Properties of a Sodium Channel (Nax) Activated by Strong Depolarization of Xenopus Oocytes

Short (<1 sec) duration depolarization of Xenopus laevis oocytes to voltages greater than +40 mV activates a sodium-selective channel (Na(x)) with sodium permeability five to six times greater than the permeability of other monovalent cations examined, including K+, Rb+, Cs+, TMA+, and Choline+. The permeability to Li+ is about equal to that of Na+. This channel was present in all oocytes examined. The kinetics, voltage dependence and pharmacology of Na(x)distinguish it from TTX-sensitive or epithelial sodium channels. It is also different from the sodium channel of Xenopus oocytes activated by prolonged depolarization, which is more highly selective for Na+, requires prolonged depolarization to be activated, and is blocked by Li+. Intracellular Mg2+ reversibly inhibits Na(x), whereas extracellular Mg2+ does not have an inhibitory effect. Intracellular Mg2+ inhibition of Na(x), is voltage dependent, suggesting that Mg2+ binding occurs within the membrane field. Eosin is also a reversible voltage-dependent intracellular inhibitor of Na(x), suggesting that a P-type ATPase may mediate the current. An additional cytoplasmic factor is involved in maintaining Na(x) since the current runs down in internally perfused oocytes and excised membrane patches. The rundown is reversible by reintroduction of the membrane patch into oocyte cytoplasm. The cytoplasmic factor is not ATP, because ATP has no effect on Na(x) current magnitude in either cut-open or inside-out patch preparations. Extracellular Gd3+ is also an inhibitor of Na(x). Na(x) activation follows a sigmoid time course. Its half-maximal activation potential is +100 mV and the effective valence estimated from the steepness of conductance activation is 1.0. Na(x) deactivates monoexponentially upon return to the holding potential (-40 mV). The deactivation rate is voltage dependent, increasing at more negative membrane potentials.     

G-protein Subunits Expressed in Catfish Olfactory Receptor Neurons

Olfactory signal transduction in a number of species has beenshown to be mediated by heterotrimeric GTP-binding proteins(G-proteins). The expression of different G-proteins in channelcatfish (Ictalurus punctatus) olfactory epithelium was investigatedusing antibodies to both the     

弱激光对大鼠海马神经元钠通道特性的影响

利用波长670nm、功率5mW的半导体激光器照射急性分离的大鼠海马CA3区锥体神经元,应用全细胞膜片钳技术研究其电压门控Na 通道的特性.实验发现:弱激光作用5min时,Na 通道激活电位和峰值电位开始向负电位方向移动,7min激光作用达稳定;激光照射对Na 通道电流峰值无影响,对照组和激光照射组峰值电流密度分别为(-383.51±26.93)pA/pF和(-368.36±33.14)pA/pF(n=8,P>0.05);激光作用降低了Na 通道的激活阈值电位和峰值电位,对照组通道电流在-40mV激活,-30mV达峰值,激光照射组通道电流在-60mV激活,-40mV达峰值;激光照射改变了Na 通道半数激活电压和斜率因子,对照组和激光照射组的半数激活电压分别为(-42.091±1.537)mV和(-54.971±1.846)mV(n=8,P<0.01),斜率因子分别为(1.529±0.667)mV和(2.634±0.519)mV(n=8,P<0.05).结果表明,弱激光照射海马神经元可改变Na 通道的激活特性,从而影响动作电位的去激化过程,进而会引起神经元细胞生理功能发生变化.     

The Lck Tyrosine Kinase Is Expressed in Brain Neurons

Abstract: The lck gene product, p56^lck, is a member of the src-related family of protein tyrosine kinases. It is known as lymphocyte specific and involved in thymocyte development and in the immune response mediated by the T cell receptor. We report that the lck gene is also expressed in adult mouse CNS and that brain p56^lck is similar to the thymus protein. In situ hybridization and immunohistochemistry show that the lck gene is expressed in neurons throughout the brain in distinct regions, including hippocampus and cerebellum. In primary cultures from fetal mouse brain, neuronal cells are immunoreactive to Lck antiserum. This suggests that the lck gene product might be involved in a new signal transduction pathway in mouse brain.     

