Molecular profile of vascular ion channels after experimental subarachnoid hemorrhage.

Cerebral vasospasm is a transient, delayed constriction of cerebral arteries that occurs after subarachnoid hemorrhage (SAH). Smooth muscle cells show impaired relaxation after SAH, which may be caused by a defect in the ionic mechanisms regulating smooth muscle membrane potential and Ca(2+) permeability. We tested this hypothesis by examining changes in expression of mRNA and protein for ion channels in the basilar arteries of dogs after SAH using quantitative real-time polymerase chain reaction (PCR) and western blotting. SAH was associated with a significant reduction in basilar artery diameter to 41 +/- 8% of pre-SAH diameter (P < 0.001) after 7 days. There was significant downregulation of the voltage-gated K(+) channel K(v) 2.2 (65% reduction in mRNA, P < 0.001; 49% reduction in protein, P < 0.05) and the beta1 subunit of the large-conductance, Ca(2+) - activated K(+) (BK) channel (53% reduction in mRNA, P < 0.02). There was no change in BK beta1 subunit protein. Changes in mRNA levels of K(v) 2.2 and the BK-beta1 subunit correlated with the degree of vasospasm (r(2) = 0.490 and 0.529 respectively, P < 0.05). The inwardly rectifying K(+) (K(ir)) channel K(ir) 2.1 was upregulated (234% increase in mRNA, P < 0.001; 350% increase in protein, P < 0.001). There was no significant change in mRNA expression of L- type Ca(2+) channels and the BK-alpha subunit. These data suggest that K(+) channel dysfunction may contribute to the pathogenesis of cerebral vasospasm.     

K(ir) and K(v) channels regulate electrical properties and proliferation of adult neural precursor cells

The functional significance of the electrophysiological properties of neural precursor cells (NPCs) was investigated using dissociated neurosphere-derived NPCs from the forebrain subventricular zone (SVZ) of adult mice. NPCs exhibited hyperpolarized resting membrane potentials, which were depolarized by the K(+) channel inhibitor, Ba(2+). Pharmacological analysis revealed two distinct K(+) channel families: Ba(2+)-sensitive K(ir) channels and tetraethylammonium (TEA)-sensitive K(v) (primarily K(DR)) channels. Ba(2+) promoted mitogen-stimulated NPC proliferation, which was mimicked by high extracellular K(+), whereas TEA inhibited proliferation. Based on gene and protein levels in vitro, we identified K(ir)4.1, K(ir)5.1 and K(v)3.1 channels as the functional K(+) channel candidates. Expression of these K(+) channels was immunohistochemically found in NPCs of the adult mouse SVZ, but was negligible in neuroblasts. It therefore appears that expression of K(ir) and K(v) (K(DR)) channels in NPCs and related changes in the resting membrane potential could contribute to NPC proliferation and neuronal lineage commitment in the neurogenic microenvironment.     

Ether-à-go-go 1 (Eag1) Potassium Channel Expression in Dopaminergic Neurons of Basal Ganglia is Modulated by 6-Hydroxydopamine Lesion

The ether à go-go (Eag) gene encodes the voltage-gated potassium (K(+)) ion channel Kv10.1, whose function still remains unknown. As dopamine may directly affect K(+) channels, we evaluated whether a nigrostriatal dopaminergic lesion induced by the neurotoxin 6-hydroxydopamine (6-OHDA) would alter Eag1-K(+) channel expression in the rat basal ganglia and related brain regions. Male Wistar rats received a microinjection of either saline or 6-OHDA (unilaterally) into the medial forebrain bundle. The extent of the dopaminergic lesion induced by 6-OHDA was evaluated by apomorphine-induced rotational behavior and by tyrosine hydroxylase (TH) immunoreactivity. The 6-OHDA microinjection caused a partial or complete lesion of dopaminergic cells, as well as a reduction of Eag1+ cells in a manner proportional to the extent of the lesion. In addition, we observed a decrease in TH immunoreactivity in the ipsilateral striatum. In conclusion, the expression of the Eag1-K(+)-channel throughout the nigrostriatal pathway in the rat brain, its co-localization with dopaminergic cells and its reduction mirroring the extent of the lesion highlight a physiological circuitry where the functional role of this channel can be investigated. The Eag1-K(+) channel expression in dopaminergic cells suggests that these channels are part of the diversified group of ion channels that generate and maintain the electrophysiological activity pattern of dopaminergic midbrain neurons.     

