白介素1β时间依赖性地抑制大鼠皮层神经元钾电流

白细胞介素1β（Interleukin-1β,IL-1β）是一种重要的促炎细胞因子,在中枢神经系统中发挥着广泛的生物学功能。大量研究表明,IL-1β的作用非常复杂,在不同的模型和条件下作用不同,包括神经损伤或者神经毒性作用。电压门控钾通道调节神经元电学性质,也参与多种中枢神经系统的病理学过程。虽然IL-1β和钾通道都在脑损伤和脑疾病过程中发挥重要作用,但目前还很少有它们之间关系的研究报导。文章作者以原代培养的大鼠皮层神经元为材料,使用全细胞膜片钳技术,研究了10 ng/mL的IL-1β在不同处理时间下对皮层神经元电压依赖性钾电流的影响。根据电流的性质,可以将记录到的钾电流分为瞬时外向电流（IA）为主的IA样电流和延迟整流电流（IK）为主的IK样电流两部分,结果显示：IL-1β处理8 h对二者没有作用;处理24 h可使IA样和IK样电流的幅度降低20%左右。以上结果提示IL-1β对大鼠皮层神经元电压门控钾电流具有抑制作用,并且这种抑制可能具有时间依赖性。     

Opioids activate both an inward rectifier and a novel voltage-gated potassium conductance in the hippocampal formation

Opioid receptors were found to activate two different types of membrane potassium conductance in acutely dissociated neurons from the CA1/subiculum regions of the adult rat hippocampal formation. Opioid-responsive neurons were distinguished based on their morphology and electrophysiological responses. In one population of neurons having a multipolar, nonpyramidal cell shape, mu-selective opioid agonists increased an inward rectifying potassium current. Opioid activation of the inward rectifying conductance resulted in small outward potassium currents at resting membrane potentials and increased inward currents at hyperpolarized potentials. In a second population of nonpyramidal neurons, mu opioid agonists increased a novel voltage-gated potassium current. This current was blocked by internal CsCl2, unaffected by external BaCl2 or CdCl2, irreversibly activated by intracellular GTP-gamma-S, and inactivated by sustained depolarization. In contrast to the inward rectifying conductance, the voltage-gated conductance was not activated at resting membrane potentials or hyperpolarized potentials. The opioid-activated, voltage-gated conductance represents a new class of G protein-regulated potassium current in the brain.     

VOCCs and TREK-1 ion channel expression in human tenocytes

Mechanosensitive and voltage-gated ion channels are known to perform important roles in mechanotransduction in a number of connective tissues, including bone and muscle. It is hypothesized that voltage-gated and mechanosensitive ion channels also may play a key role in some or all initial responses of human tenocytes to mechanical stimulation. However, to date there has been no direct investigation of ion channel expression by human tenocytes. Human tenocytes were cultured from patellar tendon samples harvested from five patients undergoing routine total knee replacement surgery (mean age: 66 yr; range: 63-73 yr). RT-PCR, Western blotting, and whole cell electrophysiological studies were performed to investigate the expression of different classes of ion channels within tenocytes. Human tenocytes expressed mRNA and protein encoding voltage-operated calcium channel (VOCC) subunits (Ca alpha(1A), Ca alpha(1C), Ca alpha(1D), Ca alpha(2)delta(1)) and the mechanosensitive tandem pore domain potassium channel (2PK(+)) TREK-1. They exhibit whole cell currents consistent with the functional expression of these channels. In addition, other ionic currents were detected within tenocytes consistent with the expression of a diverse array of other ion channels. VOCCs and TREK channels have been implicated in mechanotransduction signaling pathways in numerous connective tissue cell types. These mechanisms may be present in human tenocytes. In addition, human tenocytes may express other channel currents. Ion channels may represent potential targets for the pharmacological management of chronic tendinopathies.     

棉铃虫幼虫神经细胞的急性分离培养及其电压门控通道的膜片钳研究

探索了棉铃虫Helicoverpa armigera幼虫神经细胞的急性分离与体外培养的条件,并利用全细胞膜片钳技术首次对棉铃虫幼虫急性分离神经细胞的电压门控性钠、钾和钙通道的基本电生理学特性进行了研究。结果表明,棉铃虫幼虫中枢神经细胞在TC-100、L-15和Grace培养基中均可贴壁生长,在DMEM培养基中基本不能存活。在TC-100培养基分别与其它三种培养基按一定比例混合形成的培养液中,TC-100与L-15等量混合培养液更适合于神经细胞的生长。全细胞电压钳条件下,可分别记录到电压门控性钠、钾和钙通道电流。钙电流特征为高电压激活、缓慢失活;钠电流对河豚毒素敏感;钾电流可被细胞外液中的氯化四乙胺和4-氨基吡啶抑制。     

