Single-channel analysis of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons

Tetrodotoxin-sensitive and tetrodotoxin-resistant single sodium channel currents were recorded from rat dorsal root ganglion neurons. The two types of sodium channel currents could be distinguished by the effects of predepolarization, 10 nM tetrodotoxin, and the inactivation during depolarization. Single-channel conductances were calculated to be 6.3 and 3.4 pS in the tetrodotoxin-sensitive and tetrodotoxin-resistant channels, respectively.     

钾通道拮抗剂IBTX对大鼠脑缺血再灌注损伤的保护作用及其机制的试验研究

目的:探讨大鼠脑缺血再灌注损伤后,海马CAl区锥体细胞caspaso-3表达及细胞凋亡的变化及使用钾通道拮抗剂IBTX(ibetiotoxin)的保护作用及其保护机制。方法:通过腹腔注射硝普钠加双侧颈总动脉夹闭建立大鼠全脑缺血一再灌模型,侧脑室注射IBTX,免疫组化法测caspase-3表达情况,TUNEL法检测细胞凋亡。结果:大鼠全脑缺血-再灌注24小时后,海马CAl区锥体细胞caspase-3阳性表达,TUNEL法检测有细胞凋亡,在用药IBTX组,caspase-3阳性表达减少,TUNEI法检测细胞凋亡亦有明显降低。结论:大鼠全脑缺血-再灌注24小时后海马CAl区锥体细胞出现细胞凋亡,使用钾通道拮抗剂IBTX可减少细胞凋亡的发生,从而发挥保护作用。     

Functional identification of an outwardly rectifying pH‐ and anesthetic‐sensitive leak K+ conductance in hippocampal astrocytes

Astrocytes function as spatial K⁺ buffers by expressing a rich repertoire of K⁺ channels. Earlier studies suggest that acid‐sensitive tandem‐pore K⁺ channels, mainly TWIK‐related acid‐sensitive K⁺ (TASK) channels, mediate part of the passive astroglial membrane conductance. Here, using a combination of electrophysiology and pharmacology, we investigated the presence of TASK‐like conductance in hippocampal astrocytes of rat brain slices. Extracellular pH shifts to below 7.4 (or above 7.4) induced a prominent inward (or outward) current in astrocytes in the presence of tetrodotoxin, a Na⁺ channel blocker, and 4,4′‐diisothiocyanatostilbene‐2,2’‐disulfonate, a co‐transporter blocker. The pH‐sensitive current was insensitive to quinine, a potent blocker of tandem‐pore K⁺ channels including TWIK‐1 and TREK‐1 channels. Voltage‐clamp analysis revealed that the pH‐sensitive current exhibited weak outward rectification with a reversal potential of ?112 mV, close to the Nernst equilibrium potential for K⁺. Furthermore, the current–voltage relationship was well fitted with the Goldman–Hodgkin–Katz current equation for the classical open‐rectifier ‘leak’ K⁺ channel. The pH‐sensitive K⁺ current was potentiated by TASK channel modulators such as the volatile anesthetic isoflurane but depressed by the local anesthetic bupivacaine. However, unlike TASK channels, the pH‐sensitive current was insensitive to Ba²⁺ and quinine. Thus, the molecular identity of the pH‐sensitive leak K⁺ channel is unlikely to be attributable to TASK channels. Taken together, our results suggest a novel yet unknown leak K⁺ channel underlying the pH‐ and anesthetic‐sensitive background conductance in hippocampal astrocytes.     

Immunolocalization of the Ca2+-activated K+ channel Slo1 in axons and nerve terminals of mammalian brain and cultured neurons