Potassium channels Kv1.3 and Kv1.5 are expressed on blood-derived dendritic cells in the central nervous system

OBJECTIVE: Potassium (K(+)) channels on immune cells have gained attention recently as promising targets of therapy for immune-mediated neurological diseases such as multiple sclerosis (MS). We examined K(+) channels on dendritic cells (DCs), which infiltrate the brain in MS and may impact disease course. METHODS: We identified K(+) channels on blood-derived DCs by whole-cell patch-clamp analysis, confirmed by immunofluorescent staining. We also stained K(+) channels in brain sections from MS patients and control subjects. To test functionality, we blocked K(v)1.3 and K(v)1.5 in stimulated DCs with pharmacological blockers or with an inducible dominant-negative K(v)1.x adenovirus construct and analyzed changes in costimulatory molecule upregulation. RESULTS: Electrophysiological analysis of DCs showed an inward-rectifying K(+) current early after stimulation, replaced by a mix of voltage-gated K(v)1.3- and K(v)1.5-like channels at later stages of maturation. K(v)1.3 and K(v)1.5 were also highly expressed on DCs infiltrating MS brain tissue. Of note, we found that CD83, CD80, CD86, CD40, and interleukin-12 upregulation were significantly impaired on K(v)1.3 and K(v)1.5 blockade. INTERPRETATION: These data support a functional role of K(v)1.5 and K(v)1.3 on activated human DCs and further define the mechanisms by which K(+) channel blockade may act to suppress immune-mediated neurological diseases.     

Stimulatory effects of chlorzoxazone,a centrally acting muscle relaxant,on large conductance calcium-activated potassium channels in pituitary GH3 cells

Chlorzoxazone, a centrally acting muscle relaxant, has been used as a marker for hepatic CYP2E1 activity. However, little is known about the mechanism of chlorzoxazone actions on ion currents in neurons or neuroendocrine cells. We thus investigated its effects on ion currents in GH(3) lactotrophs. Chlorzoxazone reversibly increased Ca(2+)-activated K(+) current (I(K(Ca))) in a concentration-dependent manner with an EC(50) value of 30 microM. The chlorzoxazone-stimulated I(K(Ca)) was inhibited by iberitoxin (200 nM) or clotrimazole (10 microM), but not by glibenclamide (10 microM) or apamin (200 nM). Chlorzoxazone (30 microM) suppressed voltage-dependent L-type Ca(2+) current. In the inside-out configuration, chlorzoxazone applied to the intracellular side of the patch did not modify single-channel conductance of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels, but did increase channel activity by increasing mean open time and decreasing mean closed time. Chlorzoxazone also caused a left shift in the activation curve of BK(Ca) channels. However, Ca(2+)-sensitivity of these channels was unaffected by chlorzoxazone. 1-Ethyl-2-benzimidazolinone (30 microM), 2-amino-5-chlorobenzoxazole (30 microM) or chlormezanone (30 microM) enhanced BK(Ca) channel activity, while 6-hydroxychlorzoxazone (30 microM) slightly increased it; however, chlorphenesin carbamate (30 microM) had no effect on it. Under the current-clamp condition, chlorzoxazone (10 microM) reduced the firing rate of action potentials. In neuroblastoma IMR-32 cells, chlorzoxazone (30 microM) also stimulated BK(Ca) channel activity. The stimulatory effects of chlorzoxazone on these channels may be responsible for the underlying mechanism of chlorzoxazone actions on neurons and neuroendocrine cells.     

Mechanisms of ATP action on motor nerve terminals at the frog neuromuscular junction