Characterization of two novel lipocalins expressed in the Drosophila embryonic nervous system

We have found two novel lipocalins in the fruit fly Drosophila melanogaster that are homologous to the grasshopper Lazarillo, a singular lipocalin within this protein family which functions in axon guidance during nervous system development. Sequence analysis suggests that the two Drosophila proteins are secreted and possess peptide regions unique in the lipocalin family. The mRNAs of DNLaz (for Drosophila neural Lazarillo) and DGLaz (for Drosophila glial Lazarillo) are expressed with different temporal patterns during embryogenesis. They show low levels of larval expression and are highly expressed in pupa and adult flies. DNLaz mRNA is transcribed in a subset of neurons and neuronal precursors in the embryonic CNS. DGLaz mRNA is found in a subset of glial cells of the CNS: the longitudinal glia and the medial cell body glia. Both lipocalins are also expressed outside the nervous system in the developing gut, fat body and amnioserosa. The DNLaz protein is detected in a subset of axons in the developing CNS. Treatment with a secretion blocker enhances the antibody labeling, indicating the DNLaz secreted nature. These findings make the embryonic nervous system expression of lipocalins a feature more widespread than previously thought. We propose that DNLaz and DGLaz may have a role in axonal outgrowth and pathfinding, although other putative functions are also discussed.     

白介素1β抑制培养的大鼠皮层神经元钠电流峰值和动作电位幅度

白介素1β(interleukin-1β,IL-1β)是重要的促炎细胞因子,在中枢神经系统的生理学和病理学过程中发挥关键作用.电压门控钠通道是可兴奋细胞电学活动的基础,控制神经元的兴奋性和动作电位.最近的研究又显示了IL-1β与电压门控通道之间的相互作用.为考察中枢神经元中IL-1β与电压门控钠通道之间的相互作用,本研...     

Pharmacological characterization of ionic currents that regulate high-frequency spontaneous activity of electromotor neurons in the weakly electric fish, Apteronotus leptorhynchus

The neural circuit that controls the electric organ discharge (EOD) of the brown ghost knifefish (Apteronotus leptorhynchus) contains two spontaneous oscillators. Both pacemaker neurons in the medulla and electromotor neurons (EMNs) in the spinal cord fire spontaneously at frequencies of 500-1,000 Hz to control the EOD. These neurons continue to fire in vitro at frequencies that are highly correlated with in vivo EOD frequency. Previous studies used channel blocking drugs to pharmacologically characterize ionic currents that control high-frequency firing in pacemaker neurons. The goal of the present study was to use similar techniques to investigate ionic currents in EMNs, the other type of spontaneously active neuron in the electromotor circuit. As in pacemaker neurons, high-frequency firing of EMNs was regulated primarily by tetrodotoxin-sensitive sodium currents and by potassium currents that were sensitive to 4-aminopyridine and kappaA-conotoxin SIVA, but resistant to tetraethylammonium. EMNs, however, differed from pacemaker neurons in their sensitivity to some channel blocking drugs. Alpha-dendrotoxin, which blocks a subset of Kv1 potassium channels, increased firing rates in EMNs, but not pacemaker neurons; and the sodium channel blocker muO-conotoxin MrVIA, which reduced firing rates of pacemaker neurons, had no effect on EMNs. These results suggest that similar, but not identical, ionic currents regulate high-frequency firing in EMNs and pacemaker neurons. The differences in the ionic currents expressed in pacemaker neurons and EMNs might be related to differences in the morphology, connectivity, or function of these two cell types.     

Study of a neuronal potassium current in different culture conditions

The potassium current of neurons in explants cultured from quail mesencephalic neural crest were studied in voltage clamp, using the whole cell recording technique. Two voltage-dependent potassium currents were identified; they differed in their sensitivity to blocking agents and to sustained depolarizing voltages. The potassium current component most sensitive to 4-aminopyridine had fast activation kinetics and inactivated quickly at sustained depolarized voltages. By analogy with a current described in other preparations, this current was called IA. The current component most sensitive to tetraethylammonium had slower activation kinetics and inactivated more slowly than IA at sustained depolarized voltages. This current was called IK. The properties of IA and IK were examined in neurons cultured in a defined medium and in neurons co-cultured with striated muscle. The rate of inactivation of IA appeared to be increased when neural crest neurons were cultured in the presence of striated muscle. The change in the properties of IA could be due to a direct effect of the co-culture with muscle on the membrane current; another possibility could be that co-culture favors the survival of a neuronal population that does not survive well when cultured in a defined medium.     

