly carried by N‐type (Ca_V2.2) Ca²⁺ channels, and a late slow Ca²⁺ current (I_CaS), possibly mediated by L‐type (Ca_V1) Ca²⁺ channels. HP reduced the amplitude and decreased the maximum (saturation level) of the Ca²⁺ currents, I_CaF being more sensitive to pressure, and may have slightly shifted the voltage dependence. HP also moderately diminished the Na⁺ action current, which contributed to the depression of VDCC currents. Computer‐based modeling was used to verify the interpretation of the currents and investigate the influence of HP on the presynaptic currents. The direct HP reduction of the VDCC currents and the indirect effect of the action potential decrease are probably the major cause of pressure depression of synaptic release.     

Effect of specific ion channel blockers on cultured schwann cell proliferation

Mitogenesis in a variety of tissues is known to be inhibited by K⁺ channel blockers such as tetraethylammonium (TEA) and 4-aminopyridine (4-AP). Using radiolabeled thymidine as a proliferation index we have examined what role, if any, specific K⁺ channels have in cultured Schwann cells that have been induced to proliferate by pre-exposure to mitogens. TEA and 4-AP are “broad-spectrum” in that they block a variety of different types of K⁺ channel. In contrast, we found that α-dendrotoxin (α-DTX), a specific blocker of the type 1 fast delayed rectifier current (the largest component of Schwann cell K⁺ current) does not affect proliferation, suggesting that type 1 current may not be involved in mitogenesis. This suggestion is supported by our finding that the values of the K_D for the mitogenic effect (722 nM, 4-AP; 13 mM, TEA) are much larger than the corresponding electrophysiological values for type 1 channels (0.1 mM, 4-AP; 0.2 mM, TEA). Charybdotoxin (200 nM) and iberiotoxin (100 nM), inhibitors of Ca²⁺-activated K⁺ channels, cesium (5 mM), an inhibitor of inward rectifier channels, and furosemide (100 μM), which blocks Na⁺/K⁺/Cl⁻ cotransport, all had no effect on proliferation. Interestingly, 4,4′-diisothiocyanatostilbene 2,2′-disulphonate (DIDS), which blocks voltage-gated Cl⁻ channels, reduced proliferation. In summary, broad-spectrum K⁺ channel blockers inhibit Schwann cell proliferation, but inhibitors specific for type 1, Ca²⁺-activated, and inward rectifier K⁺ channels do not. Whether the inhibition is mediated by type 2 K⁺ channels, by an as yet unidentified Schwann cell K⁺ channel, or by another mechanism remains unclear. GLIA 22:113–120, 1998. © 1998 Wiley-Liss, Inc.     

Na+—Ca2+ Exchange Currents in Cortical Neurons: Concomitant Forward and Reverse Operation and Effect of Glutamate

Na⁺-Ca²⁺ exchanger-associated membrane currents were studied in cultured murine neocortical neurons, using whole-cell recording combined with intracellular perfusion. A net inward current specifically associated with forward (Na⁺_o-Ca²⁺_i) exchange was evoked at -40 mV by switching external 140 mM Li⁺ to 140 mM Na⁺. The voltage dependence of this current was consistent with that predicted for 3Na⁺:1Ca²⁺ exchange. As expected, the current depended on internal Ca²⁺, and could be blocked by intracellular application of the exchanger inhibitory peptide, XIP. Raising internal Na⁺ from 3 to 20 mM or switching the external solution from 140 mM Li⁺ to 30 mM Na⁺ activated outward currents, consistent with reverse (Na⁺,-Ca²⁺_o) exchange. An external Ca²⁺-sensitive current was also identified as associated with reverse Na⁺-Ca²⁺ exchange based on its internal Na⁺ dependence and sensitivity to XIP. Combined application of external Na⁺ and Ca²⁺ in the absence of internal Na⁺ triggered a 3.3–fold larger inward current than the current activated in the presence of 3 mM internal Na⁺, raising the intriguing possibility that Na⁺-Ca²⁺ exchangers might concurrently operate in both the forward and the reverse direction, perhaps in different subcellular locations. With this idea in mind, we examined the effect of excitotoxic glutamate receptor activation on exchanger operation. After 3–5 min of exposure to 100–200 μM glutamate, the forward exchanger current was significantly increased even when external Na⁺ was reduced to 100 mM, and the external Ca²⁺-activated reverse exchanger current was eliminated.     