Ca(2+)-activated voltage-dependent K(+) channels (Slo1, KCa1.1, Maxi-K, or BK channel) play a crucial role in controlling neuronal signaling by coupling channel activity to both membrane depolarization and intracellular Ca(2+) signaling. In mammalian brain, immunolabeling experiments have shown staining for Slo1 channels predominantly localized to axons and presynaptic terminals of neurons. We have developed anti-Slo1 mouse monoclonal antibodies that have been extensively characterized for specificity of staining against recombinant Slo1 in heterologous cells, and native Slo1 in mammalian brain, and definitively by the lack of detectable immunoreactivity against brain samples from Slo1 knockout mice. Here we provide precise immunolocalization of Slo1 in rat brain with one of these monoclonal antibodies and show that Slo1 is accumulated in axons and synaptic terminal zones associated with glutamatergic synapses in hippocampus and GABAergic synapses in cerebellum. By using cultured hippocampal pyramidal neurons as a model system, we show that heterologously expressed Slo1 is initially targeted to the axonal surface membrane, and with further development in culture, become localized in presynaptic terminals. These studies provide new insights into the polarized localization of Slo1 channels in mammalian central neurons and provide further evidence for a key role in regulating neurotransmitter release in glutamatergic and GABAergic terminals.     

Cerebrovascular and neurological perspectives on adrenoceptor and calcium channel modulating pharmacotherapies

Adrenoceptor and calcium channel modulating medications are widely used in clinical practice for acute neurological and systemic conditions. It is generally assumed that the cerebrovascular effects of these drugs mirror that of their systemic effects – and this is reflected in how these medications are currently used in clinical practice. However, recent research suggests that there are distinct cerebrovascular-specific effects of these medications that are related to the unique characteristics of the cerebrovascular anatomy including the regional heterogeneity in density and distribution of adrenoceptor subtypes and calcium channels along the cerebrovasculature. In this review, we critically evaluate existing basic science and clinical research to discuss known and putative interactions between adrenoceptor and calcium channel modulating pharmacotherapies, the neurovascular unit, and cerebrovascular anatomy. In doing so, we provide a rationale for selecting vasoactive medications based on lesion location and lay a foundation for future investigations that will define neuroprotective paradigms of adrenoceptor and calcium channel modulating therapies to improve neurological outcomes in acute neurological and systemic disorders.     

Distribution of ω-Conotoxin GVIA binding sites in teleost cerebellar and electrosensory neurons

The distribution of ω-Conotoxin GVIA (CgTx) binding sites was used to localize putative N-type Ca²⁺ channels in an electrosensory cerebellar lobule, the eminentia granularis pars posterior, and in the electrosensory lateral line lobe of a gymnotiform teleost (Apteronotus leptorhynchus). The binding sites for CgTx revealed by an anti-CgTx antibody had a consistent distribution on somatic and dendritic membranes of specific cell types in both structures. The distribution of CgTx binding was unaffected by co-incubation with nifedipine or Aga Toxin IVA, blocking agents for L- and P-type Ca²⁺ channels, respectively. Incubation with CgTx in the presence of varying levels of extracellular Ca²⁺ altered the number but not the cell types exhibiting immunolabel. A punctate immunolabel was detected on somatic membranes of granule and stellate cell interneurons in both the eminentia granularis pars posterior and the electrosensory lateral line lobe. Punctate CgTx binding sites were also present on spherical cell somata and on the large presynaptic terminals of primary afferents that terminate on spherical cells in the electrosensory lateral line lobe. No label was detected in association with distal dendritic membranes of any cell class, or with parallel fibers in the respective molecular layers. Binding sites for CgTx in the eminentia granularis are consistent with the established role for N-type Ca²⁺ channels in cell migrations, an activity which is known to persist in this layer in adult Apteronotus. The distribution of labeled stellate cells with respect to topographic maps in the electrosensory lateral line lobe further suggest that N-type Ca²⁺ channels are expressed in relation to functional activity across these sensory maps. © 1996 Wiley-Liss, Inc.     

Na+ currents near and away from endplates on human fast and slow twitch muscle fibers

Fast and slow twitch muscle fibers have distinct contractile properties. Here we determined that membrane excitability also varies with fiber type. Na⁺ currents (I_NA) were studied with the loose-patch voltage clamp technique on 29 histochemically classified human intercostal skeletal muscle fibers at the endplate border and <200 μm from the endplate (extrajunctional). Fast and slow twitch fibers showed slow inactivation of endplate border and extrajunctional I_NA and had increased I_NA at the endplate border compared to extrajunctional membrane. The voltage dependencies of I_NA were similar on the endplate border and extrajunctional membrane, which suggests thatboth regions have physiclogically similar channels. Fast twitch fibers had larger I_NA on the endplate border and extrajunctional membrane and manifest fast and slow inactivation of I_NA at more negative potentials than slow twitch fibers. For normal muscle, the differences between I_NA on fast and slow twitch fibers might: (1) enable fast twitch fibers to operate at high firing frequencies for brief periods; and (2) enable slow twitch fibers to operate at low firing frequencies for prolonged times. Disorders of skeletal membrane excitability, such as the periodic paralyses and myotonias, may impact fast and slow twitch fibers differently due to the distinctive Na⁺ channel properties of each fiber type. © 1993 John Wiley & Sons, Inc.     