Pharmacological characterization of ionic currents that regulate high‐frequency spontaneous activity of electromotor neurons in the weakly electric fish,Apteronotus leptorhynchus

The neural circuit that controls the electric organ discharge (EOD) of the brown ghost knifefish (Apteronotus leptorhynchus) contains two spontaneous oscillators. Both pacemaker neurons in the medulla and electromotor neurons (EMNs) in the spinal cord fire spontaneously at frequencies of 500–1000 Hz to control the EOD. These neurons continue to fire in vitro at frequencies that are highly correlated with in vivo EOD frequency. Previous studies used channel blocking drugs to pharmacologically characterize ionic currents that control high‐frequency firing in pacemaker neurons. The goal of the present study was to use similar techniques to investigate ionic currents in EMNs, the other type of spontaneously active neuron in the electromotor circuit. As in pacemaker neurons, high‐frequency firing of EMNs was regulated primarily by tetrodotoxin‐sensitive sodium currents and by potassium currents that were sensitive to 4‐aminopyridine and κA‐conotoxin SIVA, but resistant to tetraethylammonium. EMNs, however, differed from pacemaker neurons in their sensitivity to some channel blocking drugs. Alpha‐dendrotoxin, which blocks a subset of Kv1 potassium channels, increased firing rates in EMNs, but not pacemaker neurons; and the sodium channel blocker μO‐conotoxin MrVIA, which reduced firing rates of pacemaker neurons, had no effect on EMNs. These results suggest that similar, but not identical, ionic currents regulate high‐frequency firing in EMNs and pacemaker neurons. The differences in the ionic currents expressed in pacemaker neurons and EMNs might be related to differences in the morphology, connectivity, or function of these two cell types. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2006     

Characterization of a Shaw-related potassium channel family in rat brain.

Previously, we characterized a Shaker-related family of voltage-gated potassium channels (RCK) in rat brain. Now, we describe a second family of voltage-gated potassium channels in the rat nervous system. This family is related to the Drosophila Shaw gene and has been dubbed Raw. In contrast to the RCK potassium channel family the Raw family utilizes extensive alternative splicing for expressing potassium channel subunits with variant C-termini. These alternative C-termini do not appear to influence the electrophysiological and pharmacological properties as studied in the Xenopus oocyte expression system. In situ hybridizations to sections of rat brain indicate that members of the Raw family are expressed in distinct areas of the central nervous system. Probably, Raw channels are expressed predominantly as homomultimers. Immunocytochemical experiments with antibodies against Raw3 and RCK4 proteins which form two distinct A-type potassium channels indicate that in hippocampus the two channels are expressed both in different neurons and in the same ones. In general, properties of Raw potassium channels appeared to be similar to RCK channels. However, Raw outward currents, in contrast to RCK currents, exhibit an intense rectification at test potentials higher than +20 to +40 mV. RCK and Raw channel subunits did not measurably coassemble into RCK/Raw heteromultimers after coinjecting RCK and Raw cRNA into Xenopus oocytes. These results suggest that members of the RCK and the Raw potassium channel families express potassium channels which form independent outward current systems. Combining the results of in situ hybridizations, immunocytochemical staining and expression of the cloned potassium channels in Xenopus oocytes demonstrates that unrestrained mixing of potassium channel subunits to form hybrid channels does not occur in the rat central nervous system. A single neuron is able to express multiple, independently assembled potassium channels.     

Model investigation of firing mechanisms of mammalian neurons

A model of electrical activity of mammalian neurons, based on properties of the corresponding ionic components of the transmembrane current, is suggested. Its analysis showed that two types of firing are possible for these neurons. First, the firing pattern is determined by fast sodium and delayed potassium currents, and the period of oscillations is 74±27 msec. Second, the firing pattern is determined by calcium and Ca-activated potassium currents, and the mean duration of the interspike interval is 850±40 msec. A comprehensive examination of all ionic currents enabled two mechanisms of lengthening of the interspike interval (adaptation) of the discharge to be predicted. One is due to the fast potassium current, the other to accumulation of Ca⁺⁺ ions inside the cell and the development of a Ca-activated potassium current.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 445–451, July–August, 1984.     