Gating and Conduction Properties of a Sodium-activated Cation Channel from Lobster Olfactory Receptor Neurons

The gating and conduction properties of a channel activated by intracellular Na⁺ were studied by recording unitary currents in inside-out patches excised from lobster olfactory receptor neurons. Channel openings to a single conductance level of 104 pS occurred in bursts. The open probability of the channel increased with increasing concentrations of Na⁺. At 210 mm Na⁺, membrane depolarization increased the open probability e-fold per 36.6 mV. The distribution of channel open times could be fit by a single exponential with a time constant of 4.09 msec at −60 mV and 90 mm Na⁺. The open time constant was not affected by the concentration of Na⁺, but was increased by membrane depolarization. At 180 mm Na⁺ and −60 mV, the distribution of channel closed times could be fit by the sum of four exponentials with time constants of 0.20, 1.46, 8.92 and 69.9 msec, respectively. The three longer time constants decreased, while the shortest time constant did not vary with the concentration of Na⁺. Membrane depolarization decreased all four closed time constants. Burst duration was unaffected by the concentration of Na⁺, but was increased by membrane depolarization. Permeability for monovalent cations relative to that of Na⁺ (P _X /P _Na ), calculated from the reversal potential, was: Li⁺ (1.11) > Na⁺ (1.0) > K⁺ (0.54) > Rb⁺ (0.36) > Cs⁺ (0.20). Extracellular divalent cations (10 mm) blocked the inward Na⁺ current at −60 mV according to the following sequence: Mn²⁺ > Ca²⁺ > Sr²⁺ > Mg²⁺ > Ba²⁺. Relative permeabilities for divalent cations (P _Y /P _Na ) were Ca²⁺ (39.0) > Mg²⁺ (34.1) > Mn²⁺ (15.5) > Ba²⁺ (13.8) > Na⁺ (1.0). Both the reversal potential and the conductance determined in divalent cation-free mixtures of Na⁺ and Cs⁺ or Li⁺ were monotonic functions of the mole fraction, suggesting that the channel is a single-ion pore that behaves as a multi-ion pore when the current is carried exclusively by divalent cations. The properties of the channel are consistent with the channel playing a role in odor activation of these primary receptor neurons. Received: 17 September 1996/Revised: 15 November 1996     

Mouse VDAC Isoforms Expressed in Yeast: Channel Properties and Their Roles in Mitochondrial Outer Membrane Permeability

The channel-forming protein called VDAC forms the major pathway in the mitochondrial outer membrane and controls metabolite flux across that membrane. The different VDAC isoforms of a species may play different roles in the regulation of mitochondrial functions. The mouse has three VDAC isoforms (VDAC1, VDAC2 and VDAC3). These proteins and different versions of VDAC3 were expressed in yeast cells (S. cerevisiae) missing the major yeast VDAC gene and studied using different approaches. When reconstituted into liposomes, each isoform induced a permeability in the liposomes with a similar molecular weight cutoff (between 3,400 and 6,800 daltons based on permeability to polyethylene glycol). In contrast, electrophysiological studies on purified proteins showed very different channel properties. VDAC1 is the prototypic version whose properties are highly conserved among other species. VDAC2 also has normal gating activity but may exist in 2 forms, one with a lower conductance and selectivity. VDAC3 can also form channels in planar phospholipid membranes. It does not insert readily into membranes and generally does not gate well even at high membrane potentials (up to 80 mV). Isolated mitochondria exhibit large differences in their outer membrane permeability to NADH depending on which of the mouse VDAC proteins was expressed. These differences in permeability could not simply be attributed to different amounts of each protein present in the isolated mitochondria. The roles of these different VDAC proteins are discussed. Received: 19 June 1998/Revised: 1 April 1999     