Fampridine treatment and walking distance in multiple sclerosis: A randomised controlled trial

Objective

To explore the benefits of modified-release fampridine on walking distance in MS.

Lignstrazine inhibits high voltage-gated Ca^2＋ and TTX-resistant Na^＋ channels of primary sensory neuron and thermal nociception in the rat： a study on peripheral mechanism

Objective Ligustrazine, also named as tetramethylpyrazine, is a compound purified from Ligusticum chuanxiong hort and has ever been testified to be a calcium antagonist. The present investigation was to determine the antinociceptive effect of ligustrazine and, if any, the peripheral ionic mechanism involved. Methods Paw withdrawal Latency （PWL） to noxious heating was measured in vivo and whole-cell patch recording was performed on small dorsal root ganglion （DRG） neurons. Results Intraplantar injection of ligustrazine （0.5 mg in 25 μl） significantly prolonged the withdrawal latency of ipsilateral hindpaw to noxious heating in the rat. Ligustrazine not only reversibly inhibited high-voltage gated calcium current of dorsal root ganglion （DRG） neuron in dose-dependent manner with IC50 of 1.89 mmol/L, but also decreased tetrodotoxin （TTX） -resistant sodium current in relatively selective and dose-dependent manner with IC50 of 2.49 mmol/L. Conclusion The results suggested that ligustrazine could elevate the threshold of thermal nociception through inhibiting the high-voltage gated calcium current and TTX-resistant sodium current of DRG neuron .in the rat.     

Two types of K channels in excised patches of somatic membrane of the leech AP neuron

The patch-clamp technique has been applied to the somatic membrane of the leech AP neurons. Ionic currents from single potassium channels were recorded in inside-out configuration. Two types of channels, sharing close values of conductance in symmetrical K+, were identified as distinct, according to their properties of rectification, Ca2+ sensitivity and voltage dependence. The channels designated as VCI exhibited an outward rectification and their gating was quite independent on changes of patch potential and of [Ca2+]i. The channels designated as VCD showed a linear I-V relationship and their activity was dependent on both the membrane potential and the intracellular [Ca2+].     

Gender differences in penetrance and phenotype in hypokalemic periodic paralysis

Introduction: Hypokalemic periodic paralysis (HypoPP) is an autosomal dominant skeletal muscle ion channelopathy. Sex hormones are natural ion channel regulators. Different sex hormones have different effects on ion channels. A comparison of the penetrance and phenotype between males and females with HypoPP mutations should aid in proving that sex hormones play different roles in HypoPP and also provide the basis for the development of therapies against HypoPP. Methods: We identified all mutation carriers in 4 HypoPP families using PCR sequencing techniques. All patients underwent clinical investigation. Results: There were 8 men and 7 women mutation carriers in the 4 families. Male carriers had 100% penetrance, but female penetrance was only 28.57%. The highest attack frequency was 50–150 times/year for the men, whereas it was 30–50 times/year for the women. The attacks disappeared during pregnancy. Conclusions: The penetrance and attack frequency were lower in women than in men with HypoPP mutations. Muscle Nerve, 2013     

Ion channel expression by white matter glia: I. Type 2 astrocytes and oligodendrocytes