Analysis of conditional paralytic mutants in Drosophila sarco-endoplasmic reticulum calcium ATPase reveals novel mechanisms for regulating membrane excitability

Individual contributions made by different calcium release and sequestration mechanisms to various aspects of excitable cell physiology are incompletely understood. SERCA, a sarco-endoplasmic reticulum calcium ATPase, being the main agent for calcium uptake into the ER, plays a central role in this process. By isolation and extensive characterization of conditional mutations in the Drosophila SERCA gene, we describe novel roles of this key protein in neuromuscular physiology and enable a genetic analysis of SERCA function. At motor nerve terminals, SERCA inhibition retards calcium sequestration and reduces the amplitude of evoked excitatory junctional currents. This suggests a direct contribution of store-derived calcium in determining the quantal content of evoked release. Conditional paralysis of SERCA mutants is also marked by prolonged neural activity-driven muscle contraction, thus reflecting the phylogenetically conserved role of SERCA in terminating contraction. Further analysis of ionic currents from mutants uncovers SERCA-dependent mechanisms regulating voltage-gated calcium channels and calcium-activated potassium channels that together control muscle excitability. Finally, our identification of dominant loss-of-function mutations in SERCA indicates novel intra- and intermolecular interactions for SERCA in vivo, overlooked by current structural models.     

Characterization of Dir: a putative potassium inward rectifying channel in Drosophila

Potassium channels vary in their function and regulation, yet they maintain a number of important features - they are involved in the control of potassium flow, cell volume, cell membrane resting potential, cell excitability and hormone release. The potassium (K(+)) inward rectifier (Kir) superfamily of channels are potassium selective channels, that are sensitive to the concentration of K(+) ions. They are termed inward rectifiers since they allow a much greater K(+) influx than efflux. There are at least seven subfamilies of Kir channels, grouped according to sequence and functional similarities (Curr. Opin. Neurobiol. 5 (1995) 268; Annu. Rev. Physiol. 59 (1997) 171). While numerous Kir channels have been discovered in a variety of organisms, Drosophila inward rectifier (Dir) is the first putative inward rectifier to be studied in Drosophila. In fact, there are only three genes (including Dir) encoding putative inward rectifiers in the Drosophila genome. Though there are other known potassium channels in Drosophila such as ether-a-go-go and shaker, most are voltage-gated channels. As an important first step in characterizing Kir channels in Drosophila, we initiated studies on Dir.     

Spider toxins selectively block calcium currents in Drosophila

Toxins from spider venom, originally purified for their ability to block synaptic transmission in Drosophila, are potent and specific blockers of Ca2+ currents measured in cultured embryonic Drosophila neurons using the whole-cell, patch-clamp technique. Differential actions of toxins from two species of spiders indicate that different types of Drosophila neuronal Ca2+ currents can be pharmacologically distinguished. Hololena toxin preferentially blocks a non-inactivating component of the current, whereas Plectreurys toxin blocks both inactivating and non-inactivating components. These results suggest that block of a non-inactivating Ca2+ current is sufficient to block neurotransmitter release at Drosophila neuromuscular junction.     

The A-like potassium current of a leech neuron increases with age in cell culture

Single leech neurons isolated and maintained in culture sprout and form electrical and chemical synapses, as they do in vivo, retaining most of the electrical properties of the intact membrane. However, some cells, such as Retzius, Anterior Pagoda (AP) cells and motoneurons, exhibit consistent changes of biophysical characteristics, which mimic those induced by axotomy in vivo and are reversed after reconnection. To improve our understanding of the mechanisms involved in these alterations and of their physiological significance, we investigated the early changes in outward currents developed by cultured AP neurons, using the patch-clamp technique in the whole-cell recording configuration. Different currents were isolated and a differential sensitivity to the time spent in culture and to internal calcium was observed. Three potassium currents were dissected: an A-like current, a delayed rectifier and a third unidentified component. The A-like potassium current was significantly increased with neuronal age in cell culture and was a function of the internal Ca²⁺ concentration, whereas the two other potassium currents remained unchanged. Intracellular recordings performed from axotomized neurons of cultured ganglia revealed clear-cut alterations in spike adaptation, which might be due to changes of the A-like current. Accepted: 24 September 1998     

Ion channel blockade attenuates aggregated alpha synuclein induction of microglial reactive oxygen species: relevance for the pathogenesis of Parkinson's disease