成年大鼠海马CA1区锥体神经元外向整流氯离子单通道特性

采用膜片钳内面向外式技术,在急性分离成年大鼠海马CAl区锥体细胞上记录到了外向整流氯离子通道（outwardly rectifying chloride channel,ORCC）.长时间去极化（≥60 mV）刺激后,在30%的游离膜片上记录到有外向整流特性的单通道氯电流,膜电位在－60 mV到0 mV之间的单通道电导为(16.58±1.54) pS（n=10）,而在0 mV到+60 mV之间电导为（40.92±3.17） pS.通道开放概率有明显的电压依赖性(膜电位－60 mV时,P_o=0.44±0.12;膜电位为+60 mV时,P_o=0.86±0.06, n=10).在对称Cl^－浓度（150 mmol/L）时,通道翻转电位为(－4.17±1.84) mV.当溶液中部分NaCl被葡萄糖酸钠替代后,翻转电位为：(－34.23±4.86) mV (［Cl^－］_i/［Cl^－］_o=(30 mmol/L)/(150 mmol/L)),接近氯离子通道的理论值,这表明通道具有氯离子选择性.浴槽液中分别加入氯通道阻断剂DIDS和SITS可以使+40 mV的通道开放概率从(0.83±0.06)和(0.86±0.06)分别降低到(0.12±0.05)和(0.13±0.04)（n=5）,冲洗后可使开放概率基本恢复.上述研究结果显示,在成年大鼠海马CA1神经元上存在外向整流氯离子通道.     

帕金森病多巴胺神经细胞的差异表达基因分析

帕金森病(Parkinson’s disease,PD)是一种常见的神经退行性疾病,影响群体广泛。该文分析了帕金森病多巴胺细胞的表达和DNA甲基化信息,识别出了新的表达或者DNA甲基化异常的基因,并分析了这些基因与帕金森病的关联。结果表明:相比于正常细胞,帕金森病细胞中与微管形成相关的基因表达上调,这些基因包括SLAIN1、TAGLN3和TUBB2B;天然免疫关联的基因(如LY96)下调。另一个上调基因SCG5推测与免疫应激响应相关。DNA甲基化变化在启动子区显著,除了调节基因转录,这些变化可能通过PRC1和Pc G复合物改变染色质的活性水平。此外,表达水平和DNA甲基化同时调整的基因与轴突定向、胞内运输、神经元分化及迁移等功能有关。以上结果提供了对帕金森病机理特征的新的认识。     

Correlations in Ion Channel mRNA in Rhythmically Active Neurons

Background

To what extent do identified neurons from different animals vary in their expression of ion channel genes? In neurons of the same type, is ion channel expression highly variable and/or is there any relationship between ion channel expression that is conserved?

Methodology/Principal Findings

To address these questions we measured ion channel mRNA in large cells (LCs) of the crab cardiac ganglion. We cloned a calcium channel, caco, and a potassium channel, shaker. Using single-cell quantitative PCR, we measured levels of mRNA for these and 6 other different ion channels in cardiac ganglion LCs. Across the population of LCs we measured 3–9 fold ranges of mRNA levels, and we found correlations in the expression of many pairs of conductances

Conclusions/Significance

In previous measurements from the crab stomatogastric ganglion (STG), ion channel expression was variable, but many pairs of channels had correlated expression. However, each STG cell type had a unique combination of ion channel correlations. Our findings from the crab cardiac ganglion are similar, but the correlations in the LCs are different from those in STG neurons, supporting the idea that such correlations could be markers of cell identity or activity.     