White matter is a compact structure consisting primarily of neuronal axons and glial cells. As in other parts of the nervous system, the function of glial cells in white matter is poorly understood. We have explored the electrophysiological properties of two types of glial cells found predominantly in white matter: type 2 astrocytes and oligodendrocytes. Whole-cells and single-channel patch-clamp techniques were used to study these cell types in postnatal rat optic nerve cultures prepared according to the procedures of Raff et al. (Nature, 303:390-396, 1983b). Type 2 astrocytes in culture exhibit a "neuronal" channel phenotype, expressing at least six distinct ion channel types. With whole-cell recording we observed three inward currents: a voltage-sensitive sodium current qualitatively similar to that found in neurons and both transient and sustained calcium currents. In addition, type 2 astrocytes had two components of outward current: a delayed potassium current which activated at 0 mV and an inactivating calcium-dependent potassium current which activated at -30 mV. Type 2 astrocytes in culture could be induced to fire single regenerative potentials in response to injections of depolarizing current. Single-channel recording demonstrated the presence of an outwardly rectifying chloride channel in both type 2 astrocytes and oligodendrocytes, but this channel could only be observed in excised patches. Oligodendrocytes expressed only one other current: an inwardly rectifying potassium current that is mediated by 30- and 120-pS channels. Because these channels preferentially conduct potassium from outside to inside the cell, and because they are open at the resting potential of the cell, they would be appropriate for removing potassium from the extracellular space; thus it is proposed that oligodendrocytes, besides myelinating axons, play an important role in potassium regulation in white matter. The conductances present in oligodendrocytes suggest a "modulated Boyle and Conway mechanism" of potassium accumulation.     

Mechanosensitive ion channels in interstitial cells of Cajal and smooth muscle of the gastrointestinal tract

Normal gastrointestinal (GI) motility is required to mix digestive enzymes and food and to move content along the GI tract. Underlying the complex motor patterns of the gut are electrical events that reflect ion flux across cell membranes. Smooth muscle electrical activity is directly influenced by GI interstitial cells of Cajal, whose rhythmic oscillations in membrane potential in part determine the excitability of GI smooth muscle and its response to neuronal input. Coordinated activity of the ion channels responsible for the conductances that underlie ion flux in both smooth muscle and interstitial cells is a requisite for normal motility. These conductances are regulated by many factors, including mechanical stress. Recent studies have revealed mechanosensitivity at the level of the ion channels, and the mechanosensor within the channel has been identified in many cases. This has led to better comprehension of the role of mechanosensitive conductances in normal physiology and will undoubtedly lead to understanding of the consequences of disturbances in these conductances.     

Developmental and cellular expression pattern of epithelial sodium channel alpha, beta and gamma subunits in the inner ear of the rat

Endolymphatic ion composition in the adult inner ear is characterized by high K(+) and low Na(+) concentration. This unique ion composition is essential for proper functioning of sensory processing. Although a lot has been learned in recent years about molecules involved in K(+) transport in inner ear, the molecules involved in Na(+) transport are only beginning to emerge. The epithelial Na(+) channel (ENaC) is a highly selective Na(+) channel that is expressed in many Na(+)-reabsorbing tissues. The aim of our study was to investigate whether ENaC is expressed in inner ear of rats and could account for Na(+) reabsorption from endolymph. We detected mRNA for the three channel-forming subunits (alpha, beta and gamma ENaC) in cochlea, vestibular system and endolymphatic sac. mRNA abundance increased during the first 12 days of life in cochlea and vestibular system, coinciding with decreasing Na(+) concentration in endolymph. Expression was strongest in epithelial cells lining scala media, most notably Claudius' cells. As these cells are characterized by a very negative resting potential they would be ideally suited for reabsorption of Na(+). mRNA abundance in endolymphatic sac decreased during the first 6 days of life, suggesting that ENaC might be implicated in reabsorption of endolymph in the endolymphatic sac of neonatal animals. Together, our results suggest that the epithelial Na+ channel is a good candidate for a molecule involved in Na(+) homeostasis in inner ear.     