We have shown previously that ATP inhibits transmitter release at the neuromuscular junction through the action on metabotropic P2Y receptors coupled to specific second messenger cascades. In the present study we recorded K(+) or Ca(2+) currents in motor nerve endings or blocked K(+) or Ca(2+) channels in order to explore the nature of downstream presynaptic effectors. Endplate currents were presynaptically depressed by ATP. Blockers of Ca(2+)-activated K(+)-channels, such as iberiotoxin, apamin or tetraethylammonium, did not change the depressant action of ATP. By contrast, K(+) channel blocker 4-aminopyridine (4-AP) and raised extracellular Ca(2+) attenuated the effect of ATP. However, these effects of 4-AP and high Ca(2+) were reversed by Mg(2+), suggesting Ca(2+)-dependence of the ATP action. Ba(2+) promoted the depressant action of ATP as did glibenclamide, a blocker of ATP-sensitive K(+) channels, or mild depolarization produced by 7.5 mm K(+). None of the K(+) channel blockers affected the depressant action of adenosine. Focal recording revealed that neither ATP nor adenosine affected the fast K(+) currents of the motor nerve endings. However, unlike adenosine, ATP or UTP, an agonist of P2Y receptors, reversibly reduced the presynaptic Ca(2+)-current. This effect was abolished by suramin, an antagonist of P2 receptors. Depressant effect of ATP on the endplate and Ca(2+)-currents was mimicked by arachidonate, which precluded the action of ATP. ATP reduced acetylcholine release triggered by ionomycin or sucrose, suggesting inhibition of release machinery. Thus, the presynaptic depressant action of ATP is mediated by inhibition of Ca(2+) channels and by mechanism acting downstream of Ca(2+) entry.     

Overexpression of mouse IsK protein fused to green fluorescent protein induces apoptosis of human astroglioma cells

Intracellular K(+) plays an important role in controlling ion homeostasis for maintaining cell volume and inhibiting activity of pro-apoptotic enzymes. Cytoplasmic K(+) concentration is regulated by K(+) uptake via Na(+) -K(+) -ATPase and K(+) efflux through K(+) channels in the plasma membrane. The IsK (KCNE1) protein is known to co-assemble with KCNQ1 (KvLQT1) protein to form a K(+) channel underlying the slowly activating delayed rectifier K(+) outward current which delays voltage activation. In order to further study the activity and cellular localization of IsK protein, we constructed a C-terminal fusion of IsK with EGFP (enhanced green fluorescent protein). Expression of the fusion protein appeared as clusters located in the plasma membrane and induced degeneration of both transiently or stably transfected cells.     

Involvement of Na(+)-K(+)-2Cl(-) cotransporters in hypertonicity-induced rise in intracellular calcium concentration

A hypertonic saline containing propylene glycol facilitates calcium (Ca(2+)) influx through voltage-dependent Ca(2+) channels. The present study performed experiments to elucidate the mechanism by which Na(+)-K(+)-2Cl(-) cotransporters participate in the rise in the intracellular calcium concentration ([Ca(2+)]i) under the hypertonic condition. Both furosemide and ethacryonic acid significantly decreased the [Ca(2+)]i raised by hypertonicity. Similarly, Na(+)-, K(+)-, or Cl(-)-free saline also reduced it. Both norepinephrine and dopamine significantly enhanced the rise in [Ca(2+)]i. In conclusion, the findings obtained indicate that the Na+-K+-2Cl- cotransporters evoke cell depolarization and that this depolarization raises the [Ca(2+)]i by activating voltage-dependent Ca(2+) channels.     

Inwardly rectifying and voltage-gated outward potassium channels exhibit low sensitivity to methylmercury

The concentration- and time-dependence of effects of methylmercury (MeHg) on voltage-gated outward K(+) (Kv) channels, inwardly rectifying K(+) (Kir) channels, voltage-gated Ca(2+) channels and GABA(A) receptor activated channels were compared in cerebellar granule cells in culture using whole cell patch clamp recording techniques. The objective was to determine if MeHg equally affects different types of ion channels. Under similar experimental conditions, these four ion channel types displayed markedly different sensitivity to MeHg. At 0.1-1 microM, MeHg caused apparent inhibition of Ca(2+)-channel and GABA(A) receptor-mediated currents, but did not cause any significant effect on Kv or Kir channels. Among the four channel types examined, GABA(A) receptors appeared to be the most sensitive to MeHg. The Kv channels, particularly the delayed rectifiers (DRs), appeared to be relatively resistant to MeHg compared with GABA(A) receptors and Ca(2+) channels. Kir channels were virtually unaffected by MeHg in the concentration range of 10-100 microM. The differential sensitivity of GABA(A) receptors and Kv channels to MeHg was also observed in granule and Purkinje cells in freshly isolated cerebellar slices of rat. The insensitivity of Kir channel to MeHg was also seen in Xenopus laevis oocytes expressing cloned Kir7.1 channels. Thus, these appear to be general properties of these channels as opposed to distinct effects associated with granule cells in culture. These results suggest that MeHg does preferentially affect certain types of ion channels. Hence, the effects of MeHg on membrane ion channels are not due simply to nonspecific actions on the membrane. Furthermore, at least certain types of Kir channels appear to be the most resistant type of ion channel reported to date to effects of MeHg.     