Sodium and calcium mechanisms of rhythmic bursting in excitatory neural networks of the pre‐Bötzinger complex: a computational modelling study

The neural mechanisms generating rhythmic bursting activity in the mammalian brainstem, particularly in the pre‐Bötzinger complex (pre‐BötC), which is involved in respiratory rhythm generation, and in the spinal cord (e.g. locomotor rhythmic activity) that persist after blockade of synaptic inhibition remain poorly understood. Experimental studies in rodent medullary slices containing the pre‐BötC identified two mechanisms that could potentially contribute to the generation of rhythmic bursting: one based on the persistent Na⁺ current (I_NaP), and the other involving the voltage‐gated Ca²⁺ current (I_Ca) and the Ca²⁺‐activated nonspecific cation current (I_CAN), activated by intracellular Ca²⁺ accumulated from extracellular and intracellular sources. However, the involvement and relative roles of these mechanisms in rhythmic bursting are still under debate. In this theoretical/modelling study, we investigated Na⁺‐dependent and Ca²⁺‐dependent bursting generated in single cells and heterogeneous populations of synaptically interconnected excitatory neurons with I_NaP and I_Ca randomly distributed within populations. We analysed the possible roles of network connections, ionotropic and metabotropic synaptic mechanisms, intracellular Ca²⁺ release, and the Na⁺/K⁺ pump in rhythmic bursting generated under different conditions. We show that a heterogeneous population of excitatory neurons can operate in different oscillatory regimes with bursting dependent on I_NaP and/or I_CAN, or independent of both. We demonstrate that the operating bursting mechanism may depend on neuronal excitation, synaptic interactions within the network, and the relative expression of particular ionic currents. The existence of multiple oscillatory regimes and their state dependence demonstrated in our models may explain different rhythmic activities observed in the pre‐BötC and other brainstem/spinal cord circuits under different experimental conditions.     

Effects of myasthenia gravis patients' sera with different autoantibodies on slow K+ current at mouse motor nerve terminals

Abstract

The antibodies against pre-synaptic membrane receptor (PsmR) and acetylcholine receptor (AChR) in serum samples of myasthenia gravis (MG) patients and healthy donors were tested by enzyme-linked immunosorbent assays (ELISA). The serum samples of eight MG patients with different autoantibodies and those of six healthy donors without these two kinds of autoantibodies were collected to investigate their effects on the peri-neurially recorded membrane currents at mouse motor nerve terminals. After inhibition of both fast and Ca²⁺-dependent K⁺ currents by tetra-ethylammonium (TEA), a positive wave was revealed, which was a balance of the slow K⁺(I_K,s) and Ca²⁺ currents (I_Ca). Application of anti-PsmR antibody negative MG sera and healthy donor sera, whether anti-AChR antibody positive or negative, did not affect the positive wave. However, the positive wave shifted to prolonged Ca²⁺-plateau when adding two of four anti-PsmR antibody positive serum samples from MG patients, indicating an inhibition of I_K,s by anti-PsmR antibody positive sera. Meanwhile, all serum samples derived from either patients or healthy donors did not affect I_Na.     

Endogenous voltage-gated potassium channels in human embryonic kidney (HEK293) cells

Endogenous voltage-gated potassium currents were investigated in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells using whole-cell voltage clamp recording. Depolarizing voltage steps from −70 mV triggered an outwardly rectified current in nontransfected HEK293 cells. This current had an amplitude of 296 pA at +40 mV and a current density of 19.2 pA/pF. The outward current was eliminated by replacing internal K⁺ with Cs⁺ and suppressed by the K⁺ channel blockers tetraethylammonium and 4-aminopyridine. Raising external K⁺ attenuated the outward current and shifted the reversal potential towards positive potentials as predicted by the Nernst equation. The current had a fast activation phase but inactivated slowly. These features implicate delayed rectifier (I_K)-like channels as mediators of the observed current, which was comparable in size to I_K currents in many other cells. A small native inward rectifier current but no transient outward current I_A, the M current I_M, or Ca²⁺-dependent K⁺ currents were detected in HEK293 cells. In contrast to these findings in HEK293 cells, little or no I_K-like current was detected in CHO cells. The difference in endogenous voltage-activated currents in HEK293 and CHO cells suggest that CHO cell lines are a preferred system for exogenous K⁺ channel expression. J. Neurosci. Res. 52:612–617, 1998. © 1998 Wiley-Liss, Inc.     