The Synaptic Vesicle-Associated G Protein o-rab3 Is Expressed in Subpopulations of Neurons

Abstract: The distribution of o-rab3—a synaptic vesicle-associated low-molecular-weight GTP-binding protein—was studied in various neural tissues of the electric ray Torpedo marmorata. o-rab3 was shown to be associated selectively with isolated cholinergic synaptic vesicles derived from the electric organ. Gel filtration of cholinergic synaptic vesicles using Sephacryl S-1000 column chromatography demonstrated a copurification of o-rab3 with the synaptic vesicle content marker ATP and with SV2—a synaptic vesicle transmembrane glycoprotein. Indirect immunofluorescence using antibodies against o-rab3 and SV2 and a double labeling protocol revealed an identical distribution of both antigens in the cholinergic nerve terminals within the electric organ and at neuromuscular junctions. An immunoelectron microscopic analysis demonstrated the presence of o-rab3 at the surface of the synaptic vesicle membrane. In the CNS immunofluorescence of o-rab3 and SV2 overlap only in small and distinct areas. Whereas SV2 has an overall distribution in nerve terminals of the entire CNS, o-rab3 is restricted to a subpopulation of nerve terminals in the dorsolateral neuropile of the rhombencephalon and in the dorsal horn of the spinal cord. Our results demonstrate that the synaptic vesicle-associated G protein o-rab3 is specifically expressed only in subpopulations of neurons in the Torpedo CNS.     

G-protein {beta}{gamma} Subunit Genes Expressed in Olfactory Receptor Neurons

The expression of genes encoding G-protein ß subunitswas investigated in isolated olfactory receptor neurons fromchannel catfish. DNA sequencing of PCR products showed thatthe ß1, ß2, 2 and 3 genes were expressedin the neurons. Western blotting showed that at least threeof these subunit proteins were expressed. This first analysisof the expression of ß genes in olfactory receptorneurons suggests that these subunits may be involved in a varietyof transduction events in these cells. Chem. Senses 22: 587–592,1997.     

Physiological characterization of two genes for Na+ exclusion in durum wheat, Nax1 and Nax2

Durum wheat (Triticum turgidum L. subsp. durum Desf.) Line 149 contains two novel major genes for excluding Na(+) from leaf blades, named Nax1 and Nax2. The genes were separated into families containing a single gene and near-isogenic homozygous lines were selected. Lines containing either Nax1 or Nax2 had lower rates of Na(+) transport from roots to shoots than their near-isogenic pairs due to lower rates of net loading of the xylem, not to lower rates of net uptake from the soil or higher rates of retranslocation in the phloem. Nax1 and Nax2 lines also had higher rates of K(+) transport from root to shoot, resulting in an enhanced discrimination of K(+) over Na(+). Lines containing Nax1 differed from those containing Nax2 by unloading Na(+) from the xylem as it entered the shoot so that Na(+) was retained in the base of the leaf, leading to a high sheath to blade ratio of Na(+) concentration. Gradients in tissue concentrations of Na(+) along the leaf suggested that Na(+) was continually removed from the xylem. The Nax2 line did not retain Na(+) in the base of the leaf, suggesting that it functioned only in the root. The Nax2 gene therefore has a similar function to Kna1 in bread wheat (Triticum aestivum).     

Properties of Connexin26 Hemichannels Expressed in Xenopus Oocytes

1. Hemichannels formed by connexin26 (Cx26) on the horizontal cell dendrites that invaginate cone terminals in the vertebrate retina have been implicated in the feedback mechanism by which horizontal cells regulate transmitter release from cone photoreceptors. However, their membrane properties had not been studied previously, and it was unclear whether they could subserve their purported function at the membrane potentials over which horizontal cells operate. 2. We used the two-electrode voltage clamp technique to record the membrane currents and pharmacological properties of Cx26 hemichannels formed in the Xenopus oocyte expression system. 3. Oocytes expressing Cx26 exhibited large membrane conductances over a broad range of hyperpolarizing and depolarizing membrane potentials, and displayed little evidence of voltage-dependent gating, indicating that the hemichannels are constitutively open. The Cx26-mediated nonjunctional currents were relatively insensitive to quinine, a cinchona alkaloid that opens hemichannels formed by several other connexins. However, the hemichannel currents were blocked by carbenoxolone, a rise in extracellular calcium, or lowering intracellular pH. The currents could also be suppressed by reducing extracellular pH, and by the chloride channel blocker NPPB through its direct interaction with Cx26 hemichannels. 4. These findings provide a basis with which to evaluate the in situ pharmacological studies that attempt to assess the putative role of Cx26 hemichannels in the feedback pathway in the distal retina.     