Ion channels and calcium signaling in cerebral arteries following subarachnoid hemorrhage

Entry of Ca(2+) through voltage-dependent calcium channels (VDCCs) is critical to the regulation of intracellular free calcium concentration ([Ca(2+)](i)) in vascular smooth muscle and thus the control of cerebral artery diameter. Increased VDCC activity in cerebral artery myocytes may contribute to decreased cerebral blood flow and the accompanying neurological deficits associated with subarachnoid hemorrhage (SAH). This review will focus on the impact of SAH on VDCCs and K(+)-selective ion channels, two important classes of ion channels located in the plasma membrane of cerebral artery myocytes. SAH may act through a variety of direct and indirect mechanisms to increase the activity of VDCCs promoting cerebral artery constriction and reduced cerebral blood flow. Further, SAH may lead to suppression of K(+) channel activity to cause membrane potential depolarization to enhance VDCC activity. The ability of VDCC blockers or K(+) channel activators to alleviate SAH-induced vasospasm will also be examined.     

Differential effects of K(+) channel blockers on frequency-dependent action potential broadening in supraoptic neurons

Recordings were made from magnocellular neuroendocrine cells dissociated from the supraoptic nucleus of the adult guinea pig to determine the role of voltage gated K(+) channels in controlling the duration of action potentials and in mediating frequency-dependent action potential broadening exhibited by these neurons. The K(+) channel blockers charybdotoxin (ChTx), tetraethylammonium (TEA), and 4-aminopyridine (4-AP) increased the duration of individual action potentials indicating that multiple types of K(+) channel are important in controlling action potential duration. The effect of these K(+) channel blockers was almost completely reversed by simultaneous blockade of voltage gated Ca(2+) channels with Cd(2+). Frequency-dependent action potential broadening was exhibited by these neurons during trains of action potentials elicited by membrane depolarizing current pulses presented at 10 Hz but not at 1 Hz. 4-AP but not ChTx or TEA inhibited frequency-dependent action potential broadening indicating that frequency-dependent action potential broadening is dependent on increasing steady-state inactivation of A-type K(+) channels (which are blocked by 4-AP). A model of differential contributions of voltage gated K(+) channels and voltage gated Ca(2+) channels to frequency-dependent action potential broadening, in which an increase of Ca(2+) current during each successive action potential is permitted as a result of the increasing steady-state inactivation of A-type K(+) channels, is presented.     

Inhibition of potassium and calcium currents in neurones by molecularly-defined P2Y receptors

Messenger RNAs and cDNAs for individual cloned P2Y(1), P2Y2 and P2Y(6) nucleotide receptors have been expressed by micro-injection into dissociated rat superior cervical sympathetic neurones and the effects of stimulating the expressed receptors on voltage-activated N-type Ca(2+) currents and M-type K(+) currents recorded. Both currents were reduced by stimulating all three receptors, with the following mean IC(50) values: P2Y(1) (agonist: ADP) - I(K(M)) 6.9 nM, I(Ca) 8.2 nM; P2Y(2) (agonist: UTP) - I(K(M)) 1.5 microM, I(Ca) 0.5 microM; P2Y(6) (agonist: UDP) - I(K(M)) 30 nM, I(Ca) 5.9 nM. Inhibition of I(K(M)) was voltage-independent and insensitive to Pertussis toxin; inhibition of I(Ca) showed both voltage-sensitive and insensitive, and Pertussis toxin-sensitive and insensitive components. It is concluded that these P2Y receptors can couple to more than one G protein and thereby modulate more than one ion channel. It is suggested that these effects on K(M) and Ca(N) channels may induce both postsynaptic excitory and presynaptic inhibitory responses.     

Syntaxin modulation of calcium channels in cortical synaptosomes as revealed by botulinum toxin C1.