Changes in Na(+) channel expression and nodal persistent Na(+) currents associated with peripheral nerve regeneration in mice

Patients with peripheral neuropathy frequently suffer from positive sensory (pain and paresthesias) and motor (muscle cramping) symptoms even in the recovery phase of the disease. To investigate the pathophysiology of increased axonal excitability in peripheral nerve regeneration, we assessed the temporal and spatial expression of voltage-gated Na(+) channels as well as nodal persistent Na(+) currents in a mouse model of Wallerian degeneration. Crushed sciatic nerves of 8-week-old C57/BL6J male mice underwent complete Wallerian degeneration at 1 week. Two weeks after crush, there was a prominent increase in the number of Na(+) channel clusters per unit area, and binary or broad Na(+) channel clusters were frequently found. Excess Na(+) channel clusters were retained up to 20 weeks post-injury. Excitability testing using latent addition suggested that nodal persistent Na(+) currents markedly increased beginning at week 3, and remained through week 10. These results suggest that axonal regeneration is associated with persistently increased axonal excitability resulting from increases in the number and conductance of Na(+) channels.     

Neuronal voltage-gated ion channels are genetic modifiers of generalized epilepsy with febrile seizures plus

Mutations in the neuronal voltage-gated sodium channel genes SCN1A and SCN2A are associated with inherited epilepsies, including genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome (severe myoclonic epilepsy of infancy). The clinical presentation and severity of these epilepsies vary widely, even in people with the same mutation, suggesting the action of environmental or genetic modifiers. To gain support for the hypothesis that genetic modifiers can influence clinical presentation in patients with SCN1A-derived GEFS+, we used mouse models to study the effect of combining the human GEFS+ mutation SCN1A-R1648H with SCN2A, KCNQ2, and SCN8A mutations. Knock-in mice heterozygous for the R1648H mutation (Scn1a(RH/+)) have decreased thresholds to induced seizures and infrequent spontaneous seizures, whereas homozygotes display spontaneous seizures and premature lethality. Scn2a(Q54) transgenic mice have a mutation in Scn2a that results in spontaneous, adult-onset partial motor seizures, and mice carrying the Kcnq2-V182M mutation exhibit increased susceptibility to induced seizures, and rare spontaneous seizures as adults. Combining the Scn1a-R1648H allele with either Scn2a(Q54) or Kcnq2(V182M/+) results in early-onset, generalized tonic-clonic seizures and juvenile lethality in double heterozygous mice. In contrast, Scn8a mutants exhibit increased resistance to induced seizures. Combining the Scn1a-R1648H and Scn8a-med-jo alleles restores normal thresholds to flurothyl-induced seizures in Scn1a(RH/+) heterozygotes and improved survival of Scn1a(RH/RH) homozygotes. Our results demonstrate that variants in Scn2a, Kcnq2, and Scn8a can dramatically influence the phenotype of mice carrying the Scn1a-R1648H mutation and suggest that ion channel variants may contribute to the clinical variation seen in patients with monogenic epilepsy.     

Expression of sodium channel subtypes during development in rat skeletal muscle.

This study contrasts the developmental patterns of expression of 2 subtypes of the voltage-dependent sodium channel in rat muscle that are differentiated by their immunoreactivity with monoclonal antibodies raised to the purified muscle sodium channel protein. One subtype is found in the transverse tubular (T) system of slow twitch fibers as well as the plasma membrane of fast and slow twitch fibers in the anterior tibial and soleus muscles. The second is present in the plasma membrane in all fibers of both muscles. The transverse tubular subtype exhibits 2 immunocytochemical staining patterns within muscle fibers, reticular and homogeneous, which may represent labeling of the developing T tubular system and of a cytoplasmic pool of alpha subunits of the sodium channel respectively. The reticular pattern eventually disappears in fast twitch fibers but persists into the adult stage in slow twitch fibers. The homogeneous pattern is also seen with antibodies to the plasma membrane subtype and disappears in early development as immunoreactivity to both subtypes gradually appears in the surface membrane. A reticular pattern is never seen with the plasma membrane subtype. The factors that modulate the expression of these subtypes is unknown.     