Brain mononuclear phagocyte (perivascular macrophage and microglia, MG) inflammatory neurotoxins play a principal role in the pathogenesis of Parkinson's disease; chief among these are reactive oxygen species (ROS). We posit that aggregated, misfolded and oxidized alpha-synuclein (a major constituent of Lewy bodies), released or secreted from dying dopaminergic neurons, induces microglial ROS production that is regulated by ion channels and as such affects disease progression. To address this hypothesis, we performed patch clamp recordings of outward ionic currents in murine microglia and characterized their links to ROS production during alpha-synuclein stimulation. Aggregated nitrated alpha-synuclein induced ROS production in a dose-dependent manner that was inhibited by voltage-gated potassium current blockade, and to a more limited degree, by chloride current blockade. Interestingly, ROS produced in MG primed with tumor necrosis factor alpha and activated with phorbol myristate acetate was attenuated by voltage-gated potassium current blockade and more completely by chloride current blockade. In contrast, amyloid beta or cell membrane extract failed to induce microglial ROS production. Similar results were obtained using bone marrow-derived macrophages. The association of ROS production with specific plasma membrane ion currents provides a link between regulation of microglial ion transport and oxygen free radical production. Understanding these linkages may lead to novel therapeutics for Parkinson's disease where modulation of redox-related stress may slow disease progression.     

Interleukin-1β inhibits voltage-gated sodium currents in a time- and dose-dependent manner in cortical neurons

Interleukin-1β (IL-1β) is a multifunctional proinflammatory cytokine that plays a key role in the injuries and diseases of the central nervous system (CNS). A voltage-gated Na⁺ channel is essential for the excitability and electrical properties of neurons. However, it is not known whether IL-1β directly affects the central Na⁺ channels. In the present study, we examined the effects of IL-1β on Na⁺ currents in cultured cortical neurons using patch-clamp recording. Our results showed that IL-1β suppressed Na⁺ currents through its receptor in a time- and dose-dependent manner, but did not alter the voltage-dependent activation and inactivation. PKC and then p38 MAPK were involved in this inhibition. The spike amplitude was also inhibited by IL-1β in the doses that decreased the Na⁺ currents. Our findings revealed the inhibition of chronic IL-1β treatment on voltage-gated Na⁺ channels in the CNS, and showed that the action potential (AP) amplitude was reduced by IL-1β due to a decrease of Na⁺ currents.     

Pandinus imperator scorpion venom blocks voltage-gated potassium channels in GH3 cells

We examined the effects of Pandinus imperator scorpion venom on voltage-gated potassium channels in cultured clonal rat anterior pituitary cells (GH3 cells) using the gigohm-seal voltage-clamp method in the whole-cell configuration. We found that Pandinus venom blocks the voltage-gated potassium channels of GH3 cells in a voltage-dependent and dose-dependent manner. Crude venom in concentrations of 50-500 micrograms/ml produced 50-70% block of potassium currents measured at -20 mV, compared with 25-60% block measured at +50 mV. The venom both decreased the peak potassium current and shifted the voltage dependence of potassium current activation to more positive potentials. Pandinus venom affected potassium channel kinetics by slowing channel opening, speeding deactivation slightly, and increasing inactivation rates. Potassium currents in cells exposed to Pandinus venom did not recover control amplitudes or kinetics even after 20-40 min of washing with venom-free solution. The concentration dependence of crude venom block indicates that the toxins it contains are effective in the nanomolar range of concentrations. The effects of Pandinus venom were mimicked by zinc at concentrations less than or equal to 0.2 mM. Block of potassium current by zinc was voltage dependent and resembled Pandinus venom block, except that block by zinc was rapidly reversible. Since zinc is found in crude Pandinus venom, it could be important in the interaction of the venom with the potassium channel. We conclude that Pandinus venom contains toxins that bind tightly to voltage-dependent potassium channels in GH3 cells. Because of its high affinity for voltage-gated potassium channels and its irreversibility, Pandinus venom may be useful in the isolation, mapping, and characterization of voltage-gated potassium channels.     

K+ channel expression in primary cell cultures mediated by vaccinia virus

A recombinant vaccinia virus (VV) was used to express functional Drosophila Shaker H4 K+ channels in primary cell cultures from rat heart (atrial and ventricular myocytes, fibroblasts), autonomic ganglia (SCG neurons) and CNS (hippocampal neurons, cerebral astroglia). In most cells the expressed currents possessed the typical characteristics of the native Drosophila muscle A currents; a few cells showed evidence of hetero-oligomers with new properties. The maximum current density corresponded to a channel density of 2-3/microns 2. Voltage recordings in heart cells showed altered action potential waveforms after successful infection. VV vectors thus are useful for studying altered excitability and cell-specific processing of ion channel proteins.