When the presynaptic membrane protein syntaxin is coexpressed in Xenopus oocytes with N- or P/Q-type Ca(2+) channels, it promotes their inactivation (Bezprozvanny et al., 1995; Wiser et al., 1996, 1999; Degtiar et al., 2000) (I. B. Bezprozvanny, P. Zhong, R. H. Scheller, and R. W. Tsien, unpublished observations). These findings led to the hypothesis that syntaxin influences Ca(2+) channel function in presynaptic endings, in a reversal of the conventional flow of information from Ca(2+) channels to the release machinery. We examined this effect in isolated mammalian nerve terminals (synaptosomes). Botulinum neurotoxin type C1 (BoNtC1), which cleaves syntaxin, was applied to rat neocortical synaptosomes at concentrations that completely blocked neurotransmitter release. This treatment altered the pattern of Ca(2+) entry monitored with fura-2. Whereas the initial Ca(2+) rise induced by depolarization with K(+)-rich solution was unchanged, late Ca(2+) entry was strongly augmented by syntaxin cleavage. Similar results were obtained when Ca(2+) influx arose from repetitive firing induced by the K(+)-channel blocker 4-aminopyridine. Cleavage of vesicle-associated membrane protein with BoNtD or SNAP-25 with BoNtE failed to produce a significant change in Ca(2+) entry. The BoNtC1-induced alteration in Ca(2+) signaling was specific to voltage-gated Ca(2+) channels, not Ca(2+) extrusion or buffering, and it involved N-, P/Q- and R-type channels, the high voltage-activated channels most intimately associated with presynaptic release machinery. The modulatory effect of syntaxin was not immediately manifest when synaptosomes had been K(+)-predepolarized in the absence of external Ca(2+), but developed with a delay after admission of Ca(2+), suggesting that vesicular turnover may be necessary to make syntaxin available for its stabilizing effect on Ca(2+) channel inactivation.     

Characterization of cholera toxin B subunit-induced Ca(2+) influx in neuroblastoma cells: evidence for a voltage-independent GM1 ganglioside-associated Ca(2+) channel

The role of endogenous GM1 ganglioside in neurite outgrowth has been studied in N18 and NG108-15 neuroblastoma cells with the GM1-specific ligand cholera toxin B subunit (Ctx B), which stimulates Ca(2+) influx together with neuritogenesis. Our primary goal has been to identify the nature of the calcium channel that is modulated by GM1. An L-type voltage-operated Ca(2+) channel (VOCC) was previously proposed as the mediator of this phenomenon. This investigation, employing fura-2 fluorescent measurements and specific channel blockers and other agents, revealed that GM1 modulates a hitherto unidentified Ca(2+) channel not of the L type. It was opened by Ctx B; was permeable to Ca(2+) and Ba(2+) but not Mn(2+); and was blocked by Ni(2+), Cd(2+), and La(3+). Although most dihydropyridines inhibited Ctx B-induced Ca(2+) influx as well as neurite outgrowth at higher concentrations, they and other VOCC blockers at normally employed concentrations failed to do so, suggesting uninvolvement of VOCC. In addition, Ca(2+) influx induced by Ctx B was not mediated by cGMP-dependent or G-protein-coupled nonselective cation channels, as demonstrated by the cGMP antagonist Rp-cGMPS or the G-protein/receptor uncoupling agent suramin, respectively. Finally, Ca(2+) influx was unlikely to be due to inhibition or reversal of Na(+)-Ca(2+) exchanger via Ctx B induction of Na(+) uptake, insofar as no effect was seen on blocking Na(+) channels, inhibiting Na(+)-K(+)-ATPase, or eliminating extracellular Na(+). The results suggest that this novel channel is gated by interaction with GM1, which, when associated with the channel and bound by appropriate ligand, promotes Ca(2+) influx. This in turn induces signaling for the onset of neuritogenesis.     

Gabapentin actions on Kir3 currents and N-type Ca2+ channels via GABAB receptors in hippocampal pyramidal cells