The bradykinin receptor — a putative receptor-operated channel in PC12 cells: studies of neurotransmitter release and inositol phosphate accumulation

Bradykinin (BK) induced [³H]norepinephrine ([³H]NE) release and phosphatidylinositol turnover were investigated in PC12 cells. Induction of [³H]NE release by BK is mediated by activation of BK-B₂-receptors, as determined using type specific BK receptor antagonists. BK induces [³H]NE release with a half maximal effective concentration of30 ± 0.5nM, and reaches maximal net fractional release of9.0 ± 1% with 200 nM BK. The BK-induced release is Ca²⁺ dependent, reaching maximal release at 1.0 mM Ca²⁺, is pertussis toxin insensitive (1 μg/ml), slightly increased by a dibutyryl cAMP (1 mM) and not affected by inhibitors of the cyclooxygenase or lipoxygenase pathways. Voltage-sensitive Ca²⁺ channel blockers, verapamil (10 μM), nifedipine (10 μM), and ω-conotoxin (CgTx 10 nM), do not block the BK-induced release. However, a considerable inhibitory effect was obtained by divalent cations Co²⁺ (ED₅₀ = 0.2mM) and Ni²⁺ (ED_50²⁺ = 1mM). These results indicate the involvement of a Ca²⁺ channel in the BK-mediated release which is different from the L- or N-type voltage sensitive calcium channels. Whereas [Ca²⁺]_ex is essential for the BK-induction of catecholamine release, the rise in level of InsP's induced by BK in the presence or in the absence of [Ca²⁺]_ex is similar up to concentration of 1 μM. This indicates that the rise in InsP's induced by BK is not sufficient to cause neurotransmitter release. Moreover, subsequent addition of Ca²⁺ to BK-stimulated cells in Ca²⁺-free medium yields no release. Hence, no activity triggered by BK alone could be further stimulated by Ca²⁺ for induction of release. Protein kinase C inhibitors polymyxin B, K_252a, sangivamicin, and Ara-A, do not affect release induced by BK, indicating that also the diacylglycerol pathway activated by phospholipase C is not involved in the BK-mediated release. Since (a) the receptor-mediated release is absolutely calcium-dependent, with no release detected when Ca²⁺ is omitted from the extracellular medium, and (b) the receptor-triggered release of Ca²⁺ from intracellular stores is independent of [Ca²⁺]_ex⁷, it appears that calcium influx, and not Ca²⁺ released from intracellular stores, is the signal for stimulating release. Therefore, it is suggested that the primary signal stimulating release is Ca²⁺ influx via a specific calcium channel, and that the BK receptor may be coupled to this channel, which could be classified as a receptor-operated channel.     

Differential inhibition of catecholamine secretion by amitriptyline through blockage of nicotinic receptors,sodium channels,and calcium channels in bovine adrenal chromaffin cells

We investigated the effects of amitriptyline, a tricyclic antidepressant, on [³H]norepinephrine ([³H]NE) secretion and ion flux in bovine adrenal chromaffin cells. Amitriptyline inhibited [³H]NE secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) and 70 mM K⁺. The half maximal inhibitory concentration (IC₅₀) was 2 μM and 9 μM, respectively. Amitriptyline also inhibited the elevation of cytosolic calcium ([Ca²⁺]_i) induced by DMPP and 70 mM K⁺ with IC₅₀ values of 1.1 μM and 35 μM, respectively. The rises in cytosolic sodium ([Na⁺]_i) and [Ca²⁺]_i induced by the Na⁺ channel activator veratridine were also inhibited by amitriptyline with IC₅₀ values of 7 μM and 30 μM, respectively. These results suggest that amitriptyline at micromolar concentrations inhibits both voltage-sensitive calcium (VSCCs) and sodium channels (VSSCs). Furthermore, submicromolar concentrations of amitriptyline significantly inhibited DMPP-induced [³H]NE secretion and [Ca²⁺]_i rise, but not veratridine- or 70 mM K⁺-induced responses, suggesting that nicotinic acetylcholine receptors (nAChR) as well as VSCCs and VSSCs can be targeted by amitriptyline. DMPP-induced [Na⁺]_i rise was much more sensitive to amitriptyline than the veratridine-induced rise, suggesting that the influx of Na⁺ and Ca²⁺ through the nAChR itself is blocked by amitriptyline. Receptor binding competition analysis showed that binding of [³H]nicotine to chromaffin cells was significantly affected by amitriptyline at submicromolar concentrations. The data suggest that amitriptyline inhibits catecholamine secretion by blocking nAChR, VSSC, and VSCC. Synapse 29:248–256, 1998. © 1998 Wiley-Liss, Inc.