The Pannexin 1 Channel Activates the Inflammasome in Neurons and Astrocytes

The inflammasome is a multiprotein complex involved in innate immunity. Activation of the inflammasome causes the processing and release of the cytokines interleukins 1β and 18. In primary macrophages, potassium ion flux and the membrane channel pannexin 1 have been suggested to play roles in inflammasome activation. However, the molecular mechanism(s) governing inflammasome signaling remains poorly defined, and it is undetermined whether these mechanisms apply to the central nervous system. Here we show that high extracellular potassium opens pannexin channels leading to caspase-1 activation in primary neurons and astrocytes. The effect of K⁺ on pannexin 1 channels was independent of membrane potential, suggesting that stimulation of inflammasome signaling was mediated by an allosteric effect. The activation of the inflammasome by K⁺ was inhibited by the pannexin 1 channel blocker probenecid, supporting a role of pannexin 1 in inflammasome activation. Co-immunoprecipitation of neuronal lysates indicates that pannexin 1 associates with components of the multiprotein inflammasome complex, including the P2X7 receptor and caspase-1. Moreover antibody neutralization of the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) blocked ATP-induced cell death in oocytes co-expressing P2X7 receptor and pannexin 1. Thus, in contrast to macrophages and monocytes in which low intracellular K⁺ has been suggested to trigger inflammasome activation, in neural cells, high extracellular K⁺ activates caspase-1 probably through pannexin 1.Pannexin 1 is a vertebrate ortholog of the invertebrate innexin gap junction proteins (1), but it does not appear to form functional gap junctions in vivo. Instead pannexin 1 acts as a membrane channel that carries ions and signaling molecules between the cytoplasm and the extracellular space (2, 3). As such, it is a candidate ATP release channel in various cell types, including erythrocytes, astrocytes, bronchial epithelial cells, and taste cells. Various functional roles have been ascribed to pannexin 1 including local vascular perfusion control and propagation of intercellular calcium waves (4–6). Recently pannexin 1 was also shown to form the large pore of the P2X7 purinergic receptor (7, 8). P2X7 plays a major role in inflammation, and its activation by extracellular ATP results in release of interleukin (IL)²-1β from macrophages, probably involving pannexin 1 as a signaling molecule (7).IL-1β production and maturation are tightly regulated by caspase-1 incorporated into large protein complexes termed inflammasomes (9–11). The molecular composition of the inflammasome depends on the identity of the NOD-like receptor (NLR) family member serving as a scaffold protein in the complex (12). The members of the cytosolic NLR family appear to recognize conserved microbial and viral components termed pathogen-associated molecular patterns in intracellular compartments (13). The bipartite adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) bridges the interaction between NLR proteins and inflammatory caspases and plays a central role in the assembly of inflammasomes and the activation of caspase-1 in response to a broad range of pathogen-associated molecular patterns and intracellular pathogens (14). In addition, the inflammasome can be activated by danger-associated molecular patterns, molecules endogenous to the organism that signal stress or injury, including extracellular ATP acting at ionotropic P2X7 receptors, fibronectin, or monosodium urate crystals (15, 16). Moreover it has been suggested that a rapid K⁺ efflux through ATP-activated P2X7 receptors induces inflammasome assembly (17–20).Despite the recent advances in the understanding of accessory proteins required for full activation of caspase-1, little is known about the signaling pathways that trigger inflammasome activation, particularly in the central nervous system (CNS). Recently we reported that spinal cord neurons contain the NLRP1/NALP1 inflammasome consisting of NLRP1, ASC, caspase-1, caspase-11, and the X-linked inhibitor of apoptosis protein (XIAP) and that spinal cord injury induces rapid activation of the inflammasome, causing processing and secretion of IL-1β and IL-18. Moreover antibody neutralization of ASC reduces caspase-1 activation and IL-1 cytokine processing, leading to significant tissue sparing and functional improvement (21). In this study, we focused on signaling events coupling pannexin 1 and P2X7 receptors to rapid caspase-1 activation in primary neurons and astrocytes. We provide compelling evidence that high extracellular K⁺ opens the pannexin 1 channel and activates inflammasomes in neurons and astrocytes, but not THP-1 cells, thus leading to caspase-1 activation. This signaling pathway in neurons is mediated through protein interactions between pannexin 1 and inflammasome proteins. We also provide evidence that ATP acting on P2X7 induces rapid cell death and that antibody neutralization of ASC blocks ATP-induced cell death. Thus, contrary to the widely accepted view in macrophages and monocytes that low intracellular K⁺ triggers inflammasome activation, high extracellular K⁺ surrounding cells such as neurons and astrocytes opens pannexin 1 channels and induces processing of caspase-1.     