Gabapentin is a clinically effective anticonvulsant with an unclear mechanism of action. It was described as a GABA(B(1a,2)) receptor subtype-selective agonist, activating postsynaptic K(+) currents and inhibiting postsynaptic Ca(2+) channels in CA1 pyramidal cells, but without presynaptic actions. These activities appeared controversial and we therefore sought to further clarify gabapentin actions in rat hippocampal slices by characterizing K(+) currents and Ca(2+) channels targeted by gabapentin using whole-cell recording and multiphoton Ca(2+) imaging. 1) We found that gabapentin and baclofen induced inwardly rectifying K(+) currents (K(Gbp) and K(Bac), respectively), sensitive to Ba(2+) and Cs(+). 2) A constitutively active K(IR) current, independent of GABA(B) receptor activation and sensitive to Ba(2+) and Cs(+) was also present. 3) K(Gbp), K(Bac), and K(IR) currents showed some differences in sensitivity to Ba(2+) and Cs(+), indicating the possible activation of distinct Kir3 currents, independent of K(IR), by gabapentin and baclofen. 4) Gabapentin inhibition of Ca(2+) channels was abolished by omega-conotoxin GVIA, but not by omega-agatoxin IVA and nimodipine, indicating a predominant action of gabapentin on N-type Ca(2+) channels. 5) Gabapentin actions were linked to activation of pertussis toxin-sensitive G-proteins since N-ethylmaleimide (NEM) blocked K(Gbp) activation and Ca(2+) channel inhibition by gabapentin. 6) Finally, gabapentin reduced epileptiform discharges in slices via GABA(B) receptor activation. The anticonvulsant actions of gabapentin in hippocampal cells may thus involve GABA(B) receptor coupling to G-proteins and modulation of Kir3 and N-type Ca(2+) channels. Moreover, gabapentin and baclofen activation of GABA(B) receptors may couple to distinct cellular targets.     

Fluoxetine dilates isolated small cerebral arteries of rats and attenuates constrictions to serotonin, norepinephrine, and a voltage-dependent Ca(2+) channel opener.

BACKGROUND AND PURPOSE: Recent clinical observations question that the antidepressant effect of fluoxetine (Prozac) can be explained solely with serotonin reuptake inhibition in the central nervous system. We hypothesized that fluoxetine affects the tone of vessels and thereby modulates cerebral blood flow. METHODS: A small branch of rat anterior cerebral artery (195+/-15 microm in diameter at 80 mm Hg perfusion pressure) was isolated, cannulated, and pressurized (at 80 mm Hg), and changes in diameter were measured by videomicroscopy. RESULTS: Fluoxetine dilated small cerebral arteries with an EC(50) of 7.7+/-1.0x10(-6) mol/L, a response that was not affected by removal of the endothelium or application of 4-aminopyridine (an inhibitor of aminopyridine-sensitive K(+) channels), glibenclamide (an inhibitor of ATP-sensitive K(+) channels), or tetraethylammonium (a nonspecific inhibitor of K(+) channels). The presence of fluoxetine (10(-6) to 3x10(-5) mol/L) significantly attenuated constrictions to serotonin (10(-9) to 10(-5) mol/L) and norepinephrine (10(-9) to 10(-5) mol/L). Increasing concentrations of Bay K 8644 (a voltage-dependent Ca(2+) channel opener, 10(-10) to 10(-6) mol/L) elicited constrictions, which were markedly reduced by 2x10(-6) and 10(-5) mol/L fluoxetine, whereas 3x10(-5) mol/L fluoxetine practically abolished the responses. CONCLUSIONS: Fluoxetine elicits substantial dilation of isolated small cerebral arteries, a response that is not mediated by endothelium-derived dilator factors or activation of K(+) channels. The finding that fluoxetine inhibits constrictor responses to Ca(2+) channel opener, as well as serotonin and norepinephrine, suggests that fluoxetine interferes with the Ca(2+) signaling mechanisms in the vascular smooth muscle. We speculate that fluoxetine increases cerebral blood flow in vivo, which contributes to its previously described beneficial actions in the treatment of mental disorders.     

Role of L-type Ca2+ channels in neural stem/progenitor cell differentiation

Ca(2+) influx through voltage-gated Ca(2+) channels, especially the L-type (Ca(v)1), activates downstream signaling to the nucleus that affects gene expression and, consequently, cell fate. We hypothesized that these Ca(2+) signals may also influence the neuronal differentiation of neural stem/progenitor cells (NSCs) derived from the brain cortex of postnatal mice. We first studied Ca(2+) transients induced by membrane depolarization in Fluo 4-AM-loaded NSCs using confocal microscopy. Undifferentiated cells (nestin(+)) exhibited no detectable Ca(2+) signals whereas, during 12 days of fetal bovine serum-induced differentiation, neurons (beta-III-tubulin(+)/MAP2(+)) displayed time-dependent increases in intracellular Ca(2+) transients, with DeltaF/F ratios ranging from 0.4 on day 3 to 3.3 on day 12. Patch-clamp experiments revealed similar correlation between NSC differentiation and macroscopic Ba(2+) current density. These currents were markedly reduced (-77%) by Ca(v)1 channel blockade with 5 microm nifedipine. To determine the influence of Ca(v)1-mediated Ca(2+) influx on NSC differentiation, cells were cultured in differentiative medium with either nifedipine (5 microm) or the L-channel activator Bay K 8644 (10 microm). The latter treatment significantly increased the percentage of beta-III-tubulin(+)/MAP2(+) cells whereas nifedipine produced opposite effects. Pretreatment with nifedipine also inhibited the functional maturation of neurons, which responded to membrane depolarization with weak Ca(2+) signals. Conversely, Bay K 8644 pretreatment significantly enhanced the percentage of responsive cells and the amplitudes of Ca(2+) transients. These data suggest that NSC differentiation is strongly correlated with the expression of voltage-gated Ca(2+) channels, especially the Ca(v)1, and that Ca(2+) influx through these channels plays a key role in promoting neuronal differentiation.     