Ionic currents on type-I cells of the rabbit carotid body measured by voltage-clamp experiments and the effect of hypoxia

Type-I cells (from rabbit embryos) in primary culture were studied in voltage-clamp experiments using the whole cell arrangement of the patch-clamp technique. With a pipette solution containing 130 mM K⁺ and 3 mM Mg-ATP, large outward currents were obtained positive to a threshold of about −30 mV by clamping cells from −50 mV to different test pulses (−80 to 50 mV). Negative to −30 mV, the slope conductance was low (outward rectification). The outward currents were blocked by external Cs⁺ (5 mM) and partially blocked by TEA (5 mM) and Co²⁺ (1 mM). The initial part of the outward currents during depolarizing voltage pulses exhibited a transient Ca²⁺ inward component partially superimposed to a Ca²⁺-dependent outward current. Inward currents were further characterized by replacing K⁺ with Cs⁺ in the intra- and extracellular solution in order to minimize the outward component and by using 1.8 mM Ca²⁺ or 10.8 mM Ba²⁺ as charge carrier. Slow-inactivating inward currents were recorded at test potentials ranging from −50 to 40 mV (holding potential −80 mV). The maximal amplitude, measured at 10 mV in the U-shaped I–V curve, amounted to 247 ± 103pA(n = 3). This inward current was insensitive to 3 μM TTX, but blocked by 1 mM Co²⁺ and partially reduced by 10 μM D600 and 3 μM PN 200-110. In contrast to outward currents, the inward currents exhibited a ‘run-down’ within about 10 min. Lowering the pO₂ from the control of 150 Torr (air-gassed medium) to 28 Torr had no apparent effect on inward currents, but depressed reversibly outward currents by 28%. In conclusion, it is suggested that type-I cells possess voltage-activated K⁺ and Ca²⁺ channels which might be essential for chemoreception in the carotid body.     

Identified glial cells in the early postnatal mouse hippocampus display different types of Ca2+ currents

Based on their typical pattern of membrane currents, four populations of glial cells could be identified in thin brain slices of the postnatal hippocampus. In the present study, we applied the patch-clamp technique to glial cells in the hippocampal CA1 region, which are characterized by a complex pattern of different Na⁺ and K⁻ currents (“complex” cells). These cells were identified as non-neuronal cells, most likely astrocytes, by their glutamine synthetase immunoreactivity. Two types of glial Ca²⁺currents could be identified that differed in their kinetics and pharmacological properties. A low-voltage activated (LVA), fast inactivating component was activated at membrane potentials positive to −60 mV and reached maximum current amplitudes at about −20 mV. This current was sensitive to amiloride and thus displayed properties of neuronal LVA currents. The threshold potential of the second Ca²⁺ current component was at about −40 mV, and peak currents were observed at 0 mV. In contrast to the LVA component, the inactivation of these high-voltage activated (HVA) currents slowed down with increasing depolarizations. This current was sensitive to low concentrations of Cd²⁺ but was not affected by amiloride. A small fraction of the HVA currents was sensitive to nifedipine, and ω-conotoxin GVIA (ω-CgTx) was also found to reduce the glial HVA component. The study provides electrophysiological and pharmacological characterization of different types of Ca²⁺ currents in gray matter glial cells in situ. © 1996 Wiley-Liss, Inc.     

Two types of TTX-resistant and one TTX-sensitive Na+ channel in rat dorsal root ganglion neurons and their blockade by halothane

The clinically employed general anaesthetic halothane was shown to exert action on the peripheral nervous system by suppressing spinal reflexes, but it is still unclear which mechanisms underlie this action. The present study addressed the question whether blockade of tetrodotoxin-sensitive (TTXs) and -resistant (TTXr) Na⁺-channels in rat dorsal root ganglia (DRG) neurons by halothane could explain its peripheral effects. Two types of TTXr Na⁺-currents, fast and slow, with distinct activation and inactivation kinetics were found in small (< 25 μm) and medium sized (25–40 μm) DRG neurons. These currents were blocked by halothane with IC₅₀ values of 5.4 and 7.4 mmol/L, respectively. Additionally, in a concentration-dependent manner halothane accelerated the inactivation kinetics of both currents and shifted the inactivation curves to more hyperpolarized potentials. Neither the activation curves of both TTXr Na⁺-currents were influenced by halothane nor a voltage-dependent block at test potentials of the currents was seen. In contrast to that of fast current, the time-to-peak for slow current was changed in the presence of halothane. The TTXs Na⁺-current which prevailed in large neurons (> 40 μm) was blocked by halothane with an IC₅₀ of 12.1 mmol/L. Its inactivation curve was also shifted to more hyperpolarized potentials and the inactivation kinetics accelerated with increasing halothane concentration. Similarly to TTXr Na⁺-currents, the activation curve of TTXs Na⁺-current and its time-to-peak were not influenced by halothane. It is suggested that two types of TTXr Na⁺-currents can explain the heterogeneity in kinetic data for TTXr Na⁺-currents. Furthermore, the incomplete blockade of Na⁺-currents might underlie the incomplete reduction of spinal reflexes at clinically used concentrations of halothane.