Determinants of Voltage-Gated Potassium Channel Surface Expression and Localization in Mammalian Neurons

Neurons strictly regulate expression of a wide variety of voltage-dependent ion channels in their surface membranes to achieve precise yet dynamic control of intrinsic membrane excitability. Neurons also exhibit extreme morphological complexity that underlies diverse aspects of their function. Most ion channels are preferentially targeted to either the axonal or somatodendritic compartments, where they become further localized to discrete membrane subdomains. This restricted accumulation of ion channels enables local control of membrane signaling events in specific microdomains of a given compartment. Voltage-dependent K⁺ (Kv) channels act as potent modulators of diverse excitatory events such as action potentials, excitatory synaptic potentials, and Ca²⁺ influx. Kv channels exhibit diverse patterns of cellular expression, and distinct subtype-specific localization, in mammalian central neurons. Here we review the mechanisms regulating the abundance and distribution of Kv channels in mammalian neurons and discuss how dynamic regulation of these events impacts neuronal signaling.     

Prostatic Acid Phosphatase Is Expressed in Peptidergic and Nonpeptidergic Nociceptive Neurons of Mice and Rats

Thiamine monophosphatase (TMPase, also known as Fluoride-resistant acid phosphatase or FRAP) is a classic histochemical marker of small- to medium-diameter dorsal root ganglia (DRG) neurons and has primarily been studied in the rat. Previously, we found that TMPase was molecularly identical to Prostatic acid phosphatase (PAP) using mice. In addition, PAP was expressed in a majority of nonpeptidergic, isolectin B4-binding (IB4⁺) nociceptive neurons and a subset of peptidergic, calcitonin gene-related peptide-containing (CGRP⁺) nociceptive neurons. At the time, we were unable to determine if PAP was present in rat DRG neurons because the antibody we used did not cross-react with PAP in rat tissues. In our present study, we generated a chicken polyclonal antibody against the secretory isoform of mouse PAP. This antibody detects mouse, rat and human PAP protein on western blots. Additionally, this antibody detects PAP in mouse and rat small- to medium-diameter DRG neurons and axon terminals in lamina II of spinal cord. In the rat, 92.5% of all PAP⁺ cells bind the nonpeptidergic marker IB4 and 31.8% of all PAP⁺ cells contain the peptidergic marker CGRP. Although PAP is found in peptidergic and nonpeptidergic neurons of mice and rats, the percentage of PAP⁺ neurons that express these markers differs between species. Moreover, PAP⁺ axon terminals in the rat partially overlap with Protein kinase Cγ (PKCγ⁺) interneurons in dorsal spinal cord whereas PAP⁺ axon terminals in the mouse terminate dorsal to PKCγ⁺ interneurons. Collectively, our studies highlight similarities and differences in PAP localization within nociceptive neurons of mice and rats.