A novel calcium-sensitive potassium conductance is coupled to P2X3 subunit containing receptors in myenteric neurons of guinea pig ileum

This study characterized P2X receptors in guinea pig ileum myenteric S neurons (n = 124) in vitro using electrophysiological methods. ATP or alpha,beta-methylene ATP (alpha,beta-mATP), an agonist at P2X(1) and P2X(3) subunit containing receptors, depolarized 103 neurons (85%). Pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid (10 micromol L(-1)) blocked ATP- and alpha,beta-mATP-induced depolarizations. ATP-induced depolarizations and fast excitatory postsynaptic potentials (fEPSPs) were reduced by trinitrophenyl-ATP (10 micromol L(-1)), an antagonist that can block P2X(3) receptors. Ivermectin (10 micromol L(-1)), a modulator of P2X(4) and P2X(4/6) receptors, had no effect on alpha,beta-mATP-induced depolarizations. In 58% of neurons, the alpha,beta-mATP induced-depolarization was followed by an afterhyperpolarization (AHP) (P2X-AHP). Under voltage clamp, alpha,beta-mATP induced an inward current followed by an outward current which reversed polarity at 0 and -80 mV respectively. The P2X-AHP was reduced in low extracellular Ca(2+) solutions. Blockers of large, intermediate and small conductance Ca(2+)-activated K(+) channels or voltage-gated K(+) channels did not inhibit the P2X-AHP. Half of the neurons exhibiting the P2X-AHP contained nitric oxide synthase (NOS)-immunoreactivity (ir). In summary, NOS-ir S neurons express P2X(3) subunit containing P2X receptors. P2X receptors couple to activation of a Ca(2+)-activated K(+) conductance that mediates an AHP. As P2X receptors contribute to fEPSPs, the P2X-AHP may modulate S neuron excitability during purinergic synaptic transmission.     

HIV-1 gp120 enhances outward potassium current via CXCR4 and cAMP-dependent protein kinase A signaling in cultured rat microglia

Microglia are critical cells in mediating the pathophysiology of neurodegenerative disorders such as HIV-associated neurocognitive disorders. We hypothesize that HIV-1 glycoprotein 120 (gp120) activates microglia by enhancing outward K(+) currents, resulting in microglia secretion of neurotoxins, consequent neuronal dysfunction, and death. To test this hypothesis, we studied the effects of gp120 on outward K(+) current in cultured rat microglia. Application of gp120 enhanced outward K(+) current in a dose-dependent manner, which was blocked by voltage-gated K(+) (K(v) ) channel blockers. Western blot analysis revealed that gp120 produced an elevated expression of K(v) channel proteins. Examination of activation and inactivation of outward K(+) currents showed that gp120 shifted membrane potentials for activation and steady-state inactivation. The gp120-associated enhancement of outward K(+) current was blocked by either a CXCR4 receptor antagonist T140 or a specific protein kinase A (PKA) inhibitor H89, suggesting the involvement of chemokine receptor CXCR4 and PKA in gp120-mediated enhancement of outward K(+) current. Biological significance of gp120-induced enhancement of microglia outward K(+) current was demonstrated by experimental results showing the neurotoxic activity of gp120-stimulated microglia, evaluated by TUNEL staining and MTT assay, significantly attenuated by K(v) channel blockers. Taken together, these results suggest that gp120 induces microglia neurotoxic activity by enhancing microglia outward K(+) current and that microglia K(v) channels may function as a potential target for the development of therapeutic strategies.